Romina Oli ei a Sil a
Mu a ional analysis o KRAS and NRAS in me as a ic colo ec al
cance
Disse ação de candida u a ao g au de Mes e em
Oncologia – especialização em Oncologia Molecula
subme ida ao Ins i u o de Ciências Biomédicas de Abel
Salaza da Uni e sidade do Po o.
O ien ado : Manuel An ónio Rod igues Teixei a, MD, PhD
Di e o do Se iço de Gené ica e Cen o de In es igação
Ins i u o Po uguês de Oncologia do Po o
P o esso Ca ed á ico Con idado do Depa amen o de
Pa ologia e Imunologia Molecula
Ins i u o de Ciências Biomédicas de Abel Salaza –
Uni e sidade do Po o
Coo ien ado : Isabel Ma ia da Sil a Veiga dos San os,
MSc
Assesso a da ca ei a dos écnicos supe io es de saúde,
no amo de Gené ica, no Se iço de Gené ica
Ins i u o Po uguês de Oncologia do Po o
“The u u e belongs o hose who belie e in he beau y o hei d eams.”
Eleano Roose el
I
AGRADECIMENTOS
Ao meu o ien ado , P o . Manuel Teixei a, po me e dado a opo unidade de ealiza
es e abalho no seu g upo de in es igação. Ob igada po oda a paciência,
disponibilidade e o ien ação que con ibuí am pa a o meu desen ol imen o enquan o
in es igado a.
À minha coo ien ado a, Isabel, po e pa ilhado comigo oda a sua expe iência e
conhecimen o e po me e ajudado a supe a odos os obs áculos que se o am
colocando no meu caminho, semp e com boa disposição e aleg ia.
À P o . Be a, a ual di e o a do Mes ado, e a odos os docen es po e em pa ilhado
connosco odo o seu conhecimen o sob e es a as a á ea que é a Oncologia.
A odo o Se iço de Gené ica do IPO-Po o, po me e em ecebido da melho o ma
possí el e po oda a o ça que me de am ao longo des a e apa. Um ag adecimen o
especial à Ani a e à Susana Biza o po e em ac edi ado em mim e nas minhas
capacidades; à Ca a ina, à Paula, ao Hen ique, à Sa a e ao Rui po me e em
p opo cionado as melho es ho as de almoço de que enho memó ia, semp e com as
his ó ias mais hila ian es e as isadas mais con agian es; e à Ca la, à Manuela e à
Pa ícia po e em sido os meus “anjos da gua da” du an e oda es a ase, po me e em
enco ajado quando me sen ia p es es a desis i e po me e em ajudado quando eu não
sabia que p ecisa a de ajuda.
À Ca a ina A aújo, pela ajuda, comp eensão e companhei ismo ao longo des e
pe cu so. Po se ap esen a odos os dias com um so iso na ca a e ca inho em cada
ab aço. Ob igada, p incipalmen e, po me mos a es que as melho es amizades podem
su gi quando menos espe amos.
Às meninas da molecula do GDPN: Ma a, Diana, Cláudia Ma ins, Na ália, Isa,
Liliana, Elsa e A iana. Ob igada po me e em in eg ado logo no osso g upo, po oda a
paciência com os meus ho á ios malucos e po odo o apoio nes a ase mais complicada
da minha ida.

II
A odos aqueles que con ibuí am pa a que nos úl imos 5 anos eu i esse a melho
expe iência académica que pode ia e ido. Um ag adecimen o especial aos amigos que
iz du an e a licencia u a que, de uma o ma ou de ou a, es a ão semp e comigo pa a
onde que que eu á; aos “Sacaninhas da P esiden e”, po e em sido os meus
companhei os nes a a en u a e po oda a di e são, iso a e momen os pa ilhados ao
longo des es dois anos; e às meninas da Residência, po me e em ecebido de b aços
abe os e me e em p opo cionado uma expe iência que nunca esquece ei, semp e com
um pouco de loucu a e boa disposição à mis u a.
Aos meus 11 magní icos: Ana, Ca ina, Ca los, Ca i, Jeje, Joana, João, Má cia, Ma i,
TG e Ri inha. Ob igada po oda a cumplicidade, amizade, loucu a, di e são, ca inho,
comp eensão, dedicação, união, paciência, a e o e p esença cons an e. Po a u a em o
pio e acei a em o melho que há em mim. Já nada é, nem nunca mais se á, o mesmo
sem ocês.
Ao Ch is e à Inês, dos melho es amigos que a ida me pode ia e dado, pela o ça,
pelos so isos, pela b incadei a e pela con iança. Mas, p incipalmen e, po me e em dado
a opo unidade de p o a que a desculpa de que o empo e a dis ância des oem
amizades, é apenas isso, uma desculpa.
À minha amília, que semp e me acompanhou e es e e p esen e em cada e apa da
minha ida, e es a não oi exceção. Ob igada po odo o apoio e o ça que, dia iamen e,
me azem alcança odos os meus obje i os e supe a odas as ad e sidades.
Às minhas i mãs, os meus ca amelos epe idos, que an as do es de cabeça me dão
mas sem as quais a minha ida não se ia a mesma. Ob igada po odas as “cusquices”,
iso as e pale mices, mas p incipalmen e, po o na em a minha ida udo menos
monó ona.
Aos meus pais, a quem de o udo aquilo que sou e aos quais não enho pala as
su icien es pa a ag adece udo o que me o am p opo cionando ao longo da ida.
Ob igada po odas as pala as de enco ajamen o, pela o ça e apoio incondicionais ao
longo de cada e apa e decisão da minha ida. Ob igada po simplesmen e es a em lá
pa a me ajuda em a o na -me na melho pessoa que posso se . Es e abalho é
dedicado a eles.
III
ABSTRACT
The epide mal g ow h ac o ecep o (EGFR) is a ansmemb ane y osine kinase
ecep o ha , upon ac i a ion, igge s se e al pa hways, such as he RAS/RAF/ERK
pa hway, ha a e o en de egula ed in colo ec al ca cinomas. Two monoclonal an ibodies
a ge ing EGFR, ce uximab and pani umumab, ha e p o en o be e ec i e in he
ea men o me as a ic colo ec al cance (mCRC). Howe e , i was disco e ed ha
pa ien s wi h ac i a ing mu a ions in exon 2 (codons 12/13) o he KRAS gene do no
espond o his he apy, es ablishing hem as he i s nega i e p edic o s o esponse o
an i-EGFR he apy.
The KRAS gene, an e ec o o EGFR signaling h ough he RAS/RAF/ERK pa hway, is
mu a ed in exon 2 in abou 40% o all mCRC. In KRAS exon 2 wild- ype pa ien s, only 40
o 60% achie e a esponse when ecei ing his he apy, some hing ha sugges s ha
al e a ions in o he EGFR downs eam e ec o s may also be associa ed wi h he lack o
esponse o his he apy. Recen ly, he impo ance o less equen KRAS and NRAS
(RAS) mu a ions has been unco e ed based on new esul s om ecen clinical ials,
which epo ed ha pa ien s wi h a e ac i a ing RAS mu a ions also do no bene i om
an i-EGFR he apy. This in o ma ion shows he need o iden i y new p edic i e bioma ke s
ha migh help o selec pa ien s who a e mos likely o bene i om an i-EGFR.
In a consecu i e se ies o 241 mCRC samples, wild- ype o KRAS codons 12 and 13,
we sea ched o less equen RAS mu a ions ha migh ac as p edic o o esponse o
an i-EGFR he apy, namely mu a ions in less equen mu a ional ho spo s in KRAS (exon
3 and 4) and NRAS (exons 2, 3 and 4).
Abou 19% (46/241) o he cases e alua ed had a mu a ion in he analyzed egions. All
mu a ions we e ound in he e egozi y and we e mu ually exclusi e. Thi y cases (12.4%;
30/241) had a KRAS mu a ion and six een (6.6%; 16/241) had a NRAS mu a ion, wi h he
ollowing dis ibu ion: hi een mu a ions we e ound in KRAS exon 3 (28.3%; 13/46),
se en een in KRAS exon 4 (37.0%; 17/46), eigh in NRAS exon 2 (17.4%; 8/46) and eigh
in NRAS exon 3 (17.4%; 8/46). No mu a ions we e ound in exon 4 o he NRAS gene.
One no el poin mu a ion, no p e iously desc ibed in CRC, was ound in exon 3 o he
NRAS gene in wo cases. KRAS mu a ions we e mo e equen in ea lie han in la e
s ages a diagnosis (P=0.001).
In conclusion, nea ly one- i h o mCRC pa ien s wild- ype o KRAS exon 2 (codons
12/13) p esen o he , less equen , RAS mu a ions ha migh be associa ed wi h lack o
esponse o an i-EGFR he apy. Howe e , u he s udies a e necessa y o con i m hese
mu a ions as nega i e p edic o s o esponse o his he apy in he pa ien s o ou se ies.
