UNIVERSITAT POLITÈCNICA DE
VALÈNCIA
DEPARTAMENTO DE BIOTECNOLOGÍA
Mu a ional analysis o RAS genes in me as a ic
colo ec al cance : conco dance be ween umou
issue and liquid biopsy
TRABAJO FIN DE MÁSTER EN BIOTECNOLOGÍA BIOMÉDICA
ALUMNO: BORJA LAFUENTE GUTIÉRREZ
TUTORA: ELOISA JANTUS LEWINTRE
DIRECTORA (Co- u o ex e no): SILVIA CALABUIG FARIÑAS
Cu so Académico: 2014/2016
VALENCIA, JULIO DE 2016
DATOS DEL ALUMNO/A - Dades de l'alumne/a - S uden ’s iden i ica ion
TÍTULO DEL TRABAJO FIN DE MÁSTER - Tí ol del T eball Fi de Màs e - Ti le o he Final Mas e ’s deg ee hesis
Conside o que el TFM es á inalizado y se puede acep a pa a la de ensa del mismo SI
Conside e que el TFM es à inali za i es po acep a la de ensa d’aques
I conside ha he TFM is comple ed and can accep o he de ense
E aluación de la calidad del TFM y la labo del es udian e.
A aluació de la quali a del TFM i la labo del es udian .
E alua ion o he quali y o he Final Mas e ’s deg ee hesis and he wo k o he s uden
DEPARTAMENTO DE BIOTECNOLOGÍA
INFORME TUTOR TRABAJO FIN DE MÁSTER (pa a cualquie a de las modalidades)
In o me del u o (pe a qualse ol de les modali a s del eball in de mas e )
TUTOR’s REPORT ( o any ype o inal Mas e ’s deg ee hesis)
CURSO - Cu s - Academic yea 2015-2016
Más e (Màs e –Mas e 's deg ee):
Bio ecnología Biomédica
Apellidos (Cognoms - Su name(s)):
La uen e Gu ié ez
Nomb e (Nom – Name):
Telé ono (Telè on - Phone n .): 662275327
DNI (ID ca d n .):
21007055M
Co eo elec . (Co eu elec . – email):
bo jala uen e.biomedi[email p o ec ed]m
Bo ja
Mu a ional analysis o RAS genes in me as a ic colo ec al cance : conco dance be ween umo
issue and liquid biopsy
Tu o /a (Tu o ):
Co u o /a (Co-Tu o ):
Sil ia Calabuig Fa iñas
Eloísa Jan us Lewin e
El abajo p esen ado iene un cla o obje i o aslacional, alo ando la posibilidad de usa mues as de plasma
pa a analiza mu aciones en genes RAS p esen es en el umo , especí icamen e a a és de una écnica de muy
al a sensibilidad como lo es la PCR-digi al. En es e caso, el es udio ha mos ado una al a conco dancia en e las
écnicas compa adas, y po lo an o su posible implemen ación en la p ác ica clínica. El es udian e ha pa icipado
ac i amen e en el abajo expe imen al y ambién en el análisis y discusión de da os, habiendo adqui ido las
compe encias eque idas pa a un g ado de mas e .
FECHA (Da a) Da e: 08 de Julio de 2016
Uni e si a Poli ècnica de València
Depa amen o de Bio ecnología
Edi icis 3J bajo. Camí de Ve a, s/n, 46022 València
Tel. +34 96 387 74 20 • Fax +34 96 387 74 29
depb c@up .es
DEPARTAMENTO DE BIOTECNOLOGÍA
ESCUELA TÉCNICA SUPERIOR DE INGENIERÍA
AGRONÓMICA Y DEL MEDIO NATURAL
AUTORIZACIÓN DEL COTUTOR PARA PRESENTACIÓN DEL TRABAJO FIN DE MÁSTER
DE BIOTECNOLOGÍA BIOMÉDICA
DEPARTAMENTO DE BIOTECNOLOGÍA.E.T.S.I.A.M.N
Camino de Ve a, s/nº.46022VALENCIA●Tel.+34963877420●Fax+34963877429
LA COTUTORA:
D.ª SILVIA CALABUIG FARIÑAS Adsc i a al o ganismo FUNDACIÓN PARA
LA INVESTIGACIÓN DEL HOSPITAL GENERAL UNIVERSITARIO DE VALENCIA (FIHGUV) donde se ha ealizado
el T abajo Fin de Más e i ulado:
Mu a ional analysis o RAS genes in me as a ic colo ec al cance : conco dance be ween umo issue and liquid biopsy
Del que es AUTOR:
D. BORJA LAFUENTE GUTIÉRREZ
AUTORIZA la p esen ación del TRABAJO FIN DE MÁSTER pa a su de ensa.
Valencia, Julio 2016

INDEX
1. INTRODUCTION.........................................................................................................1
1.1. The concep o cance ........................................................................................1
1.2. Colo ec al cance ................................................................................................2
1.2.1. Epidemiology.................................................................................................3
1.2.2. Diagnosis and s aging....................................................................................3
1.3. CRC classi ica ion: om his ology o molecula biology.................................4
1.3.1. Indi idual soma ic mu a ions.........................................................................5
1.3.2. De ec ion o RAS mu a ions in CRC...............................................................6
1.3.3. Di ec Sequencing: p inciples o py osequencing..........................................7
1.4. T ea men o CRC.................................................................................................9
1.5. Liquid biopsy......................................................................................................11
1.5.1. Digi al PCR: BEAMing sys em.......................................................................13
2. OBJECTIVES..............................................................................................................15
3. MATERIALS AND METHODS....................................................................................16
3.1. S udy design and pa ien s................................................................................16
3.2. Biological samples: FFPE and blood.................................................................16
3.3. DNA ex ac ion..................................................................................................16
3.4. Py osequencing..................................................................................................17
3.5. BEAMing de e mina ion o RAS mu a ions…………………………...........................18
3.6. S a is ical analysis..............................................................................................20
4. RESULTS AND DISCUSSION......................................................................................21
4.1. Pa ien cha ac e is ics.......................................................................................21
4.2. RAS mu a ional analysis in issue and c DNA..................................................22
4.2.1. RAS analysis in FFPE samples.................................................................22
4.2.2. RAS analysis in c DNA.............................................................................24
4.3. Conco dance be ween plasma and issue RAS mu a ional s a us.................28
4.4. Associa ion o RAS mu a ional s a us wi h clinicopa hological
cha ac e is ics....................................................................................................33
4.5. Su i al analysis acco ding o RAS mu a ional s a us.....................................36
5. CONCLUSIONS.........................................................................................................39
6. REFERENCES.............................................................................................................40
7. APPENDICES.............................................................................................................46
1
1. INTRODUCTION
1.1. The Concep o Cance
Cance is a g oup o ela ed diseases, in ol ing wo common phenomena: uncon olled
p oli e a ion and he po en ial o in ade su ounding issues o e en sp ead o o he o gans o
issues (me as asize). The p ocess o ca cinogenesis in ol es dynamic changes in he genome
o no mal cells, e en ually leading o he ans o ma ion in o umou cells (Hanahan &
Weinbe g 2000). This allows umou cells o escape om homeos a ic mechanisms ha
con ol p oli e a ion.