V
RESUMO
O ece o de a o de c escimen o epidé mico (EGFR) é uma i osina cinase
ansmemb ana que, após a i ação, inicia di e sas ias de ansdução de sinal, ais
como a ia RAS/RAF/ERK, cuja a i idade es á equen emen e des egulada em
ca cinomas colo- e ais. Dois an ico pos monoclonais di igidos ao EGFR, ce uximab e
pani umumab, p o a am se e icazes no a amen o do canc o colo- e al me as á ico. No
en an o, oi descobe o que indi íduos com mu ações a i an es nos codões 12 e 13 do
gene KRAS não bene iciam des e a amen o, o que as o na no p imei o bioma cado
p edi i o de ausência de espos a à e apia an i-EGFR.
O gene KRAS, um e e o do EGFR a a és da ia de ansdução de sinal
RAS/RAF/ERK, es á mu ado no exão 2 em ce ca de 40% dos ca cinomas colo- e ais
me as á icos. Dos doen es sem mu ação no exão 2 do KRAS, só 40 a 60% espondem ao
a amen o, o que suge e que al e ações nou as p o eínas e e o as do EGFR pode ão
es a en ol idas nes a ausência de espos a. Recen emen e, oi e elada a impo ância
de mu ações menos equen es nos genes KRAS e NRAS (RAS) em ensaios clínicos,
que epo a am que indi íduos com es as mu ações não bene iciam do a amen o com
an i-EGFR.
Numa sé ie consecu i a de 241 casos de ca cinoma colo- e al me as á ico, sem
mu ações nos codões 12 e 13 do KRAS, p ocu ámos mu ações RAS menos equen es
que possam se usadas como bioma cado es p edi i os de espos a à e apia com
ce uximab e pani umumab. Em pa icula , o am pesquisadas mu ações nou os codões
do KRAS (exões 3 e 4) e do NRAS (exões 2, 3 e 4).
Ce ca de 19% (46/241) dos casos analisados ap esen a am uma mu ação nas egiões
analisadas. Todas as mu ações o am encon adas em he e ozigo ia e e am mu uamen e
exclusi as. T in a casos (12,4%; 30/241) inham uma mu ação no KRAS e dezasseis
(6,6%; 16/241) no NRAS, com a seguin e dis ibuição: eze mu ações no exão 3 (28,3%;
13/46) e dezasse e no exão 4 (37,0%; 17/46) do KRAS; oi o no exão 2 (17,4%; 8/46) e
oi o no exão 3 (17,4%; 8/46) do NRAS. Não o am encon adas mu ações no exão 4 do
gene NRAS. Uma mu ação pon ual no a, não desc i a em canc o colo- e al, oi
encon ada no exão 3 do NRAS em dois casos. As mu ações no KRAS o am mais
equen es nos es ádios mais p ecoces do que nos mais a dios na al u a do diagnós ico
(p=0,001).
Em conclusão, ap oximadamen e um quin o dos doen es com canc o colo- e al
me as á ico, sem mu ações nos codões 12 e 13 do KRAS, ap esen am ou as mu ações
RAS menos equen es que podem es a associadas à ausência de espos a à e apia

XIII
RELEVANT ABREVIATIONS
AKT – -ak mu ine hymoma i al oncogene homolog
APC – Adenoma ous polyposis coli
ARAF – - a mu ine sa coma 3611 i al oncogene homolog
BAX – BCL2-associa ed X p o ein
BRAF – - a mu ine leukemia i al oncogene homolog B1
CCND1 – Cyclin D1 encoding gene
CDK8 – Cyclin-dependen kinase 8
c-RAF1 – - a -1 mu ine leukemia i al oncogene homolog 1
CRC – Colo ec al cance
CTNNB1 – Ca enin be a 1
DCC – Dele ed in colo ec al cance
DNA – Deoxy ibonucleic acid
dNTP – Deoxy ibonucleo ide iphospha e
EGF – Epide mal g ow h ac o
EGFR – Epide mal g ow h ac o ecep o
ERBB1 – see HER1
ERBB2 – see HER2
ERBB3 – see HER3
ERBB4 – see HER4
ERK – elk- ela ed y osine kinase
ERK1 – elk- ela ed y osine kinase 1
ERK2 – elk- ela ed y osine kinase 2
FISH – Fluo escen in si u hyb idiza ion
G-domain – Guanine nucleo ide-binding domain
GDP – Guanosine diphospha e
G-p o ein – Guanine nucleo ide-binding p o ein
G b2 – G ow h ac o ecep o -bound p o ein 2
GTP – Guanosine iphospha e
HER1 – Epide mal g ow h ac o ecep o
HER2 – -e b-b2 e y h oblas ic leukemia i al oncogene homolog 2
HER3 – -e b-b2 e y h oblas ic leukemia i al oncogene homolog 3
HER4 – -e b-b2 e y h oblas ic leukemia i al oncogene homolog 4
HRAS – -H- as Ha ey a sa coma i al oncogene homolog
IgG1 – Immunoglobulin G subclass 1
XIV
IgG2 – Immunoglobulin G subclass 2
IGFIIR – Insulin-like g ow h ac o 2 ecep o
JAK – Janus kinase
KRAS – Ki s en a sa coma-2 i al ( -Ki- as2) oncogene
MAPK – Mi ogen-ac i a ed p o ein kinase
mCRC – Me as a ic colo ec al cance
MEK – Mi ogen-ac i a ed p o ein kinase kinase
MEK1 – Mi ogen-ac i a ed p o ein kinase kinase 1
MEK2 – Mi ogen-ac i a ed p o ein kinase kinase 2
MGMT – O(6)-me hylguanine-DNA me hyl ans e ase
MLH1 – mu L homolog 1
MSH2 – mu S homolog 2
MSH3 – mu S homolog 3
MSH6 – mu S homolog 6
mTo – Mechanis ic a ge o apamycin
NRAS – Neu oblas oma RAS i al ( - as) oncogene homolog
PCR – Polyme ase chain eac ion
PI3K – Phosphoinosi ide-3-kinase
PIK3CA – Phosphoinosi ide-3-kinase, ca aly ic, alpha polypep ide
PTEN – Phospha ase and ensin homolog
PLCϒ – Phospholipase C gamma
SH2 – S c homolog 2
SMAD2 – SMAD amily membe 2
SMAD4 – SMAD amily membe 4
SOS – Son o se enless
STAT – S e ol O-acyl ans e ase 1
TCF – T cell- ac o
TGF-

– T ans o ming g ow h ac o 
TGF-β – T ans o ming g ow h ac o β
TGFβRII – T ans o ming g ow h ac o β ecep o ype II
TP53 – Tumo p o ein p53
VEGF – Vascula endo helial g ow h ac o
VEGFR – Vascula endo helial g ow h ac o ecep o
I
NTRODUCTION
INTRODUCTION
3
I. INTRODUCTION
Wi h o e 14 million new cance cases and 8.2 million cance dea hs es ima ed o ha e
occu ed in 2012 (IARC, 2013), cance is among he leading causes o dea h in he wo ld.
The bu den o his malignancy seems o be inc easing in economic de eloping coun ies,
mos ly due o popula ion aging and g ow h, as well as a esul o an inc easing adop ion o
cance -associa ed beha io s, such as smoking (Jemal e al., 2011). Despi e inc easing
awa eness, colo ec al cance (CRC) emains as one o he mos common cance s
wo ldwide.
Epidemiology
CRC is a majo cause o mo bidi y and mo ali y h oughou he wo ld, wi h o e 1.3
million new cases diagnosed in 2012 (Figu e 1). Eu ope and No h Ame ica a e among
he egions wi h he highes incidence a es o his ype o cance . This a e is apidly
inc easing in se e al a eas ha a e conside ed as low isk a eas, such as Eas e n Asia.
This migh be he e lec ion o changes in die a y and li es yle ac o s associa ed wi h
“wes e niza ion”, like smoking o obesi y. In con as o hese high incidence ends, he
occu ence o his pa hology seems o be dec easing in se e al pa s o he wo ld,
including he Uni ed S a es, p obably due o popula ion sc eening schemes ha allow
ea ly de ec ion o CRC and emo al o p ecance ous lesions (Jemal e al., 2011; Fe lay e
al., 2013; IARC, 2013).

MUTATIONAL ANALYSIS OF KRAS AND NRAS IN METASTATIC COLORECTAL CANCER
4
In 2012, CRC was he second mos common malignancy in Eu ope (excluding non-
melanoma skin cance s), wi h 464.000 newly diagnosed cases es ima ed o ha e
occu ed, which accoun s o 12.1% o all cance cases. I had he hi d highes incidence
in men, ollowing p os a e and lung cance , and he second in women, only su passed by
b eas cance . This malignancy is sligh ly mo e inciden in men han in women. I was
also he second mos equen cause o dea h by cance , wi h almos 215 000 dea hs
es ima ed, which accoun s o 12.2% o all cance dea hs (Fe lay e al., 2013).
In e ms o CRC incidence and mo ali y, Po ugal ollows he same pa e ns o Eu ope
(Figu e 2). In 2012, i was he highes inciden malignancy, wi h 7129 new cases
diagnosed. Da a analysis by sex demons a ed ha CRC has he second highes
incidence in bo h sexes, a e p os a e (male) and b eas cance ( emale). I was also he
leading cause o dea h by cance , wi h 3797 dea hs, which accoun s o 15.7% o all
cance dea hs (Fe lay e al., 2013; IARC, 2013).
Figu e 1. Es ima ed age-s anda dised incidence and mo ali y a es o
CRC o male and emale, in he wo ld [Globocan, 2012 (IARC, 2013)].
INTRODUCTION
5
Risk Fac o s
CRC is a e y complex and he e ogeneous disease and se e al e iologic ac o s
con ibu e o he appea ance o his malignancy.
The isk o de eloping CRC inc eases wi h age, p e e en ially a e he age o 40. I is
es ima ed ha mo e han 90% o he pa ien s diagnosed wi h his malignancy a e aged 50
o olde (Ame si e al., 2005; Hagga & Boushey, 2009).
CRC usually occu s in one o h ee pa e ns: inhe i ed, amilial o spo adic. Inhe i ed
o ms a e esponsible o abou 5-10% o all CRC cance s, and a e ela ed o ecognized
he edi a y condi ions (Figu e 3). The mos common a e amilial adenoma ous polyposis
(FAP) and he edi a y nonpolyposis colo ec al cance (HNPCC), also know as Lynch
synd ome, which a e esponsible o 1% and 2%-4% o all CRC cases, espec i ely
(Ame si e al., 2005; Rus gi, 2007; Hagga & Boushey, 2009; Jaspe son e al., 2010).
O he inhe i ed diseases ha lead o an inc eased isk o CRC a e MUYTH-associa ed
polyposis (MAP), Peu z-Jeghe s synd ome (PJS) and ju enile polyposis synd ome (JPS)
(Rus gi, 2007; Jaspe son e al., 2010).
Familial cases a e de ined as amilies wi h inc eased p edisposi ion o cance , p obably
due o an he edi a y basis wi h he in ol emen o genes ha a e less pene an and/o
he sign o sha ed en i onmen al and li es yle ac o s. I is es ima ed ha abou 20-30% o
all CRC cases occu in his con ex (Rus gi, 2007; Jaspe son e al., 2010).
A
B
Figu e 2. Es ima ed age-s anda dised incidence and mo ali y a es o CRC o male and emale, in A)
Eu ope and B) Po ugal [Globocan, 2012 (IARC, 2013)].
MUTATIONAL ANALYSIS OF KRAS AND NRAS IN METASTATIC COLORECTAL CANCER
6
Al hough gene ics, amily his o y and suscep ibili y ac o s play an impo an ole in
he de elopmen o his disease, he majo i y o CRCs a e spo adic (~70%), wi h no p io
amily his o y (Hagga & Boushey, 2009). Se e al epidemiological s udies ha e con i med
he in luence o nume ous en i onmen al and die a y ac o s in he e iology o his
disease, such as a die high in a and low in ibe , a seden a y li es yle, obesi y, diabe es,
ciga e e smoking and alcohol abuse (Hagga & Boushey, 2009; Chan & Gio annucci,
2010; Ging as & Beli eau, 2011; Colussi e al., 2013). Physical ac i i y, on he o he hand,
is hough o lowe CRC isk. In ac , se e al s udies epo ha highe o e all le els o
physical ac i i y a e associa ed wi h a dec eased isk o CRC o ~20% (Huxley e al.,
2009). Ano he isk ac o is he p esence o ch onic in lamma o y bowel diseases (IBD),
such as C ohn’s disease o ulce a i e coli is (Ilyas e al., 1999; Colussi e al., 2013).
CRC Diagnosis and S aging
CRC diagnosis is usually made a e he onse o he symp oma ology o he disease,
bu mos o he symp oms ( ec al bleeding, blood in he s ools, change in bowel habi s)
a e non-speci ic and consis en wi h o he condi ions besides CRC. This, along wi h he
ac ha mos pa ien s wi h an ea ly-s age disease a e asymp oma ic, p esen s di icul ies
when ying o diagnose his disease. The mos common sc eening echniques a e he
ecal occul blood es and/o colonoscopy, he la e allowing di ec inspec ion o he
en i e colon and same-session biopsy necessa y o his opa hological diagnosis. Pa ien s
should also unde go a physical examina ion and may pe o m compu ed omog aphy
(CT), magne ic esonance imaging (MRI) o luo odeoxyglucose-posi on emission
Figu e 3. Ci cle g aph depic ing he geno ypic and pheno ypic he e ogenei y in CRC
[adap ed om (Lynch e al., 2009)].
INTRODUCTION
7
omog aphy (FDG-PET), which allow he iden i ica ion and cha ac e iza ion o a possible
me as a ic disease (Le in e al., 2008; Van Cu sem, No dlinge , e al., 2010; Cummings &
Coope , 2011).
Once diagnosis is made, i is necessa y o s age he umo acco ding o i s pa hological
cha ac e is ics. S aging assessmen is a key ac o used o de ine ea men and o
es ima e he chance o a success ul ea men ou come. The mos common s aging
sys em o CRC is he TNM sys em (Cen elles, 2012). Each o he h ee le e s o he TNM
sys em (Table 1), acco ding o he Ame ican Join Commi ee on Cance (AJCC), s and
o a speci ic meaning in ega d o he cha ac e is ics o he umo : T - size and/o ex en
( each) o he p ima y umo ; N - amoun o sp ead o nea by lymph nodes; and M -
p esence o me as asis o seconda y umo s caused by he sp ead o cance cells o o he
pa s o he body (Edge e al., 2010; Cen elles, 2012). I is also impo an o epo he
iming o he s aging assessmen . Clinical s aging – o cTNM – includes any in o ma ion
ob ained be o e ini ia ion o any kind o p eope a i e ea men . Pa hological s aging –
pTNM – is de ined by he in o ma ion ob ained a e he examina ion o he su gically
emo ed issues (Edge e al., 2010).
Table 1. AJCC cance s anging o colon and ec al ca cinomas [adap ed om (Shia e al., 2012)].
P ima y Tumo (T)
Tx
P ima y umo canno be assessed
T0
No e idence o p ima y umo
Tis
Ca cinoma in si u: in aepi helial o in asion o lamina p op ia
T1
Tumo in ades submucosa
T2
Tumo in ades muscula is p op ia
T3
Tumo in ades muscula is p op ia in o pe icolo ec al issues
T4a
Tumo pene a es o he su ace o he isce al pe ino eum
T4b
Tumo di ec ly in ades o is adhe en o o he o gans o s uc u es
Regional Lymph Nodes (N)
Nx
Regional lymph nodes canno be assessed
N0
No egional lymph node me as asis
N1
Me as asis in 1 o 3 egional lymph nodes
N1a
Me as asis in 1 egional lymph node
N1b
Me as asis in 2-3 egional lymph nodes
N1c
Tumo deposi (s) in he subse osa, mesen e y, o nonpe i onealized pe icolic o pe i ec al issues
wi hou egional nodal me as asis
N2
Me as asis in 4 o mo e egional lymph nodes
N2a
Me as asis in 4-6 egional lymph nodes
N2b
Me as asis in 7 o mo e egional lymph nodes
Dis an Me as asis (M)
M0
No dis an me as asis (no pa hological M0; use clinical M o comple e s age g oup)
M1
Dis an me as asis
M1a
Me as asis con ined o 1 o gan o si e (eg. li e , lung, o a y, non egional node)
M1b
Me as ases in mo e han 1 o gan/si e o he pe i oneum
MUTATIONAL ANALYSIS OF KRAS AND NRAS IN METASTATIC COLORECTAL CANCER
14
O he pa hways in ol ed in CIN
SMAD2, SMAD4 and DCC genes a e all loca ed in he long a m o ch omosome 18
(18q21.1). SMAD2 and SMAD4 a e ansc ip ion ac o s in ol ed in he TGF-β signaling
pa hway ha egula es g ow h as well as apop osis. The DCC gene codes o a la ge
memb ane ecep o p o ein, om he immunoglobulin supe amily, ha p omo es
apop osis in he absence o i s ligand (ne in-1) (Ilyas e al., 1999; Wo hley e al., 2007).
LOH o 18q is associa ed wi h nega i e p ognosis and is epo ed in up o 60% o CRCs
(Wo hley e al., 2007; Colussi e al., 2013).
Mu a ions in he phosphoinosi ide-3 kinase gene (PIK3CA), de ec ed in app oxima ely
a hi d o CRCs, o en occu simul aneously wi h APC mu a ions, and cause inc eased
AKT signaling e en wi hou he p esence o g ow h ac o s. They also in e ac wi h a
cen al egula o o cell g ow h and me abolism (mTOR) and wi h KRAS. Addi ionally, he
phospha ase and ensin homolog (PTEN), which ac s as a umo supp esso gene in his
pa hway due o i s inhibi o y e ec on PI3K-AKT signaling, is silenced in nea ly 30% o
CRC (Kan han e al., 2012; Yu e al., 2014).