Hanahan and Weinbe g p oposed ha he e a e en essen ial cha ac e is ics, known as he
“hallma ks o cance ”, o he de elopmen o cance disease (Hanahan & Weinbe g 2011)
(Figu e 1). Tumou cells p esen genomic ins abili y, esul ing in he accumula ion o genomic
mu a ions. The di e en s eps in ol ed in umou p og ession a e a succession o clonal
expansions p oduced by he accumula ion o mu a ions ha gene a e selec i ely
ad an ageous neoplas ic cells.
Figu e 1: The en hallma ks o cance , modi ied om Hanahan &
Weinbe g 2011.
In addi ion o cance cells, umou s possess ano he dimension o complexi y: hey con ain a
epe oi e o ec ui ed cells ha a e ac i e playe s in he umou “mic oen i onmen ”, one o
he main hallma k ai s. In he mic oen i onmen (Figu e 2), epi helial neoplas ic cells and he
umou -associa ed s oma o m wo well di e en ia ed compa men s, wi h dis inc cell ypes
and molecules ha modula e umou g ow h and in asi eness.
2
Figu e 2: In luence o mic oen i onmen in p ima y umou and me as asis (Ko kaya e al.
2011).
1.2. Colo ec al Cance
Colo ec al cance (CRC) is a o m o cance o he in es inal gland cells ha s a s in he colon
o he ec um. Bo h en i onmen al and gene ic ac o s play key oles in i s e iology. Gene ic
suscep ibili y anges om well-de ined inhe i ed synd omes ( amilial adenoma ous polyposis,
Lynch synd ome, e c.), o less de ined amilial agg ega ions.
CRC a ises a e accumula ion o acqui ed gene ic and epigene ic changes ha ans o m
no mal glandula epi helial cells in o in asi e adenoca cinomas. The classic CRC p og ession
model (depic ed in Figu e 3) p oposed by Vogels ein and colleagues desc ibes he
ans o ma ion o no mal colonic epi helium in o ca cinomas (Vogels ein e al. 1988). A
numbe o key pa hways a e in ol ed in he oncogenesis o colon cance , wi h he
clinicopa hological ea u es o speci ic subg oups being d i en by unde lying molecula
changes.
9
Gly12Se (G12S)
GGT  AGT
Gly12Se (G12S)
GGT  AGT
Gly12Ala (G12A)
GGT  GCT
Gly12Ala (G12A)
GGT  GCT
Gly12A g (G12R)
GGT  CCT
Gly12A g (G12R)
GGT  CGT
Codon 13
Codon 13
Gly13Asp (G13D)
GCG  GAC
Gly13Se (G13S)
GGT  AGT
Codon 61 (CAA)1
Gly13Cys (G13C)
GGT  TGT
Gln61His (Q61H)
TTG  GTG
Gly13A g (G13R)
GGT  CGT
Gln61Leu (Q61L)
TTG  TAG
Gly13Asp (G13D)
GGT  GAT
Gln61A g (Q61R)
TTG  TCG
Gly13Val (G13V)
GGT  GTT
Gln61His (Q61H)
TTG  ATG
Gly13Ala (G13A)
GGT  GCT
Gln61Glu (Q61E)
TTG  TTC
Codon 59
Codon 59
Ala59Th (A59T)
GCT  ACT
Ala59Th (A59T)
GCA  ACA
Ala59P o (A59P)
GCT  CCT
Ala59Se (A59S)
GCA  TCA
Ala59Gly (A59G)
GCT  GGT
Ala59Gly (A59G)
GCA  GGA
Ala59Asp (A59D)
GCT  GAT
Ala59Glu (A59E)
GCA  GAA
Ala59Val (A59V)
GCT  GTT
Ala59Leu (A59L)
GCA  GTA
Codon 61
Codon 117
Gln61Lys (Q61K)
CAA  AAA
Lys117Glu (K117E)
AAA  GAA
Gln61A g (Q61R)
CAA  CGA
Lys117Gln (K117Q)
AAA  CAA
Gln61Leu (Q61L)
CAA  CTA
Lys117Asn (K117N)
AAA  AAC
Gln61His (Q61H)
CAA  CAT
Lys117Asn (K117N)
AAA  AAT
Gln61His (Q61H)
CAA  CAC
Codon 146
Codon 117
Ala146Th (A146T)
GCA  ACA
Lys117Asn (K117N)
AAG  AAC
Ala146P o (A146P)
GCA  CCA
Lys117Asn (K117N)
AAG  AAT
Ala146Val (A146V)
GCA  GTA
Codon 146
Ala146Gly (A146G)
GCA  GGA
Ala146Th (A146T)
GCC  ACC
Ala146P o (A146P)
GCC  CCC
Ala146Se (A146S)
GCC  TCC
Ala146Val (A146V)
GCC  GTC
Ala146Gly (A146G)
GCC  GGC
1KRAS codon 61 is assayed in he e e se di ec ion by Py osequencing. Mu a ions co e ed
by OncoBEAM RAS CRC ki a e highligh ed in yellow.
1.4. T ea men o CRC
The managemen o CRC depends mainly on clinicopa hological cha ac e is ics o he pa ien s,
umou s age and also on he molecula al e a ions ound in umou cells.
The SEOM guidelines (A anda e al. 2015) ecommend, o mos pa ien s wi h good
pe o mance s a us (PS) and no signi ican como bidi ies, he combina ion o 5-FU/leuco o in
(5-FU/LV) wi h ei he oxalipla in (FOLFOX) o i ino ecan (FOLFIRI) as backbone o i s -line

10
ea men . O he op ion includes capeci abine, an o al luo opy imidine wi h simila e icacy in
i s -line ea men o mCRC (Van Cu sem e al. 2004).
On he o he hand, i s -line a ge ed he apies include he an i- ascula endo helial g ow h
ac o (VEGF) agen be acizumab and he an i-EGFR d ugs ce uximab and pani umumab.
An appa en lack o esponse o a ge ed he apy wi h an i-EGFR monoclonal an ibodies in
a ound 40-50% o he pa ien s wi h KRAS exon 2 wild- ype (WT) umou s obse ed in some
clinical ials boos ed he sea ch o addi ional p edic i e bioma ke s. The e ec o mu a ions
in o he membe s o he EGFR signaling pa hway like BRAF, PIK3CA and NRAS was analysed in
mul iple s udies (Ka ape is e al. 2014; Sa o e-Bianchi e al. 2009).
I was ound ha he e a e a ound 5% o CRC pa ien s who ha e mu a ions in KRAS exons 3 o
4, and a u he 5% wi h mu a ions in NRAS exons 2, 3 o 4. These RAS mu a ions p e iously
sugges ed we e es ed by Sange sequencing: KRAS exon 3 (codons 59 and 61) and exon 4
(codons 117 and 146), and NRAS exon 2 (codons 12 and 13), exon 3 (codons 59 and 61), exon 4
(codons 117 and 146) (see Figu e 8).
Figu e 8: F equency o RAS mu a ions beyond KRAS codon 2 in CRC (Hech e al. 2015).
Re ospec i e analyses o se e al phase III ials indica ed ha all RAS mu a ions we e
ega ded as a nega i e p edic i e ac o o an i-EGFR he apy: one example was he PRIME
ial (Douilla d e al. 2013). These mu a ions we e associa ed wi h in e io p og ession- ee
su i al (PFS) and o e all su i al (OS) wi h pani umumab-FOLFOX4 ea men , like KRAS exon
2 mu a ions. The e idence suppo ed he addi ion o all hese mu a ions o he ou inely
es ed KRAS mu a ion analysis, in o de o u he imp o e he selec ion o pa ien s o an i-
EGFR he apy.