Mic osa elli e ins abili y (MSI) pa hway
Mic osa elli es a e sho epea nucleo ide sequences loca ed h oughou he genome,
in bo h coding and non-coding egions. Because o hei epe i i e s uc u e, hey a e
p one o e o s ha occu du ing DNA eplica ion. Those e o s a e ecognized and
epai ed by he DNA Misma ch Repai (MMR) sys em du ing eplica ion, which ensu es a
co ec DNA syn hesis (Al-Sohaily e al., 2012; Kan han e al., 2012). The MMR sys em is
composed o mul iple in e ac ing p o eins, such as MSH2 and MLH1, and mu a ions in he
genes encoding hese p o eins lead o he inac i a ion o he MMR sys em and he
accumula ion o se e al DNA eplica ion e o s, esul ing in MSI (Ilyas e al., 1999;
Ma kowi z & Be agnolli, 2009; Al-Sohaily e al., 2012; Kan han e al., 2012).
MSI is he hallma k o he HNPCC synd ome, o igina ed by MMR ge mline mu a ions.
This pa hway is also in ol ed in he genesis o app oxima ely 15% o spo adic CRC cases
and is mos ly caused by epigene ic silencing o he MLH1 gene p omo e (Al-Sohaily e
al., 2012; Kan han e al., 2012; Colussi e al., 2013; Bogae & P enen, 2014). Tumo s ha
de elop h ough his pa icula pa hway p esen a di e en pheno ype om CIN posi i e
CRCs: hey a e mo e likely o a ise in he p oximal colon, a e poo ly di e en ia ed, o en
exhibi lymphocy ic in il a ion and, in gene al, pa ien s a ec ed by MSI-high (MSI-H)
CRCs p esen be e p ognosis and su i al. Spo adic MSI-H umo s a e also
cha ac e ized by a low equency o APC, CTNNB1 and KRAS mu a ions and a high

INTRODUCTION
15
equency o BRAF mu a ions, a membe o he RAF amily in ol ed in he media ion o
cellula esponse h ough he RAS-RAF-ERK pa hway.
Se e al o he genes, such as TGFβRII and BAX, a e also mu a ed in hese CRCs.
TGFβRII inac i a ing mu a ions a e ound in mo e han 80% o all MSI-H CRCs and a e
in ol ed in he adenoma ansi ion o high-g ade dysplasia o me as a ic ca cinoma.
Mu a ions in he SMAD2 and SMAD4 genes, in ol ed in he deac i a ion o TGFβ
signaling, a e also common in MSI-H CRCs. The p o-apop o ic umo supp esso gene
BAX is mu a ed in 50% o CRCs cases and allows umo cells o e ade he in insic
apop osis mechanisms. Addi ionally, mu a ions in o he genes, such as MSH3 and MSH6,
Insulin G ow h Fac o Type 2 Recep o (IGFIIR) o CCND1, a e also equen ly p esen in
MSI-H CRCs, al hough a a lowe equency hen he ones men ioned abo e (Colussi e
al., 2013).
CpG island me hyla o pheno ype (CIMP)
This hi d pa hway, p esen in app oxima ely 20 o 30% o CRC, consis s o he
abe an hype me hyla ion o he CpG dinucleo ide sequences localized in he p omo e
egions o genes in ol ed in se e al unc ions, such as cell cycle egula ion, apop osis,
DNA epai and in asion. This hype me hyla ion esul s in gene silencing, which p o ides
an al e na i e mechanism o loss o unc ion o umo supp esso genes. In ac , he
epigene ic silencing o a gene is biologically equi alen o acqui ing an inac i a ing
mu a ion, so i can occu as a i s , second o bo h hi s o inhibi gene exp ession
(Wo hley e al., 2007; Ma kowi z & Be agnolli, 2009; Al-Sohaily e al., 2012; Colussi e
al., 2013).
CIMP umo s a e classi ied as CIMP-high (CIMP-H) o CIMP-low (CIMP-L), based on
he numbe o me hyla ed ma ke s (Al-Sohaily e al., 2012; Colussi e al., 2013). CIMP-H
CRCs o en con ain BRAF gene mu a ions, which a e associa ed wi h inc eased cell
g ow h and p og ession o ca cinogenesis. BRAF V600E (Val600Glu) mu a ion is p esen
in 80 o 90% o CRC cases wi h sessile se a ed adenomas (SSA) bu is mos ly absen in
con en ional adenomas. Addi ionally, BRAF mu a ions a e p esen in ea ly hype plas ic
polyps ( he se a ed p ecu so s) o in la e dysplas ic se a ed adenomas ha equen ly
ha e CIMP-H and MSI-H ea u es, which leads o he hypo hesis ha he se a ed
pa hway is in ol ed in he spo adic CIMP CRCs de elopmen (Wo hley e al., 2007;
Legge & Whi ehall, 2010; Colussi e al., 2013).
Clinically, CIMP-H umo s ha e a pa icula ly poo p ognosis and a e usually loca ed in
he p oximal si e o he colon, simila o MSI umo s (Wo hley e al., 2007; Bogae &
P enen, 2014). On he o he hand, CIMP-L umo s ha e a low le el o DNA me hyla ion
MUTATIONAL ANALYSIS OF KRAS AND NRAS IN METASTATIC COLORECTAL CANCER
16
and, ins ead o he BRAF mu a ions obse ed in CIMP-H umo s, hey a e usually
associa ed wi h KRAS and MGMT mu a ions (Wo hley e al., 2007; Colussi e al., 2013;
Bogae & P enen, 2014).
KRAS and NRAS mu a ional s a us and i s impo ance in
CRC ea men
The disclosu e ha pa ien s wi h ac i a ing KRAS gene mu a ions do no bene i om
an i-EGFR he apy (ce uximab o pani umumab) su aced a e a se ies o ini ial
e ospec i e analyses. This made KRAS mu a ions eme ge as he only nega i e
bioma ke p edic o o esponse o his he apy. To be e unde s and how KRAS
ac i a ing mu a ions in luence an i-EGFR he apy’s e icacy, i is necessa y o unde s and
he link be ween EGFR and RAS in CRC.
EGFR signaling pa hways in CRC
EGFR, also known as HER1/ERBB1, belongs o he E bB amily o ecep o TKs,
which comp ises h ee o he membe s: HER2 (ERBB2/neu), HER3 (ERBB3) and HER4
(ERBB4). All p o eins o his amily a e ancho ed in he cy oplasmic memb ane and sha e
a simila s uc u e composed by an ex acellula ligand-binding domain, a single
hyd ophobic ansmemb ane domain and a cy oplasmic TK-con aining domain (Spano e
al., 2005; No manno e al., 2006; Scal i i & Baselga, 2006). In no mal cells, EGFR
signaling pa hway is ac i a ed in a ligand-dependen manne . E bB amily membe s can
be ac i a ed by se e al known ligands, such as epide mal g ow h ac o (EGF),
ans o ming g ow h ac o α (TGF-α), amphi egulin and epi egulin, ha bind speci ically o
EGFR (Hynes & MacDonald, 2009).
Figu e 6. Gene ic ins abili y pa hways and hei o e lapping ela ionships
[adap ed om (Ma kowi z & Be agnolli, 2009)].
INTRODUCTION
17
EGFR ac i a ion by ligand binding induces he dime iza ion o he ecep o wi h
o ma ion o homo- and he e odime s ha leads o au ophospho yla ion o speci ic
y osine esidues wi hin he cy oplasmic ail o he ecep o s, ini ia ing in acellula
signaling ia se e al pa hways, namely RAS/RAF/ERK, PI3K/AKT, JAK/STAT and PLCϒ
(Figu e 7) (No manno e al., 2006; Scal i i & Baselga, 2006). These signal ansduc ion
cascades a e esponsible o di e se cellula esponses, such as p oli e a ion, mig a ion,
di e en ia ion and apop osis. Cons i u ional ac i a ion o hese pa hways can be achie ed
by ecep o o e exp ession o ac i a ing mu a ions, which a e common in se e al
malignancies, including CRC (No manno e al., 2006; Robe s & De , 2007).
The RAS/RAF/ERK pa hway
The RAS/RAF/ERK pa hway is one o he mos de egula ed signaling pa hways in
human cance . In his pa hway, RAS ac i a ion leads o a sequen ial ac i a ion o h ee
MAPKs (RAF, MEK and ERK), which in u n gene a e signals ha p omo e egula ion o
se e al cellula esponses ha es ablish cell p oli e a ion, su i al and di e en ia ion
(Dhillon e al., 2007; Robe s & De , 2007).
Figu e 7. EGFR signaling pa hways [adap ed om (Scal i i & Baselga, 2006)].