As o he e ec o mu a ions beyond an i-EGFR ea men e icacy, mu an KRAS and BRAF
ha e been independen ly associa ed wi h wo se o e all su i al in me as a ic CRC pa ien s. In
he la ges s udy, pa ien s wi h CRC ha ha bo ed a KRAS mu a ion had a wo se o e all
su i al (OS) bu simila p og ession- ee su i al (PFS) compa ed o pa ien s wi h umou s
bea ing WT KRAS (Richman e al. 2009), as seen in Figu e 9.
11
Figu e 9: P ognos ic impac o KRAS and BRAF mu a ions in A) p og ession- ee su i al (PFS)
and B) o e all su i al (OS), compa ed o non-mu a ed pa ien s, in any ea men a m.
Modi ied om Richman e al. 2009.
1.5.Liquid biopsy
Since he ad en o a ge ed he apies, inc eased su i al pe iods and imp o ed quali y o li e
a e achie ed o pa ien s whose cance s ha bo speci ic molecula al e a ions. Howe e ,
a ge ed he apies ha e b ough new challenges: high cos s, po en ial mo bidi y o he
necessa y biopsies, lack o e ec i e d ugs agains mos genomic abe a ions, echnical
limi a ions and egula o y obs acles. In addi ion, almos all umou s de elop esis ance
mechanisms h ough umou he e ogenei y, clonal e olu ion and selec ion. The e o e, new
me hodologies a e needed, ha allow us o o e come hese di icul ies. Liquid biopsies appea
o be a eliable al e na i e o con en ional biopsies. They can p o ide bo h p ecise molecula
da a use ul o imp o ing he clinical managemen o mCRC cance pa ien s, and a less in asi e
way o moni o ing umou beha io .
Mul iple s udies ha e shown ha i is possible o econs uc umou genomes om plasma
DNA (Thie y 2016; Go o e al. 2016). T aces o umou DNA (ci cula ing umou DNA, c DNA)
can be ound in he cell- ee ac ion o blood, oge he wi h DNA agmen s om no mal cells
(c DNA).
A e i s desc ip ion o agmen s o DNA exis ing in he blood (Mandel & Me ais 1948),
highe le els o so called ci cula ing ee DNA (c DNA) we e iden i ied in cance pa ien s
compa ed o heal hy con ols, sugges ing ha his co ela ed wi h malignancy and umou
s age (Leon e al. 1977).
To da e, wo main mechanisms o eleasing ci cula ing umou DNA (c DNA), “passi e” and
“ac i e”, ha e been pos ula ed. The passi e mechanism in ol es he elease o nucleic acids
di ec ly om apop o ic and nec o ic umou cells in o he bloods eam o indi ec ly by
nec o ic umou cells engul ed by mac ophages (Diehl e al. 2005). This was u he suppo ed
by measu ing he size dis ibu ion o DNA agmen s (Jah e al. 2001; Hei ze e al. 2013). In
con as , agmen s o c DNA can also be “ac i ely” sec e ed in o he ci cula ion, pe haps in
associa ion wi h a p o ein complex o ac as an in e cellula messenge o so s (Pe e s &
P e o ius 2012).
12
Conside ing ha eleasing o DNA in o he bloods eam is no an exclusi e p ocess o p ima y
o me as a ic si es, c DNA can p o ide a be e o e all ep esen a ion o he malignan disease
as a whole (Kuo e al. 2014). In cance pa ien s, c DNA le els can a y acco ding o umou
bu den and s age, ana omical p oximi y o ascula u e, and biological ea u es like apop o ic
a e and me as a ic po en ial.
The clinical applica ions o c DNA (see Figu e 10) can be di ided in o h ee main ca ego ies: a)
ea ly diagnosis and p ognosis; b) p o iling and molecula cha ac e iza ion o umou genomic
al e a ions, and c) moni o ing ea men esponse and de ec ion o esis ance mechanisms.
Figu e 10: Uses o liquid biopsies in clinical oncology (Si a egna & Ba delli 2014).
Fi s , i has been p oposed ha moni o ing umou -speci ic changes may be a use ul ool o
ea ly cance de ec ion and/o p ognosis. A ecen example o diagnos ic app oach in ol ed
blood-based CRC sc eening es using he SEPT9 bioma ke ha speci ically de ec s a majo i y
o CRCs o all s ages and colo ec al loca ions (Chu ch e al. 2014). The simple measu emen o
he quan i y o c DNA in plasma by quan i a i e PCR is posi i ely co ela ed wi h umou
bu den in mCRC (Schmi e al. 2012; Spindle e al. 2012).
Second, as a p ognos ic bioma ke , se e al s udies ha e demons a ed ha ci cula ing- ee
DNA le els (c DNA) o he numbe o ci cula ing umou cells (CTCs) a e posi i ely co ela ed
wi h pa ien ou come in mCRC. In su gically esec ed CRC pa ien s, he de ec ion o c DNA
a e su ge y was ela ed o an inc eased elapse a e (Diehl e al. 2008). Fu he mo e, i was
shown ha high concen a ions o c DNA and KRAS mu a ion we e clea indica o s o poo
ou come o ad anced CRC pa ien s (Spindle e al. 2012).
13
Finally, one o he mos widesp ead applica ions o liquid biopsy is moni o ing esponse o
he apy, pa icula ly o hose he apies wi h known esis ance mechanisms. Se e al s udies
ha e epo ed ha an i-EGFR esis an clones a e p esen in he ci cula ion mon hs be o e
p og ession was clinically ob ious (Misale e al. 2014; Mohan e al. 2014).
In summa y, he de e mina ion o molecula al e a ions enables o he selec ion o adequa e
a ge ed he apies o each pa ien , and allows he clinician o make apid he apeu ic
decisions i esis an clones a e de ec ed in ci cula ion. Al hough liquid biopsy is e y use ul
and ad an ageous compa ed o umou issue biopsies, he de ec ion o sca ce c DNA mixed
wi h ela i ely abundan WT c DNA equi es inno a i e ul a-sensi i e echniques, such as
digi al PCR.
1.5.1. Digi al PCR: BEAMing sys em
Gene ally, he e a e wo app oaches o he analysis o c DNA. A a ge ed app oach: analysis
o a small se o equen ly occu ing d i e mu a ions wi h implica ions o he apy decisions,
such as mu a ions in KRAS o EGFR. The second in ol es an un a ge ed app oach wi hou
knowledge o any speci ic changes p esen in he p ima y umou (Hei ze e al. 2015). Gi en
he small p opo ion o c DNA p esen in he o al c DNA samples, i is impo an o selec he
co ec me hods o i s analysis; se e al highly sensi i e echniques ha e been de eloped o
he la e , anging om eal ime PCR-based o mo e complex digi al-PCR based echnologies
BEAMing (Beads-Emulsion-Ampli ica ion-Magne ics, by Sysmex Inos ics) is a a ge ed,
quan i a i e digi al PCR echnology ha employs bead-based ampli ica ion in wa e -in-oil
emulsions, and allele-speci ic hyb idiza ion ollowed by low cy ome y, o he de ec ion o
small amoun s o mu a ed DNA eleased by umou s in o he blood ci cula ion. BEAMing is
highly sensi i e, able o de ec mu an c DNA in e y low p opo ion (as low as 0.01% o o al
DNA agmen s; see Figu e 11) in a backg oud o no mal (WT) DNA.
Figu e 11: Me hodologies o de ec ing ci cula ing umou DNA (c DNA). Py ophospho olysis-
ac i a ed polyme iza ion (PAP); agged-amplicon deep sequencing (TAM-Seq) (Díaz J e al.
2014).
14
Ideally, a e p eampli ica ion, a single DNA agmen is cap u ed in a single magne ic bead,
and each bead alls in o one hyd ophilic d ople o he wa e -oil emulsion. The ampli ica ion
PCR akes place wi h TaqMan p obes designed o co e mu a ional ho spo s in exons 2, 3 and
4 o bo h genes (KRAS and NRAS).
The mu an ac ion canno be in e p e ed as he ac ion o cance cells ha ha bo a
pa icula mu a ion. While c DNA quan i ica ion by quan i a i e PCR is posi i ely co ela ed
wi h umou bu den, mu an ac ion may ep esen a combina ion o clones p oduced by
di e en umou lesions.
In he con ex o CRC, RAS WT umou s a e o en sensi i e o EGFR blockade wi h ce uximab o
pani umumab, bu almos all pa ien s de elop esis ance wi hin a ew mon hs (Ka ape is e al.
2008). Liquid biopsy can subs i u e se ial umou issue sampling, and may p o ide a global
and e ol ing pic u e o he disease. Se e al s udies ha e epo ed ha an i-EGFR esis an
clones a e p esen in he ci cula ion mon hs be o e p og ession was clinically ob ious (Misale
e al. 2014; Mohan e al. 2014). Figu e 12 shows he e olu ion o mu an c DNA ac ion in a
pa ien ; he inc ease in concen a ion p edic ed he eme gence o seconda y esis ance o
ce uximab ea men (Misale e al. 2014).
Figu e 12: Quan i a i e analysis o KRAS Q61H mu a ion in plasma by BEAMing
(Misale e al. 2014).
P o en use ul in cance esea ch (Tabe ne o e al. 2015), BEAMing is now being alida ed o
diagnos ic de e mina ion o mu a ions in KRAS and NRAS in plasma o mCRC pa ien s. Ou
labo a o y had a chance o collabo a e in his en e p ise, and in his s udy we will de e mine
whe he blood-based RAS mu a ion es ing is an app op ia e su oga e o issue-based RAS
es ing o assess eligibili y o mCRC pa ien s o an i-EGFR he apy by compa ing he deg ee o
conco dance o plasma and issue-based RAS es ing in me as a ic CRC pa ien s.

15
2. OBJECTIVES
Tumou issue is cu en ly used o RAS es ing in mCRC pa ien s, bu he de ec ion o
ci cula ing umou DNA (c DNA) is being ac i ely in es iga ed as a new me hod o he
de ec ion o ac ionable mu a ions in plasma samples. The e o e, he main objec i e o his
wo k is o e alua e he conco dance o RAS mu a ional s a us by compa ison o esul s om
c DNA and issue-based es ing in a coho o mCRC pa ien s.
The speci ic aims o his s udy a e he ollowing:
a) To analyse conco dances and disc epancies in RAS (KRAS and NRAS) mu a ional s a us
be ween blood samples and issue samples in a small coho o newly diagnosed mCRC
pa ien s (n=30).
b) To s udy co ela ions be ween he mu a ions de ec ed in c DNA and some ele an
clinicopa hological ea u es.
c) To in eg a e he esul s and de e mine whe he RAS c DNA es ing is a easible
al e na i e o umou issue-based RAS es ing.
16
3. MATERIALS AND METHODS
3.1. S udy design and pa ien s
This is a e ospec i e analysis in 30 he apy-naï e pa ien s wi h his ologically o cy ologically
documen ed me as a ic colo ec al cance . Pa ien s ha ing a his o y o ano he malignancy o
ha ing ecei ed any p e ious ea men (chemo he apy o a ge ed he apy) we e excluded.
We en iched ou coho wi h pa ien s wi h de ec ed mu a ions in RAS genes (KRAS and NRAS),
so ha we could pe o m he conco dance s udy.
The s udy was conduc ed in acco dance wi h he Decla a ion o Helsinki, and he ins i u ional
e hical e iew boa d app o ed he p o ocol.
3.2. Biological samples: FFPE and blood
Fi s o all, pa ien s mus sign an in o med consen documen , by which hey a e gi en all he
in o ma ion ega ding he samples ha hey will dona e, how hey will be aken and he
ele ance ha hey may ha e on ea men and/o p ognosis.
A o al o 30 o malin- ixed, pa a in-embedded (FFPE) CRC samples ob ained by colonoscopy
o su gical esec ion we e p o ided by he Pa hology Se ice a he Conso cio Hospi al Gene al
Uni e si a io de Valencia. A pa hology epo was a ailable o all he samples, enabling hei
cha ac e isa ion. Fo mu a ional analysis o umou issue, he specimens we e examined by a
pa hologis , and hose ha ing >5% o umou cells we e selec ed. Th ee o i e 5 µM hick
issue sec ions we e used o DNA isola ion.
Blood samples we e ob ained a he ime o diagnosis o me as a ic disease, p e ious o any
sys emic ea men . 10 mL o blood we e collec ed om each pa ien in K2 EDTA BD
Vacu aine ® ubes, and p ocessed o ob ain plasma wi hin an hou . B ie ly, a se ies o
cen i uga ions a e pe o med, a inc easing speeds, in o de o isola e and clean-up he
plasma. The isola ed plasma is s o ed in Sa s ed ™ C yoPu e ubes, a -80 °C, un il c DNA
ex ac ion.
3.3. DNA ex ac ion
FFPE: DNA was isola ed om FFPE umou issue sec ions. Fi s pa a in was emo ed by
incuba ing he samples wi h mine al oil a 95°C, ollowed by he addi ion o xylene. A e
cen i uga ion, he xylene supe na an was emo ed. Then e hanol 100% was added o clean
esidual xylene, ollowed by ano he cen i uga ion s ep. A e ha , he samples we e allowed
o ai -d y o 20-30 minu es.
The Cobas DNA Sample P epa a ion Ki (Roche®) was used o he ex ac ion o DNA om
depa a inized samples, ollowing manu ac u e ’s ecommenda ions. B ie ly, DNA Tissue Lysis
17
Bu e and P o einase K we e added. A e wo incuba ions (56 °C and 95 °C) and b ie cooling,
DNA Pa a in Binding Bu e and isop opanol we e added, wi h espec i e incuba ion pe iods,
and he con en o each ube was ans e ed o a il e ed ube. Then a se ies o
cen i uga ions wi h washing s eps in be ween we e pe o med, and inally he DNA Elu ion
Bu e was added in o de o collec DNA in he de ini i e ube.
Plasma: DNA om plasma samples was ob ained by he comme cial QIAamp® "Ci cula ing
Nucleic Acid" ki , ollowing manu ac u e 's ins uc ions. This p o ocol is based on a ini y
columns o e aining nucleic acids.
FFPE samples DNA quan i ica ion was pe o med in he NanoD op® 2000C sys em (The mo
Fishe Scien i ic). Fo plasma c DNA, quan i ica ion was pe o med using Qubi ® (Li e
Technologies) Fluo ome e : A Wo king solu ion was p epa ed, dilu ing Qubi ® eagen 1:200 in
Qubi ® Bu e . Sample DNA was dilu ed 1:200 in Wo king solu ion, and s anda d samples we e
dilu ed 1:20. The ubes we e incuba ed a oom empe a u e be o e eadings.