MUTATIONAL ANALYSIS OF KRAS AND NRAS IN METASTATIC COLORECTAL CANCER
18
RAS p o eins – KRAS, HRAS and NRAS – a e impo an componen s o a la ge amily
o small GTP-binding p o eins. These h ee membe s o he RAS amily a e composed o
a C- e minal and N- e minal egions, bo h impo an o RAS p o eins unc ions. The C-
e minal egion con ains a CAAX mo i , which is he a ge o pos - ansla ional
modi ica ions ha allow he ec ui men o he p o eins o he inne ace o he plasma
memb ane, essen ial o hei no mal unc ion (Downwa d, 2003; Ka noub & Weinbe g,
2008). On he o he hand, he N- e minal egion is an impo an egula o o he p o ein
GDP-bound and GTP-bound s a es. The s uc u al di e ences be ween hese s a es
eside in wo egions o he N- e minal, he swi ch I and swi ch II egions. Binding o he
GTP molecule al e s he con o ma ion o bo h swi ch egions and allows he RAS p o ein
o emain in an ac i e s a e. Upon he elease o he GTP’s ex a phospha e g oup, he
swi ch egions modi y hei con o ma ion and e u n o he inac i e s a e (Ka noub &
Weinbe g, 2008; San a pia e al., 2012). This GDP/GTP cyclic p ocess is ca alyzed by
guanine nucleo ide exchange ac o s (GEFs) and GTPase ac i a ing p o eins (GAPs), wi h
he o me acili a ing he exchange om GDP o GTP and he la e p omo ing he
hyd olysis o GTP o GDP (Figu e 8). The balance be ween hese p o eins is impo an o
de e mine he ac i a ion o he RAS p o ein and i s downs eam a ge pa hways
(Downwa d, 2003; San a pia e al., 2012).
In no mal cells, RAS becomes ac i a ed upon ex acellula s imuli, which ac i a e
ecep o TKs such as EGFR. The au ophospho yla ed ecep o hen binds o he SH2
domain o he adap o p o ein g ow h- ac o - ecep o -bound p o ein 2 (GRB2). Since he
SOS (GEF) is connec ed o he same adap o p o ein in he SH3 domain, he binding o
he ecep o o he GRB2 b ings SOS in close p oximi y o he RAS p o ein, leading o i s
ac i a ion (Downwa d, 2003; Robe s & De , 2007). Ac i a ed RAS in e ac s wi h h ee
closely ela ed RAF kinases (c-RAF1, BRAF and ARAF), mobilizing hem o he plasma
memb ane, whe e hey become ac i a ed. Then, Ra kinases phospho yla e MEK1 and
MEK2 (MAPKKs), which in u ns igge s he phospho yla ion and ac i a ion o MAPKs
(ERK1 and ERK2) (Figu e 9). Once ac i a ed, ERK1 and ERK2 a e ansloca ed o he
Figu e 8. Ups eam signaling o RAS and i s con ol by he GDP-
GTP cycle [adap ed om (Downwa d, 2003)].
INTRODUCTION
19
nucleus, whe e hey egula e he ac i i y o a ious ansc ip ion ac o s (Downwa d, 2003;
Robe s & De , 2007; San a pia e al., 2012).
Since his is a complex signaling pa hway ha ensu es essen ial cellula esponses, i s
de egula ion is an impo an key ac o o cance p og ession. Se e al mechanisms, such
as KRAS and BRAF ac i a ing mu a ions o EGFR o e exp ession, con ibu e o an
imp ope ac i a ion o he pa hway and ha e been desc ibed in se e al umo ypes,
including CRC. Fu he mo e, i was also iden i ied ha E k ac i a ion can induce
up egula ion o EGFR ligands, which p omo es an au oc ine g ow h loop c ucial o umo
g ow h (Robe s & De , 2007; San a pia e al., 2012).
P edic i e bioma ke s o an i-EGFR he apy esponse
As s a ed be o e, se e al e ospec i e s udies o KRAS mu a ional s a us in umo s
om pa ien s ea ed wi h an i-EGFR he apy ound ha ac i a ing mu a ions in KRAS
codons 12 and 13 we e associa ed wi h a lack o esponse o hese he apies (Table 3a
and 3b). Mu a ions in hese pa icula codons in KRAS exon 2, p esen in nea ly 40% o all
mCRC pa ien s, cause cons i u i e ac i a ion o he RAS/ /ERK pa hway, despi e EGFR
inhibi ion. Thus, sc eening o hese mu a ions is ecommended be o e he apy, since only
pa ien s wi h KRAS wild- ype mCRC will bene i om i . Howe e , among hose pa ien s
wi h KRAS exon 2 wild- ype umo s, only 40-60% esponds o an i-EGFR he apy (De
Roock e al., 2008; Lie e e al., 2008). This sugges s ha o he ac i a ing mu a ions
along his pa hway may also con e esis ance o an i-EGFR he apies. So, he e is a
Figu e 9. Oncogenic ac i a ion o he RAS/RAF/ERK signaling pa hway [adap ed om
(Robe s & De , 2007)].

MUTATIONAL ANALYSIS OF KRAS AND NRAS IN METASTATIC COLORECTAL CANCER
20
g ea need o iden i y al e na i e p edic i e bioma ke s ha will dis inguish pa ien s who
a e mos likely o bene i om his ype o he apy.
The i s a emp s o iden i y such bioma ke s led o he disco e y ha posi i e EGFR
o e exp ession (de e mined by immunohis ochemis y) has no co ela ion wi h ea men
esponse (Chung e al., 2005) and ha he associa ion be ween inc eased EGFR gene
copy numbe (de ec ed by FISH) and ea men esponse emains unce ain/con o e sial
(Mo oni e al., 2005; Lau en -Puig e al., 2009). These indings make i di icul o es ablish
EGFR al e a ions as p edic i e bioma ke s o ea men esponse. Howe e , se e al
s udies ha e been ocusing in he analysis o o he a ge s, such as o he EGFR
downs eam e ec o s (BRAF, NRAS, and PIK3CA), as well as less equen KRAS
mu a ions (De Roock e al., 2010; Guedes e al., 2013).
Recen analyses o umo s om pa ien s en olled in clinical ials demons a ed ha
o he KRAS (codons 59/61 – exon 3; codons 117/146 – exon 4) o NRAS (codons 12/13 –
exon 2; codons 59/61 – exon 3; codons 117/146 – exon 4) mu a ions lead o inc eased
le els o RAS-GTP. These ecen s udies also epo ed ha mos pa ien s ha bo ing hese
a e mu a ions did no achie e an objec i e esponse wi h an i-EGFR he apy (Douilla d
e al., 2013; Seymou e al., 2013). Gi en his, ecen guidelines o an i-EGFR CRC
ea men ecommend he e alua ion o he mu a ional s a us o he h ee exons o bo h
genes be o e ea men ini ia ion wi h an i-EGFR he apy.
Table 3. Design cha ac e is ics used in A) me a-analysis and B) he change in median p og ession- ee
su i al in KRAS wild- ype and mu an g oups o each s udy [adap ed om (Adels ein e al., 2011)].
A
INTRODUCTION
21
1(Amado e al., 2008) 2(Ka ape is e al., 2008) 3(Van Cu sem e al., 2009) 4(Van Cu sem, Lang, e al., 2010) 5(Pee e s e al.,
2010) 6(Bokemeye e al., 2009) 7(Bokemeye e al., 2011) 8(Maughan e al., 2010) 9(Douilla d e al., 2010) 10(T ei e al.,
2010) 11(Hech e al., 2009) 12(Tol e al., 2009)
B
A
AIMS OF THE
STUDY
MUTATIONAL ANALYSIS OF KRAS AND NRAS IN METASTATIC COLORECTAL CANCER
30
DNA ex ac ion om o malin- ixed pa a in-embedded issue
Whene e possible, umo a eas con aining a leas 50% o umo cells we e delimi ed, by
a pa hologis , in he hema oxylin and eosin (H&E) s ained slides o each sample. The
co esponding uns ained slides we e imme sed in xylene [SIGMA] and wice in e hanol 100%
[Me ck] o 5 minu es each. Tumo a eas, which we e p e iously delimi ed by compa ison
wi h he co esponden H&E s ained slides, we e mac odissec ed and ans e ed o a
mic ocen i uge ube. DNA was isola ed using he QIAamp® DNA FFPE Tissue Ki [QIAGEN],
ollowing manu ac u e ’s ins uc ions. Finally, DNA was quan i ied by spec opho ome y
wi h NanoD op ND-1000® [NanoD op Technologies].
Mu a ional s a us analysis
All samples we e ini ially sc eened by HRM o mu a ions in KRAS (NM_004985) exons
3 and 4 and NRAS (NM_002524.4) exons 2, 3 and 4, ollowed by au oma ed DNA Sange
sequencing o one s and ( o wa d o e e se), in o de o e alua e he p esence/absence
o DNA al e a ions. A second HRM was pe o med in all posi i e samples o he ini ial
analysis, ollowed by au oma ed DNA Sange sequencing o bo h s ands.