3.4. Py osequencing
All issue biopsies we e analysed o RAS geno yping assessmen using wo CE-IVD ma ked
comme cial ki s: “The asc een® KRAS Py o Ki ” and “The asc een® RAS Ex ension Py o Ki ”
(bo h om QIAGEN, Hilden, Ge many) acco ding o he p oduce p o ocols (“The asc een
KRAS Py o Ki Handbook”, e sion 1, July 2011, and “The asc een RAS Ex ension Py o Ki
Handbook”, e sion 1, Oc obe 2014). F om each sample, 10 ng DNA we e ampli ied o
de e mining mu a ions s a us in: KRAS 12-13, NRAS 12-13, NRAS 61, KRAS 59-61, KRAS 117,
KRAS 146, NRAS 58-59, NRAS 117 and NRAS 146.
Py osequencing was pe o med using 10 μL o each PCR p oduc wi h Py oMa k Gold Q96
eagen s (QIAGEN), S ep a idin Sepha ose (GE Heal hca e Bio-Science AB, Uppsala, Sweden),
in he Py oMa k Q24 ins umen (QIAGEN). The esul s we e analysed using Py oMa k Q24
2.0.7 so wa e (QIAGEN).
The p o ocol is based on empla e DNA immobiliza ion on Sepha ose (beaded o m o
aga ose), PCR ampli ica ion and sequencing. The e e se ampli ica ion p ime (RP) is
bio inyla ed, and so he sequencing is pe o med on he o wa d s and (excep o KRAS
codons 59/61, as seen in Figu e 13).
18
Figu e 13: Ampli ica ion p ime s (black a ows) and sequencing p ime s (whi e a ows) o
KRAS codons 12-13 (A) and 59-61 (B). The g ay ci cles ep esen bio inyla ed p ime s.
A single nucleo ide is inco po a ed in each sequencing s ep. I i is inco po a ed in o he DNA
s and, a pulse o ligh is gene a ed (see Figu e 14) and he in ensi y is egis e ed (i mo e han
one nucleo ide o he same ype is inco po a ed, he in ensi y is highe ). Finally, he
nucleo ides a e deg aded and ano he nucleo ide is inco po a ed, s a ing a new cycle.
Figu e 14: Biochemical basis o he gene a ion o ligh by DNA py osequencing. ATP,
adenosine iphospha e; ADP, adenosine diphospha e; dNDP, deoxy-nucleo idyl diphospha e;
dNMP, deoxy-nucleo idyl monophospha e; PPi, py ophospha e. (Pe osino e al. 2009).
3.5. BEAMing de e mina ion o RAS mu a ions
RAS mu a ional analysis on c DNA was done wi h BEAMing digi al-PCR (OncoBEAM™ RAS CRC
Ki Sysmex® Inos ics), a echnique based on emulsion PCR ha allows de ec ion o one mu an
allele in 10000 WT alleles. As shown in Figu e 15, DNA isola ion and p e-ampli ica ion eac ions
we e pe o med on he p e-PCR labo a o y, whe eas om emulsion-PCR s ep un il he inal
low-cy ome y analysis he expe imen al wo k was done on he pos -PCR labo a o y
(physically sepa a ed a ea), in o de o a oid c oss-con amina ion.A wo k low o he di e en
s eps in he echnique is shown in Figu e 15:
25
KRAS 13
3
10.0%
KRAS 61
2
6.7%
KRAS 117
1
3.3%
To al mu a ions iden i ied in NRAS
NRAS 12
3
10.0%
NRAS 13
1
3.3%
NRAS 61
6
20.0%
Mu a ions iden i ied in one codon
KRAS 12
9
30.0%
KRAS 13
2
6.6%
KRAS 61
1
3.3%
NRAS 13
1
3.3%
NRAS 61
1
3.3%
Mu a ions iden i ied in mo e han one codon
KRAS 12 + KRAS 117
1
3.3%
KRAS 12 + NRAS 61
2
6.6%
KRAS 12 + NRAS 12 + NRAS 61
1
3.3%
KRAS 13 + NRAS 12 + NRAS 61
1
3.3%
KRAS 12 + KRAS 61 + NRAS 12 +
NRAS 61
1
3.3%
As expec ed, KRAS codon 12 was he mos equen ly mu a ion ound in ou coho . In hese
cases, no in o ma ion on he speci ic base subs i u ion was a ailable, because BEAMing is only
able o dis inguish be ween he WT o m and all he MUT o ms o each codon. Mu an
ac ions ob ained in he MUT cases anged om 0.004% (case 11), which is ex emely low, o
25.045% in case 6. No ably, case 11 was one o he h ee cases wi h especially high c DNA yield
(25.8 ng/µL); his p obably allowed us o de ec mu a ions p esen a ex emely low
concen a ion in c DNA. In Figu e 17 some examples o high (Fig 17 A) and low (Fig 17 B and C)
mu an ac ion a e shown.
The cu o o KRAS codon 12 mu a ions was es ablished a 40 mu an beads. As seen in Figu e
17, case 15 (Fig 17 B) was jus o e he cu o alue (56 mu a ed beads), whe eas case 3 (Fig 17
C) was jus below i (22 mu a ed beads). In ac , case 3 was a disco dan case, in which issue
sample was in o med as MUT by py osequencing (KRAS Gly12Val mu a ion), al hough i
showed a ela i ely low mu an ac ion (10%).

26
Figu e 17: Examples o BEAMing esul s. A) Case 8, KRAS codon 12 MUT. B) Case 15, KRAS
codon 12 MUT. C) Case 3, KRAS codon 12 WT.
27
Table 8 con ains a summa y o all he esul s ob ained o RAS mu a ional analysis in c DNA by
BEAMing echnology:
Table 8: Summa y o he esul s o RAS plasma analysis by BEAMing (n=30).
Case
OncoBEAM
RAS
[c DNA] (ng/µL)
Mu an beads ac ion (%)
(same o de )
1
WT
0.370
2
KRAS 13
0.554
49.442
3
WT
1.640
4
KRAS 12
1.760
28.042
5
WT
0.782
6
KRAS 12
NRAS 12
NRAS 61
1.980
25.045
0.022
0.007
7
KRAS 12
0.174
2.124
8
KRAS 12
0.876
19.032
9
KRAS 12
0.296
0.791
10
KRAS 12
NRAS 61
1.880
23.254
0.013
11
KRAS 13
NRAS 12
NRAS 61
25.800
10.926
0.013
0.004
12
WT
0.262
13
KRAS 13
54.600
5.329
14
WT
0.200
15
KRAS 12
KRAS 117
0.446
0.072
0.264
16
KRAS 12
0.300
13.056
17
KRAS 12
0.666
10.515
18
WT
0.168
19
KRAS 12
0.294
6.575
20
WT
0.362
21
KRAS 12b
KRAS 61
NRAS 12
NRAS 61
3.820
0.013
0.016
0.018
0.021
22
In alida
0.224
23
KRAS 12
0.230
9.843
24
WT
6.640
28
25
NRAS 61
0.526
0.262
26
KRAS 12
0.504
10.498
27
KRAS 12
NRAS 61
10.500
8.061
0.006
28
NRAS 13
87.600
29.580
29
WT
0.326
30
KRAS 61
0.226
0.122
a Sample om case 22 epo ed in alid esul sdue o low DNA amoun . b KRAS
12 mu a ion was condi ioned because he beads we e oo dispe sed.
O in e es , we ound 6 MUT cases displaying coexis en RAS mu a ions in c DNA (Table 8;
cases: 6, 10, 11, 15, 21, and 27). This was no en i ely unexpec ed due o he high sensi i i y o
BEAMing, bu he biological and he apeu ic ele ance o hese mu a ed subclones ha a e
p esen in e y low p opo ion needs o be u he in es iga ed. May be, is exac ly in his poin
whe e he ad an ages o he high-sensi i i y blood-based RAS es ing pla o ms will ha e mo e
ele ance in he clinical p ac ice, since i is e y well known ha in me as a ic pa ien s, a single
umo issue biopsy may no ep esen he e ol ing RAS mu a ional s a us o he disease. In
pa ien s whose umou s show ex ensi e he e ogenei y, a comp ehensi e su eying o RAS
s a us by es ing mul iple issue samples would be desi able; howe e , his is imp ac ical and
un easible. Ins ead, se ial blood sampling and BEAMing analysis o RAS s a us could be he
solu ion.
Finally, i is wo h poin ing ou ha up un il now, almos all s udies ha included BEAMing
analysis had been pe o med cen alized in he Sysmex acili ies in Hambu g. The Molecula
Oncology Labo a o y a FIHGUV is one o he i s labo a o ies using he BEAMing echnology
ou o he cen al labo a o y o Sysmex, , as pa o a pilo p ojec conduc ed on 8 Spanish
cen e s.
4.3. Conco dance be ween plasma and issue RAS mu a ional s a us
De e mina ions o RAS mu a ional s a us in c DNA by BEAMing and in issue by
py osequencing ha e been summa ized in Table 9.
Table 9: Summa y o RAS mu a ional s a us as de e mined in umou issue by
py osequencing, and in plasma c DNA by BEAMing.
Case
RAS issue
RAS c DNA
Case
RAS issue
RAS c DNA
1
WT
WT
16
KRAS G12V
KRAS G12A
KRAS 12
2
KRAS G13D
KRAS 13
17
KRAS G12D
KRAS 12
3
KRAS G12V
WT
18
WT
WT
4
KRAS G12V
KRAS 12
19
KRAS G12V
KRAS 12
5
KRAS G12D
WT
20
WT
WT
6
KRAS G12V
KRAS 12
21
WT
KRAS 12b
29
NRAS 12
NRAS 61
KRAS 61
NRAS 12
NRAS 61
7
KRAS G12C
KRAS 12
22
KRAS G13D
NAa
8
KRAS G12S
KRAS 12
23
KRAS G12V
KRAS 12
9
KRAS G12D
KRAS 12
24
WT
WT
10
KRAS G12V
KRAS 12
NRAS 61
25
NRAS Q61R
NRAS 61
11
KRAS G13D
KRAS 13
NRAS 12
NRAS 61
26
KRAS G12V
KRAS 12
12
WT
WT
27
KRAS G12A
KRAS 12
NRAS 61
13
KRAS G13D
KRAS 13
28
NRAS G13R
NRAS 13
14
WT
WT
29
WT
WT
15
KRAS G12C
KRAS 12
KRAS 117
30
KRAS Q61H
KRAS 61
O he 30 pai ed issue/plasma samples analysed, he e was one case ha had o be excluded
om he analysis due o low concen a ion o DNA in plasma, which esul ed in in alid RAS
geno yping by BEAMing. O he 29 emaining cases, 21 we e RAS MUT in FFPE (72.4%) whe eas
in c DNA we ound 20 RAS MUT cases (69.0%). RAS mu a ion posi i e samples we e called
abo e mu an allelic ac ion h esholds o 0.02% in plasma and 5% in issue.
The ag eemen be ween BEAMing sys em in plasma samples and he de e mina ion by
py osequencing in issue samples was es ima ed by calcula ing he aw ag eemen and
pe o ming he conco dance es . O e all ag eemen was ob ained, along wi h posi i e and
nega i e ag eemen (Table 10)
Table 10: Conco dance o plasma and issue o RAS mu a ional s a us.
Tissue RAS s a us
Plasma RAS s a us
Posi i e (Mu )
Nega i e (WT)
To al
Posi i e (Mu )
19
1
20
Nega i e (WT)
2
7
9
To al
21
8
29
O e all ag eemen = 0.896  89.6 %
Posi i e ag eemen = 0.905  90.5 %
Nega i e ag eemen = 0.875  87.5 %
30
These esul s show a good conco dance be ween BEAMing and py osequencing, wi h an
o e all ag eemen (OA) o 89.6%. Al hough he e a e ew s udies in he li e a u e, conco dance
be ween liquid biopsy, using highly sensi i e me hods such as digi al PCR and umou
geno yping ha e usually esul ed in conco dance. One example is a epo using da ase s wi h
76 pai ed issue-blood samples om wo clinical ials (OPUS and CRYSTAL), whe e OA
be ween issue (RAS DNA sequencing o FFPE umou samples) and plasma (BEAMing RAS 33
Mu a ion Panel) was 93.4% (Jones e al. 2015). In o he umou s, like b eas and lung cance ,
Higgins e al ound a 100% OA in he e ospec i e assessmen o PI3KCA mu a ions in b eas
cance samples (BEAMing c DNA s. sequencing umou issue); in he p ospec i e analysis,
howe e , only a 72.5% o conco dance was achie ed (Higgins e al. 2012). Finally, Ka lo ich
and colleagues ound an OA o 67% when assessing T790M mu a ion by cobas® Tumou es
and BEAMing plasma c DNA on non-small cell lung cance samples (Ka lo ich e al. 2016).
Al hough highly conco dan , we had 3 disco dan cases, which a e summa ized in Table 11:
Table 11: Disco dan cases summa y.
Case
Tumou si e
Plasma esul
Tissue esul
#3
Rec o-sigmoid
junc ion
WT
KRAS exon 2
G12V
#5
T ans e se
colon
WT
KRAS exon 2
G12D
#21
Rec um
KRAS 12, 61 +
NRAS 12, 61
WT
Among he wo pa ien s in which RAS mu a ion was iden i ied in issue bu no in plasma, we
in es iga ed possible causes. Fi s , we e-examined FFPE samples by py osequencing,
con i ming p e ious esul s.
Second, p eanaly ical p ocedu es o plasma p ocessing could also explain he lack o
conco dance. Since his is a e ospec i e s udy, cha ac e is ics o he collec ion ube, ime
om collec ion un il p ocessing samples and plasma s o age condi ions migh ha e a ec ed
he yield and/o quali y o c DNA ob ained. In bo h cases (#3 and #5), blood samples we e
collec ed in s anda d K2-EDTA ubes wi hou any nucleic-acid s abilizing agen . Apa om his,
samples we e s o ed a -80 °C o mo e han 5yea s.
Thi d, he e is also a possible co ela ion be ween umou bu den, numbe and loca ion o
me as a ic disease lesions and he amoun o plasma DNA eco e ed. In e es ingly, case #3
had a unique me as a ic lesion (low umou bu den), e en hough he amoun o plasma
ob ained was in he median yield. On he con a y, case #5 was a pa ien wi h high umou
bu den a he ime o diagnosis (p ima y umou and se e al li e me as asis, he g ea e
measu ing mo e han 90 mm). Consequen ly, we would expec highe c DNA le els, bu in his
case he yield was hal o ha ob ained in case #3.