High Resolu ion Mel ing
PCR ampli ica ion and HRM analysis we e bo h pe o med on a Ligh Cycle -480 II
Real-Time Sys em [Roche Diagnos ics]. The PCR eac ion mix u e added o each well, o a 96
well pla e, was composed o a pai o p ime s ( o wa d and e e se), DNA o each sample
and PCR eagen s (Table 4). To p e en con amina ion and/o e apo a ion, 15µL o
mine al oil we e added o each well. The pla e was hen sealed wi h sealing ilm and
cen i uged a 2000 pm o 2 minu es.
Table 4. Componen s o he PCR eac ion mix u e.
PCR eac ion mix u e componen s
2,5x Ligh Scanne ® Mas e Mix [Idaho]
4.0µL
Fo wa d p ime [ ilabo]
350nM
Re e se p ime [ ilabo]
350nM
DNA
20-100ng
Reagen g ade wa e [Idaho]
4.9µL
To al eac ion olume
10µL
The p ime pai s used in his s udy o NRAS exons 2, 3 and 4 and KRAS 3 and 4 we e
all designed wi h p ime -BLAST so wa e (h p://www.ncbi.nlm.nih.go / ools/p ime -blas )
and a e a ailable upon eques o he Depa men o Gene ics o IPO-Po o.

MATERIALS AND METHODS
31
PCR ampli ica ion and HRM condi ions we e he same o all NRAS exons and o
KRAS exon 3. KRAS exon 4 condi ions di e ed om hose applied o he exons
men ioned abo e. Howe e , all exons we e subjec ed o an ini ial dena u a ion, ollowed
by 35-40 cycles o ampli ica ion. A e ha , one cycle o he e oduplex and one o mel ing
we e done be o e he pla e was cooled o 40ºC. The de ailed condi ions a e desc ibed,
sepa a ely, below:
I. Fo all NRAS exons and o KRAS exon 3: An ini ial dena u a ion a 95ºC o 15
minu es was ollowed by 35 cycles o 10 seconds a 95ºC, 20 seconds a 69ºC and
30 seconds a 72ºC. A e ha , one he e oduplex cycle was done a 97ºC o 1
minu e and 40ºC o 2 minu es, ollowed by one mel ing cycle om 70ºC o 95ºC
wi h 25acquisi ions/ºC. The pla e was inally cooled o 40ºC o 1 minu e wi h a
amp a e o 2.2ºC/second.
II. Fo KRAS exon 4: Ini ial dena u a ion was done a 95ºC o 10 minu es and
ollowed by 35 cycles o 20 seconds a 95ºC, 20 seconds a 65ºC and 20 seconds
a 72ºC, wi h a inal ex ension o 10 minu es a 72ºC. One he e oduplex and one
mel ing cycle we e done a e ha , wi h he samples being dena u ed wi h an ini ial
hold o 5 minu es a 95ºC and 1 minu e a 40ºC (he e oduplex cycle), ollowed by a
mel ing p o ile om 70ºC o 90ºC wi h 25acquisi ions/ºC (mel ing cycle). The pla e
was cooled o 40ºC in he same condi ions as desc ibed be o e o all NRAS exons
and KRAS exon 3.
Ampli ica ion and mel ing cu es we e ob ained and analyzed using he Ligh Cycle ®
480 Gene Scanning so wa e 1.5 [Roche diagnos ics].
Figu e 10. High esolu ion mel ing analysis o KRAS exon 3. A)
No malized and B) di e ence g aph, wi h wild- ype (blue) and mu a ed
(g een and ed) samples.
A
B
MUTATIONAL ANALYSIS OF KRAS AND NRAS IN METASTATIC COLORECTAL CANCER
32
DNA Sequencing
Be o e sequencing, all PCR ampli ica ion p oduc s we e pu i ied o emo e excess o
p ime s, sal s, enzymes and dNTPs om he p e ious eac ion. Fo ha pu pose, Illus a
GFX PCR DNA and Gel Bad Pu i ica ion Ki [GE Heal hca e Li e Sciences] and NZYGelpu e Ki
[nzy ech] we e used, acco ding o he manu ac u e ’s p o ocol.
A e ha , 1µL o each sample p oduc was used o he sequencing eac ion, which
also con ained 0.5µL o Big Dye® Te mina o 1.1 cycle sequencing Ready Reac ion Mix
[Applied Biosys ems], 3.4µL o Big Dye® Te mina o 1.1, 1.3 5x sequencing bu e [Applied
Biosys ems], 350nM o one o he p ime s ( o wa d o e e se) and 4.78µL o bides illed s e ile
wa e [B. B aun], o a o al olume o 10µL. Samples we e hen subjec ed o an ini ial
dena u a ion a 95ºC o 4 minu es, ollowed by 35 cycles o 95ºC o 10 seconds, 50ºC o
10 seconds and 60ºC o 2 minu es, wi h a inal ex ension o 60ºC o 10 minu es.
PCR sequencing p oduc s we e pu i ied using Illus a Sephadex® G-50 ine [GE Heal hca e
Li e Sciences] and added o 12µL o Hi-DiTM Fo mamide [Applied Biosys ems]. The p oduc s we e hen
un in ei he an ABI PRISMTM 310 Gene ic Analyze [Applied Biosys ems] o a 3500 Gene ic
Analyze [Applied Biosys ems]. Elec ophe og ams o each sample we e analyzed wi h he
Sequencing Analysis So wa e 5.4 [Applied Biosys ems]. All o hem we e ead a leas wice,
e iewed manually and wi h he Mu a ion Su eyo So wa e 4.0.8.
S a is ical analysis
S a is ical analysis was pe o med using ei he Qui-squa e o Fishe ’s exac es s o
assess s a is ical di e ences be ween he a ian s. Associa ions we e conside ed
s a is ically signi ican when P≤0.05. S a is ical analysis was pe o med wi h he SPSS
S a is ics so wa e package .22.0.
A
B
Figu e 11. Elec ophe og am o KRAS exon 3 sequence, wi h A) a wild- ype and B) a mu a ed sample.
R
ESULTS
RESULTS
35
IV. RESULTS
DNA om a o al o 241 KRAS exon 2 wild- ype mCRC samples we e sc eened in
pa allel o mu a ions in exons 3 and 4 o KRAS and exons 2, 3 and 4 o NRAS by HRM
and au oma ed sequencing. Au oma ed sequencing o he HRM p oduc s con i med he
p esence o 46 mu a ions (19.1%) in KRAS exons 3/4 o NRAS exons 2/3/4, wi h he
emaining 80.9% (195/241) being wild- ype o all egions s udied. All mu a ions we e
ound in he e ozygosi y and as a single mu a ion.
Table 5. Mu a ional s a us o he 241 mCRC samples analyzed.
Samples
Mu a ional S a us
F equencies
Mu an
46
Wild- ype
195
To al
241
Mu a ional Type and Dis ibu ion
O e all, 12.4% (30/241) o he cases p esen ed a mu a ion in KRAS and 6.6% (16/241)
we e NRAS mu a ed. The mu a ional dis ibu ion o he 46 posi i e cases was as ollows:
65.2% (30/46) in KRAS, wi h 28.3% (13/46) in KRAS exon 3 and 37.0% (17/46) in KRAS
exon 4, and 34.8% (16/46) in NRAS, wi h 17.4% (8/46) in NRAS exon 2 and 17.4% (8/46)

MUTATIONAL ANALYSIS OF KRAS AND NRAS IN METASTATIC COLORECTAL CANCER
36
in NRAS exon 3 (Figu e 12). No mu a ions we e ound in exon 4 o NRAS. The indi idual
mu a ions ound in each gene a e p esen ed in Tables 6 and 7. Ele en di e en mu a ions
we e ound in KRAS and se en di e en mu a ions we e de ec ed in NRAS. In all bu wo
cases he mu a ions we e missense, whe eas he emaining wo cases had an in ame
duplica ion and an in ame dele ion in KRAS exon 3.
Table 6. KRAS mu a ions iden i ied a e au oma ed sequencing.
KRAS
Case
Exon
Mu a ion
N .
73
3
c.151_195dup
p.Cys51_Se 65dup
1
5
3
c.176_178del
p.Asp59del
1
216
3
c.175G>A
p.Ala59Th
1
175
3
c.179G>A
p.Gly60Asp
1
68, 87, 138, 141, 192
3
c.182A>T
p.Gln61Leu
5
39, 84, 209
3
c.183A>C
p.Gln61His
3
224
3
c.183A>T
p.Gln61His
1
78, 93, 213
4
c.351A>T
p.Lys117Asn
3
165
4
c.351A>C
p.Lys117Asn
1
19, 30, 47, 49, 108, 119, 120,
131, 173, 200, 235
4
c.436G>A
p.Ala146Th
11
149, 164
4
c.437C>T
p.Ala146Val
2
To al
30
Figu e 12. Dis ibu ion (%) o he 46 mu a ions de ec ed in all analyzed exons
in mCRC samples.
RESULTS
37
B p.Gln61His
Table 7. NRAS mu a ions iden i ied a e au oma ed sequencing
NRAS
Case
Exon
Mu a ion
N .