31
S anda dized me hods a e s ill needed in o de o minimize hei impac on mu a ion
de ec ion a es (El Messaoudi e al. 2013). Some impo an ac o s a e:
 P ocessing he blood in 4-6 hou s a e d awing is essen ial, because hal -li e o c DNA
in ci cula ion is be ween 16 minu es and 2 hou s (Diehl e al. 2008).
 Use o Cell-F ee DNA™ Blood Collec ion Tubes (S eck, Omaha, NE) is ad an ageous
o e o he collec ion ubes: hey con ain a o maldehyde- ee p ese a i e ha
p e en s whi e blood cells om b eaking, a oiding WT DNA con amina ion up o 72
hou s pos -collec ion (Xue e al. 2009; She wood e al. 2016).
The o he disco dan case was RAS MUT in plasma bu no mu a ion was de ec ed on FFPE
sample. O in e es , in his case (#21) RAS geno yping was pe o med on a FFPE sample
co esponding o a li e me as asis, since p ima y umou issue was no a ailable. Figu e 18
shows he plo s ob ained o all ou mu a ions (A-D):
Figu e 18: Plo s o all ou mu a ions de ec ed in c DNA o case 21, wi h numbe o mu a ed
beads and mu an ac ion.
Di e ences in RAS mu a ion s a us be ween plasma and issue may be a ibu ed o in a o
in e - umou molecula he e ogenei y. Fo ins ance, RAS WT in issue bu MUT in plasma, such
as in case 21, may a ise in pa ien s ha ing he e ogeneous dis ibu ion o RAS mu an clones in
32
he p ima y umou and/o in he me as ases, which a e no ep esen ed in he analysed
issue sample bu a e de ec ed sys emically by he plasma es .
In e es ingly, pa ien #21 was ea ed wi h a combina ion o FOLFOX wi h an i-EGFR agen
pani umumab o 7 cycles. No esponse was obse ed, only achie ing disease s abiliza ion. The
ea men was s opped and ou mon hs la e , he pa ien p og essed and a second line
ea men was ini ia ed. So, hese appa en ly disco dan esul s be ween plasma and issue
make sense when we look closely a he clinical e olu ion o he pa ien . P obably, i we had
ollowed he pa ien wi h se ial blood sampling we could ha e de ec ed an inc ease in he
mu an ac ion du ing ea men .
Al hough no a disco dance, he e we e 5 cases in which BEAMing de ec ed he mu a ion
obse ed by py osequencing and addi ional mu a ions p esen in low p opo ion in c DNA. The
ele ance o hese esul s is s ill unknown, and hey should be checked; analysing issue
samples using BEAMing echnology could e eal i hose mu a ions we e p esen in he issue
in such low p opo ion ha py osequencing could no de ec hem. This is being pe o med
igh now on ou labo a o y.
Finally, case #22 did no yield any esul s because o low DNA concen a ion and subsequen
ailu e o emulsion PCR. The concen a ion o c DNA a e nucleic acid ex ac ion was 0.224 ,
which is jus o e he alue we es ima e o be su icien o de ec a leas one mu a ion in
c DNA. The e o e, he bes cou se o ac ion would be o epea c DNA ex ac ion om he
same plasma sample (i s ill possible), inc easing he amoun o inpu plasma: he e is e idence
ha inc easing he amoun o inpu plasma can imp o e c DNA mu a ion de ec ion i he
p ocessing o he plasma is op imal (She wood e al. 2016).
Clinical alida ion o BEAMing has a i ed i s o umou issue analysis. O e 1200 pa ien s
om di e en clinical ials had umou issue samples es ed using he BEAMing pla o m in a
e ospec i e manne :
 In OPUS ial sample se (Bokemeye e al. 2015), mu a ion equency was b oadly
conco dan wi h hose epo ed in simila s udies using py osequencing (Douilla d e
al. 2013; Schwa zbe g e al. 2014; S in zing e al. 2012).
 In he CRYSTAL s udy, RAS mu a ions we e ound in 14.7% o e aluable pa ien s.
 Finally, esul s om he subg oup analysis o CALGB/SWOG 80405 ha e no been
published ye . P elimina y esul s show ha new RAS mu a ions we e iden i ied in
15.3% o analysed pa ien s.
In conclusion, liquid biopsy could hold he key o ea lie de ec ion and ea men o elapsed
disease, and ul ima ely imp o e he ou come o a pa ien . As blood se ial sampling is much
less in asi e and sa e han me as asis o umou biopsies, c DNA analysis o RAS mu a ional
33
s a us assessmen ep esen s a po en ial su oga e o solid biopsies. The e o e, sensi i e
echniques such as BEAMing sys em able o de ec minimal quan i ies o c DNA ca ying
ac ionable mu a ions a e desi able and will become an essen ial ool o molecula oncology
diagnos ics. Mo eo e , BEAMing echnique is e sa ile, able o analyse DNA om bo h plasma
and issue samples: an in aluable esou ce o molecula oncology esea che s and
oncologis s.
4.4. Associa ion o RAS mu a ional s a us wi h clinico-pa hological
cha ac e is ics
We analysed associa ion be ween clinicopa hological a iables and RAS mu a ional s a us as
de e mined by bo h echniques. RAS s a us was dicho omized as ollows: "RAS WT" (meaning
no de ec ion o mu a ed DNA) and "Any RAS MUT" (meaning ha a leas one KRAS o NRAS
mu a ion was de ec ed). Fi s , esul s om umou issue py osequencing a e shown in Table
12; no s a is ically signi ican co ela ion was ound be ween RAS mu a ional s a us in umou
issue and hese cha ac e is ics:
Table 12: Co ela ion analysis be ween RAS mu a ional s a us ( umou issue) and
clinicopa hological a iables o in e es in CRC using Mann-Whi ney U es .
Cha ac e is ics
All
(n=30)
All RAS
WT
(n=8;
26.7%)
Any RAS
mu a ion
(n=22;
73.3%)
p-
alue
Age a diagnosis
(yea s)
Median [ ange]
68 [43-83]
67 [43-71]
69 [47-83]
*0.270
Sex
Man
20 (66.7%)
6 (20%)
14 (46.7%)
†0.682
Woman
10 (33.3%)
2 (6.7%)
8 (26.7%)
CEA (ng/mL) a
diagnosisa
Median [ ange]
31 [2.7-
7059.5]
19.9 [6.5-
1000]
36.1 [2.7-
7059.5]
*1.000
P ima y umou
localiza ion
Ascending colon
6 (20%)
1 (3.3%)
5 (16.7%)
†0.650
T ans e se colon
3 (10%)
0 (0%)
3 (10%)
Descending colon
8 (26.7%)
2 (6.7%)
6 (20%)
Rec um
13 (43.3%)
5 (16.7%)
8 (26.7%)
P ima y umou
his ologyb
ADC
24 (85.7%)
7 (25%)
17 (60.7%)
†1.000
MUC
4 (14.3%)
1 (3.6%)
3 (10.7%)
G ade o
di e en ia ion
Well di .
9 (45%)
2 (10%)
7 (35%)
†1.000
Mode a ely di .
10 (50%)
3 (15%)
7 (35%)
Poo ly di .
1 (5%)
0 (0%)
1 (5%)
Tumou size and
in asi eness (T)
T2
2 (11.8%)
1 (5.9%)
1 (5.9%)
†1.000
T3
8 (47.1%)
3 (17.6%)
5 (29.4%)
T4
7 (41.2%)
3 (17.6%)
4 (23.5%)
Regional lymph
N0
6 (33.3%)
2 (11.1%)
4 (22.2%)
†0.350
34
nodes in ol emen
(N)
N1
6 (33.3%)
4 (22.2%)
2 (11.1%)
N2
6 (33.3%)
1 (5.6%)
5 (27.8%)
Lympha ic
in asion (L)
L0
9 (64.3%)
2 (14.3%)
7 (50%)
†0.580
L1
5 (35.7%)
2 (14.3%)
3 (21.4%)
Vascula in asion
(V)
V0
13 (86.7%)
4 (26.7%)
9 (60%)
†1.000
V1
2 (13.3%)
1 (6.7%)
1 (6.7%)
Pe ineu al
in asion (Pn)
Pn0
12 (85.7%)
4 (28.6%)
8 (57.1%)
†1.000
Pn1
2 (14.3%)
0 (0%)
2 (14.3%)
Li e me as asis
No
7 (23.3%)
3 (10%)
4 (13.3%)
†0.345
Yes
23 (76.7%)
5 (16.7%)
18 (60%)
O gans a ec ed by
me as asis
1 o gan
14 (46.7%)
6 (75%)
6 (31.6%)
†0.101
>1 o gan
16 (53.3%)
2 (25%)
13 (68.4%)
*K uskal-Wallis es ; †Mann-Whi ney U es . a CEA le els only a ailable o 17 pa ien s. b ADC =
Adenoca cinoma; MUC = Mucinous adenoca cinoma.
Second, he esul s ob ained om RAS mu a ional s a us by c DNA analysis a e shown in Table
13:
Table 13: Co ela ion analysis be ween RAS mu a ional s a us and clinicopa hological a iables
o in e es in CRC using Mann-Whi ney U es .
Cha ac e is ics
All
(n=29)
All RAS
WT
(n=9;
31%)
Any RAS
mu a ion
(n=20;
69%)
p- alue
Age a diagnosis
(yea s)
Median [ ange]
68 [43-83]
68 [43-81]
69 [47-83]
*0.850
Sex
Man
20 (69%)
6 (20.7%)
14 (48.3%)
†1.000
Woman
9 (31%)
3 (10.3%)
6 (20.7%)
CEA (ng/mL) a
diagnosisa
Median [ ange]
36.1 [2.7-
7059.5]
17.25
[2.7-1000]
41.45 [2.7-
7059.5]
*0.302
P ima y umou
localiza ion
Ascending colon
6 (20.7%)
1 (3.4%)
5 (17.2%)
†0.933
T ans e se colon
3 (10.3%)
1 (3.4%)
2 (6.9%)
Descending colon
7 (24.1%)
2 (6.9%)
5 (17.2%)
Rec um
13 (44.8%)
5 (17.2%)
8 (27.6%)
P ima y umou
his ologyb
ADC
24 (88.9%)
8 (29.6%)
16 (59.3%)
†1.000
MUC
4 (11.1%)
1 (3.7%)
2 (7.4%)
G ade o
di e en ia ion
Well di .
9 (47.4%)
3 (15.8%)
6 (31.6%)
†1.000
Mode a ely di .
9 (47.4%)
3 (15.8%)
6 (31.6%)
Poo ly di .
1 (5.3%)
0 (0%)
1 (5.3%)
Tumou size and
in asi eness (T)
T2
2 (12.5%)
0 (0%)
2 (12.5%)
†0.621
T3
8 (50%)
4 (25%)
4 (25%)
T4
6 (37.5%)
3 (18.8%)
3 (18.8%)
41
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45
7. APPENDICES
7.1. Communica ions de i ed om his s udy
VI Mee ing o Young Resea che s (RTICC). Salamanca, Spain 2016.
Mu a ional analysis o RAS genes in me as a ic colo ec al cance : Conco dance be ween
issue and liquid biopsy.
Bo ja La uen e Gu ié ez, Alejand o He e os Poma es, Sil ia Calabuig-Fa iñas, Ma ais Mosqueda, E a
Esco ihuela, Sand a Gallach, Ma ía José Sa on , Eloisa Jan us-Lewin e, Ca los Camps.
BACKGROUND: Mu a ions in p o o-oncogenes RAS (KRAS and NRAS) a e ou inely es ed in
me as a ic colo ec al cance (mCRC). Tumo s wi h ac i a ing mu a ions in RAS genes do no
espond o an i-EGFR a ge ed he apy (ce uximab and pani umumab). In consequence, hese
d ugs a e es ic ed o pa ien s wi h RAS wild- ype umo s. E en ually umo s de elop
esis ance by selec ion o RAS mu an subclones. Thus, se ial sampling o mu a ional analysis
is desi able, bu i en ails mul iple biopsies (in asi e and expensi e). Liquid biopsy sys ems, like
BEAMing, allow o con inuous mu a ional analysis in a non-in asi e, ul asensi i e manne .
OBJECTIVE: We aim o compa e ou s anda d echnique o RAS assessmen in umo issue,
Py osequencing, wi h a "liquid biopsy" app oach based on c DNA analysis: BEAMing sys em.
METHODS: We used The aSc een® KRAS Py o Ki and RAS Ex ension Py o Ki o
py osequencing o umo issue DNA, and Sysmex® OncoBEAM CRC RAS ki o BEAMing c DNA
analysis. Conco dance was de e mined by he numbe o cases epo ed as “mu an ” o “wild-
ype/no mu a ion de ec ed” in each sys em.
RESULTS: We es ed RAS mu a ional s a us in umo issue and plasma samples o 30 pa ien s.
We ound an o e all ag eemen o 89.6% (Table 1), wi h h ee disco dan cases (Table 2). One
case could no be analyzed by BEAMing because o low DNA inpu :
Table 3: Conco dance able o plasma and issue esul s o RAS mu a ional s a us.
Tissue RAS s a us
Plasma
RAS s a us
Posi i e (Mu )
Nega i e (WT)
To al
Posi i e (Mu )
19
1
20
Nega i e (WT)
2
7
9
To al
21
8
29
46
Table 4: Summa y o disco dan cases, ype o disco dance, plasma and issue esul s.
Case
Disco dan ype
Plasma esul
Tissue esul
#3
False Nega i e
WT
KRAS G12V
#5
False Nega i e
WT
KRAS G12D
#21
False Posi i e
KRAS 12, 61 + NRAS 12, 61
WT
The alse nega i es we e in es iga ed (Table 2), poin ing owa ds c DNA deg ada ion o e ime
and p eanaly ical subop imal p ocessing as he main ac o s in ol ed.
In case 21, he p esence o RAS mu an c DNA in plasma be o e an i-EGFR ea men could
ha e p edic ed ea ly disease p og ession, which occu ed jus ou mon hs a e ea men .
P obably, RAS mu an subclones began o p oli e a e and sus ain he umo , gene a ing he
seconda y esis ance o an i-EGFR ea men s.
CONCLUSIONS: Ou esul s indica e ha BEAMing analysis o RAS mu a ions in c DNA has a
high conco dance a e when compa ed o py osequencing umo issue. The e o e, BEAMing is
an op imal echnique o molecula diagnosis o RAS mu a ional s a us o mCRC pa ien s. In
addi ion, i is e y use ul in disease moni o ing as a non-in asi e, speci ic me hod o de ec ing
low p e alence RAS mu a ions in plasma, allowing ea lie in e en ions o modi y and imp o e
ea men s.
This p ojec was suppo ed by [RD12/0036/0025] om RTICC.