31, 88
2
c.34G>T
p.Gly12Cys
2
40, 64, 118, 220, 228
2
c.35G>A
p.Gly12Asp
5
139
2
c.37G>C
p.Gly13A g
1
13, 26, 227
3
c.181C>A
p.Gln61Lys
3
38, 111
3
c.182A>G
p.Gln61A g
2
124
3
c.182A>T
p.Gln61Leu
1
4, 52
3
c.183A>T
p.Gln61His
2
To al
16
No el Mu a ions
O he 11 di e en KRAS mu a ions and se en di e en NRAS mu a ions iden i ied in
his s udy, he mu a ion c.183A>T, p.Gln61His, is no el (Figu e 13) and he emaining 17
mu a ions ha e p e iously been epo ed in he COSMIC da abase (COSMIC) o in he
li e a u e.
A
Figu e 13. Elec ophe og ams o he mu a ion ound in NRAS exon 3 ha was no p e iously
desc ibed, wi h A) wild- ype and B) mu an sample.
MUTATIONAL ANALYSIS OF KRAS AND NRAS IN METASTATIC COLORECTAL CANCER
38
Clinicopa hological Associa ions
The es ablishmen o associa ions be ween he umo gene ic al e a ions and
clinicopa hological ea u es was possible in 215 ou o 241 cases.
Mu a ion equencies in his subg oup a e desc ibed below (Table 8/Figu e 14). Qui-
squa e o Fishe ’s exac es s (each one used when app op ia e) we e done o assess
di e ences be ween KRAS and NRAS mu a ion dis ibu ion and he ollowing a iables:
sex, age and s age a diagnosis, and p ima y umo si e.
Table 8. Mu a ional s a us in he subg oup o cases wi h a ailable clinical da a.
Samples
Mu a ional S a us
F equencies
KRAS mu an
28
NRAS mu an
12
Wild- ype
175
To al
215
Figu e 14. Mu a ional s a us (%) in he subg oup o cases wi h a ailable clinical da a.
RESULTS
39
Table 9. Dis ibu ion o KRAS and NRAS
mu a ions acco ding o pa ien sex.
No di e ences we e ound ega ding KRAS o NRAS mu a ion dis ibu ion by pa ien
gende : 12.1% in men s. 15.2% in women (p=0.537) o KRAS and 4.7% in men s.
7.6% in women (p=0.520) o NRAS.
Sex
KRAS
To al
Wild-Type
Mu an
Men
131
18
149
Women
56
10
66
To al
187
28
215
Sex
NRAS
To al
Wild-Type
Mu an
Men
142
7
149
Women
61
5
66
To al
203
12
215
NRAS
KRAS
p=0.537
p=0.520
Figu e 15. Dis ibu ion o KRAS and NRAS mu a ions
acco ding o pa ien sex.
MUTATIONAL ANALYSIS OF KRAS AND NRAS IN METASTATIC COLORECTAL CANCER
46
In a consecu i e se ies o 241 mCRC samples wild- ype o KRAS codons 12 and 13,
we sea ched o mu a ions in he less equen ly mu a ed KRAS mu a ional ho spo s in
exon 3 (codons 59/61) and 4 (codons 117/146) and in exon 2 (codons 12/13), 3 (codons
59/61) and 4 (codons 117/146) o NRAS. These ho spo s a e loca ed in he P-loop domain
(exon 2), swi ch II (exon 3) and G4/G5 egions (exon 4) o he highly conse ed G domain,
which is a common s uc u e among RAS p o eins (Edkins e al., 2006; Schubbe e al.,
2007). Ini ially, all samples we e sc eened by HRM o mu a ions in KRAS and NRAS.
Subsequen ly, au oma ed DNA sequencing was pe o med in all HRM p oduc s, in o de
o iden i y he al e a ions associa ed wi h each o he mu an cases. HRM was used as a
sc eening mu a ion me hod, ins ead o a egula PCR, since his echnique is a e y
accu a e, as and sensi i e me hod ha allows he de ec ion o a small ac ion o mu a ed
alleles in umo samples (~5%), h ough he e alua ion o he di e en mel ing pa e ns
ob ained om wild- ype sequences s. he e ozygo e a ian s (K ypuy e al., 2006; Pin o e
al., 2011). Fu he mo e, sequencing o HRM p oduc s inc eases sensibili y in mu a ion
de ec ion om 85% o 98% (Pin o e al., 2011). All HRM p oduc s we e sequenced due o
he ac ha we ob ained di e en a es o ampli ica ion among ou samples and because
o he use o big amplicons, such as hose o KRAS and NRAS exon 4, which migh
dec ease he sensi i i y o mu a ion de ec ion h ough HRM (K ypuy e al., 2006; Do e al.,
2008).
The equency o RAS mu a ions in his se ies (46/241 – 19.1%) is simila o ha
epo ed in ecen s udies wi h KRAS exon 2 wild- ype mCRC, which anges om
app oxima ely 15 o 20% (Vaughn e al., 2011; Douilla d e al., 2013; Neg u e al., 2014;
So ich e al., 2014). The mu a ional dis ibu ion o he 46 mu a ions is he ollowing: 12.4%
(30/241) we e ound in KRAS, 5.4% (13/241) and 7.1% (17/241) in exons 3 and 4,
espec i ely; and 6.6% (16/241) we e ound in NRAS, 3.3% (8/241) in exon 2 and 3.3%
(8/241) in exon 3. Al hough his mu a ional dis ibu ion sligh ly di e s om ha epo ed by
Neg u and collabo a o s (1.9% and 3.8% o KRAS exons 3 [codons 59/61] and 4 [codons
117/146], and 7.8% and 1.9% o NRAS exons 2 [codons 12/13] and 3 [codons 59/61],
espec i ely), i is e y simila o ha epo ed by So ich and collabo a o s in a ecen
sys ema ic e iew and me a-analysis o nine andomized con olled ials comp omising a
o al o 5948 pa ien s (4.3% and 6.7% o KRAS exons 3 [codons 59/61] and 4 [codons
117/146], and 3.8% and 4.8% o NRAS exons 2 [codons 12/13] and 3 [codons 59/61],
espec i ely) (Neg u e al., 2014; So ich e al., 2014). We did no de ec mu a ions in
NRAS exon 4 (codons 117/146), which seems o be a a e e en in CRC, as indica ed by
he epo ed equency anging om 0.2 o 1% (Douilla d e al., 2013; Neg u e al., 2014;
So ich e al., 2014).

DISCUSSION
47
Abe an RAS unc ion ound in cance cells is ypically associa ed wi h mu a ions in
codons 12, 13 o 61, since hese codons, loca ed in he P-loop (codons 12 and 13) and in
he swi ch egion II (codon 61), play an impo an ole in he main enance o he GTP-GDP
ansi ion s a e. Mu a ions in hese si es impai GTP hyd olysis and lead o he oncogenic
ac i a ion o he p o ein (Sche zek e al., 1997; Schubbe e al., 2007; P io e al., 2012).
Fu he mo e, i was demons a ed ha he subs i u ion o he Gln61 esidue by o he
amino acids abolished GAP-dependen GTPase ac i a ion, leading o a cons i u i e
ac i a ion o he RAS p o ein. This indica es ha his amino acid is essen ial o GAP
connec ion speci ici y o RAS GTPases (Nu & Ma u a, 1992). In he p esen se ies only
he NRAS gene was analyzed o codons 12 and 13 and he eigh mu a ions de ec ed
esul ed in h ee amino acid subs i u ions: p.Gly12Cys, p.Gly12Asp and Gly13A g.
Al hough he mos equen Gly12 mu an in ou se ies was he Gly12Asp (5/8; 62.5%), i s
oncogenic po en ial is smalle han ha o Gly12Val o Gly12A g mu an s (Schubbe e
al., 2007; P io e al., 2012), which we did no ind.
Codon 61 was analyzed in bo h KRAS and NRAS genes and nine mu a ions we e
ound in KRAS and eigh in NRAS, ep esen ing ou di e en amino acid subs i u ions:
p.Gln61Lys, p.Gln61A g, p.Gln61Leu and p.Gln61His. One hi d (2/6) o all p.Gln61His
mu an s we e ound in NRAS and, acco ding o he li e a u e and he COSMIC da abase,
his al e a ion has no p e iously been epo ed in his gene in CRC. Al hough he e a e no
da a conce ning i s oncogenic p op ie ies, he ac ha i is loca ed in Gln61 migh be an
indica o o i s ole in RAS ac i a ion. Jus as o Gly12, Gln61 mu an s ha e a ious
ans o ma ion e iciencies ha a y om 10 o 1000- old. One o he highes
ans o ma ion e iciencies is seen wi h he p.Gln61Leu mu an (Buh man e al., 2007),
which is also he mos equen Gln61 mu an in ou se ies (7/17; 41.2%). Howe e , in an
analysis made by Vaughn and collabo a o s, p.Gln61Leu was ound in only 17.1% (6/35)
o KRAS and NRAS codon 61 mu a ions (Vaughn e al., 2011). We also obse ed ha ,
despi e hei high deg ee o homology, he equency o mu a ions in hese h ee ho spo s
di e s be ween hese wo RAS p o eins. In KRAS, mu a ions in codons 12 and 13 a e
gene ally mo e equen han in codon 61, howe e in ou se ies mu a ions in NRAS we e
mo e equen in codon 61 han in codons 12 and 13 (50% s. 43.75% s. 6.25%,
espec i ely), which is in acco dance wi h he li e a u e (Fe nandez-Meda de & San os,
2011; P io e al., 2012).
Due o a pe sis en bias in mu a ion sc eening o e he yea s, he ole o mu a ions in
codons such as 59, 117 o 146 has been o e looked. Mu a ional analysis o hese h ee
codons was pe o med in ou se ies, and mu a ions we e ound in all o hem. Ala59
mu an s ound in ou se ies we e all loca ed in KRAS and included one poin mu a ion
(p.Ala59Th ), one in ame dele ion (p.Ala59del) and one la ge in- ame duplica ion
MUTATIONAL ANALYSIS OF KRAS AND NRAS IN METASTATIC COLORECTAL CANCER
48
(p.Cys51_Se 65dup). The e a e no su icien da a o unde s and how hese al e a ions
migh in luence RAS p o ein s uc u e and unc ion, bu he ac ha his codon is loca ed
in he swi ch egion II, he same as codon 61, indica es ha mu a ions in his codon migh
also in luence he ansi ion complex du ing GTP hyd olysis (Macaluso e al., 2002).
On he o he hand, mu a ions in codons 117 and 146, which a e in ol ed wi h guanine
base in e ac ion, a e known o inc ease he GDP o GTP exchange a e wi hou a ec ing
he GTPase ac i i y (Edkins e al., 2006). In ac , in i o exp ession o bo h mu an s
esul ed in ele a ed RAS-GTP exp ession compa ed wi h wild- ype RAS, al hough lowe
han he one obse ed wi h KRAS codons 12 and 13 alleles (Janaki aman e al., 2010). In
ou se ies, mu a ions in hese codons we e also ound only in KRAS, wi h ou mu a ions
in codon 117 and hi een in codon 146 (23.5% and 76.5%, espec i ely). These
mu a ions o igina ed h ee di e en mu an s, Lys117Asn, Ala146Val and Ala146Th , wi h
he la e being he mos equen mu an ou o he h ee (11/17; 64.7%), some hing ha
is consis en wi h he indings in o he publica ions (Janaki aman e al., 2010; Vaughn e
al., 2011).
Besides hose men ioned abo e, we ound one mo e mu a ion in KRAS exon 3,
p e iously desc ibed by Molina i and collabo a o s (Molina i e al., 2011). This mu a ion,
p.Gly60Asp, has no unc ional s udies ha can con i m i s ole as an ac i a ing mu a ion.
Howe e , his esidue is a conse ed amino acid in he supe amily o GTPases and is
known o in e ac wi h ϒ-phospha e o GTP, which is consis en wi h he hypo hesis ha a
mu a ion in his codon migh be oncogenic (Bou ne e al., 1991; Guedes e al., 2013).
I is also impo an o men ion he mu ually exclusi e dis ibu ion o mu a ions among
KRAS and NRAS exons ob ained in ou se ies, since we only ound single mu a ions in
ou pool o cases. This in o ma ion sugges s ha al e a ions in hese genes con e
o e lapping downs eam e ec s due o unc ional edundancy, which is consis en wi h
indings ac oss he li e a u e (De Roock e al., 2010; Janaki aman e al., 2010; Douilla d e
al., 2013).
In he 215 cases wi h a ailable clinical da a, we es ed o associa ion be ween RAS
mu a ions and clinicopa hological ea u es, such as gende , age and s age a diagnosis,
and p ima y umo si e. In e es ingly, an associa ion was ound be ween KRAS mu a ions
(p=0.001) and ea lie umo s ages a diagnosis, an associa ion ha was p e iously
desc ibed (Fe nandez-Meda de & San os, 2011). No o he s a is ically signi ican
associa ions we e ound, bu his migh be due o he ela i ely small sample size and
hese indings should he e o e be con i med in la ge se ies.
Al hough i had been al eady sugges ed in he pas (De Roock e al., 2010), he
impo ance o RAS mu a ions, besides hose in codons 12 and 13 o KRAS, as p edic o s
o esis ance o an i-EGFR has only ecen ly been es ablished. Douilla d and collabo a o s
DISCUSSION
49
published ecen ly he esul s o he PRIME ial, which assessed he e icacy and sa e y
o adding pani umumab o FOLFOX4 in RAS mu a ed pa ien s (Douilla d e al., 2013). O
he 1183 pa ien s who unde wen andomiza ion, 108 pa ien s (17%; 108/620) wi hou
KRAS mu a ions in exon 2 had mu a ions in o he RAS exons. In his subg oup o
pa ien s, he analysis showed ha PFS and o e all su i al (OS) obse ed we e sho e in
he pani umumab-FOLFOX4 g oup han in he FOLFOX4-alone g oup (7.3 s. 8.0 mon hs,
p=0.33; 17.1 s. 18.3 mon hs, p=0.31). Al hough he di e ence was no signi ican , hese
ou comes we e consis en wi h hose ound o he subg oup o pa ien s wi h KRAS
mu a ions in exon 2. Mo eo e , pa ien s wi hou RAS mu a ions in he pani umumab-
FOLFOX4 g oup we e associa ed wi h a signi ican imp o emen in p og ession ee
su i al (10.1 s. 7.9 mo hs, p=0.004) and o e all su i al (26.0 s. 20.2, p=0.04), when
compa ed wi h FOLFOX-alone.
Simila esul s, conce ning he addi ion o ce uximab o FOLFIRI in he ea men o
mCRC pa ien s, we e epo ed by Cia diello and collabo a o s in he la es esul s om he
CRYSTAL ial (Cia diello e al., 2014). 1198 andomized and ea ed pa ien s we e
e alua ed in his ial, and 14.7% (63/430) o hose conside ed wild- ype o KRAS codons
12 and 13 umo s had o he RAS mu a ions. The di e ences epo ed o PFS and OS in
his subg oup, be ween he ce uximab-FOLFIRI and he FOLFIRI-alone g oups, we e no
s a is ically signi ican (7.2 s. 6.9 mon hs, p=0.56; 18.2 s. 20.7 mon hs, p=0.50).
Howe e , when compa ed wi h he RAS wild- ype subg oup esul s (11.4 s. 8.4 mon hs,
p=0.0002; 28.4 s. 20.2 mon hs, p=0.0024) i is possible o conclude ha he addi ion o
ce uximab o FOLFIRI has no bene i o pa ien s wi h RAS mu a ions. All hese indings
sugges ha RAS ac i a ing mu a ions, in addi ion o KRAS exon 2 mu a ions, p edic lack
o esponse in pa ien s who ecei ed an i-EGFR he apy (ce uximab o pani umumab).
Due o he absence o in o ma ion, a he ime o w i ing, on he ou come o he RAS
mu a ed pa ien s ea ed wi h ce uximab/pani umumab, we could no e alua e he ole o
RAS mu a ions, as p edic i e bioma ke s o ea men esponse, in his se ies o pa ien s.
Howe e , conside ing he esul s ob ained in ou mu a ional analysis o 241 cases and he
indings by Douilla d and collabo a o s (Douilla d e al., 2013) and Cia diello and
collabo a o s (Cia diello e al., 2014), we can expec ha abou one- i h o pa ien s
conside ed wild- ype o KRAS exon 2 a e unlikely o bene i om an i-EGFR he apy due
o he p esence o o he RAS mu a ions.
C
ONCLUSIONS

CONCLUSIONS
53
VI. CONCLUSIONS
Taking in o accoun he esul s ob ained in his s udy, we can conclude ha :
I) HRM ollowed by au oma ed Sange sequencing o KRAS exons 3 and 4 and
NRAS exons 2, 3 and 4 allows he de ec ion o o he RAS mu a ions in abou
one- i h o 241 Po uguese mCRC pa ien s wild- ype o KRAS exon 2;
II) The 46 addi ional RAS mu a ions ound a e mu ually exclusi e and ha e he
ollowing dis ibu ion:
a. 5.4% in KRAS exon 3;
b. 7.1% in KRAS exon 4;
c. 3.3% in NRAS exon 2;
d. 3.3% in NRAS exon 3;
III) Ele en and se en di e en mu a ions we e ound in KRAS and NRAS,
espec i ely, wi h a no el NRAS exon 3 mu a ion being ound in wo cases;
IV) In his se ing, a s a is ically signi ican associa ion was ound be ween KRAS
exon 3/4 mu a ions and ea ly umo s age a diagnosis.
FUTURE
PERSPECTIVES
MUTATIONAL ANALYSIS OF KRAS AND NRAS IN METASTATIC COLORECTAL CANCER
62
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