Biology and Clinical Potential of Circulating Tumor DNA

The Liquid Biopsy in CRC

The Circulating Component of Tumors
• Circulating cell free DNA first identified
in 1948.

• Is present in higher levels among

patients with autoimmune diseases
and cancer

• Has a short half-life (10 to 15 min) and

is removed mainly by the liver

• Results from an excessive release of

DNA caused by massive cell death

• May carry genetic or epigenetic

changes that provide diagnostic,
prognostic and therapeutic
information.

• Is abundant enough to be analyzed by

a number of sensitive methods: PCR,
Real-Time PCR, Methylation-Specific
PCR and NGS.
The Circulating Component of Tumors
Proof of concept for Detection of Tumor Mutations in Plasma

Real-Time PCR methods enables non-invasive detection of KRAS

in plasma of patients with metastatic CRC with high sensitivity
and high specificity.

“We anticipate that

the method will be
employed in multiple
applications
in the clinic, including
diagnosis, cancer
recurrence
monitoring, and
treatment
Detection limit as low as 1 mutant allele in 200.000 WT copies management.”
 Towards Clinical Applications:
Circulating Tumor DNA is more abundant than CTCs
 Towards Clinical Applications:
Circulating Tumor DNA is more abundant in certain tumor types
 Towards Clinical Applications:
Circulating Tumor DNA is more abundant in advanced disease
 Towards Clinical Applications:
Circulating DNA abundance change with Therapy
Quantificação de DNA circulante (Dra. Leonor Pinto)
 Towards Clinical Applications:
Detection of Tumor Mutations in FFPE Tissue & circulating DNA
 Towards Clinical Applications:
Detection of Tumor Mutations in FFPE Tissue & circulating DNA

Plasma DNA Tumor DNA

KRAS WT 127 128  NPV= 99,2%
KRAS MUT 69 79  PPV= 88%
Not detc. 11* 0 Concordance= 95%

* Low CEA, mucinous histology,

 Towards Clinical Applications:
Detection of Tumor Mutations in FFPE Tissue & circulating DNA
COMPARAÇÃO DO STATUS MUTACIONAL RAS E BRAF ENTRE TECIDO E BIÓPSIA LIQUIDA
(Dra. Leonor
Tumor 1º Pinto)Tecido Status RAS Tecido Status RAS Status BRAF Tecido Status BRAF
Biop. Liquida Biop. Liquida
Colon Esquerdo T. Primário
WT WT WT WT
(colon)
Reto KRAS Mut (Exão 2
M. Pulmonar WT WT WT
-codão 12/13)
KRAS Mut (Exão 2
Colon Direito KRAS Mut (Exão 2
M. Hepática -codão 12/13) WT WT
-codão 12/13)

T. Primário BRAF Mut - V600E BRAF Mut - V600E

Colon Transverso
(colon) WT WT

Colon Direito Massa WT

WT WT WT
Peri-renal
T. Primário KRAS Mut (Exão 2
Colon Transverso KRAS Mut (Exão 2 WT
(colon) -codão 12/13) WT
-codão 12/13)

KRAS Mut (Exão 2

Colon Esquerdo M. Pulmonar
-codão 12/13) WT WT WT

KRAS Mut (Exão 2

Reto
M. Pulmonar -codão 12/13) WT WT WT

Colon Direito
M. Hepática WT WT WT WT

Reto T. Primário
WT WT WT WT
(colon)
 Towards Clinical Applications:
Circulating DNA mutations allows to portrait tumor heterogeneity
 Towards Clinical Applications:
Circulating DNA allows to Monitor Therapy Resistance/Response
 Towards Clinical Applications:
Circulating DNA allows to Monitor Therapy Resistance/Response
EVOLUÇÃO DO STATUS MUTACIONAL RAS E BRAF NO FOLLOW-UP de CRC POR BIÓPSIA
LIQUIDA (Dra. Inês Rêgo)
 Conclusions: The Clinical Potential of Circulating DNA

Arguments driving the concept and clinical utility of a “liquid biopsy”

- Non-invasive monitoring of disease
recurrence and emergence of resistance
mutations without repeated biopsy

- Overcome cases of insufficient material

in archival diagnostic specimens with an
actionable liquid biopsy

- Possibility to make a clonal portrait of

tumor heterogeneity

- Historical monitoring of the clonal

dynamics in response to therapy

- Re-challenging using therapies for which

tumor has become responsive
 Towards Clinical Applications:
Circulating DNA allows to Monitor Therapy Resistance/Response

STATUS MUTACIONAL RAS E BRAF NO FOLLOW-UP DE ADENOCARCINOMA DO PÂNCREAS POR

BIÓPSIA LIQUIDA (Dra. Joana Cunha Carvalho)
 Towards Clinical Applications:
Circulating DNA allows to Monitor Therapy Resistance/Response
 Towards Clinical Applications:
Circulating DNA allows to Monitor Therapy Resistance/Response
Towards Clinical Validation of Circulating Tumor DNA
 Circulating Tumor DNA in Endocrine Neoplasia

• Half-life of ctDNA is measured in

minutes/hours

• Protein markers (ex, CEA) may have a

half-life of days, with post-treatment
spikes

• Many endocrine tumors are

nonfunctional or secrete hormones
that do not cause clinical syndromes

• May provide means to differentiate

between benign and malignant lesions
without the need for a tissue biopsy
1. Husain. Clin Cancer Res. 2017;23:4716. 2. Tie. Ann Oncol. 2015;26:1715.
Circulating Tumor DNA in Endocrine Neoplasia
Circulating Tumor DNA in Endocrine Neoplasia

REVIEW ARTICLE
Front. Endocrinol., 20 November 2015 | https://doi.org/10.3389/fendo.2015.00176
Molecular Targeted Therapies of Aggressive Thyroid Cancer
Circulating Tumor DNA in Endocrine Neoplasia
Circulating Tumor DNA in Endocrine Neoplasia
 Towards Clinical Applications:
Circulating DNA allows to Monitor Response to Therapy
 Towards Clinical Applications:
Circulating DNA as a Surrogate Tumor Sample/Biopsy
 Plasma Follow-
Tissue Plasma T0 Tx until progression
up
WT WT KRAS_A146 FOLFIRI + anti EGFR durante 6 meses

KRAS_G12 KRAS_G12 KRAS_Q61 FOLFIRI + anti VEGF durante 8 meses

KRAS_G12 KRAS_G12 ---- FOLFIRI 1 mês (2 ciclos), faleceu de complicação infeciosa

WT WT --- FOLFIRI + anti- EGFR durante 6 meses,

KRAS_G12 KRAS_G12 KRAS_G12 FOLFIRI + anti VEGF durante 13 meses

KRAS_G13 KRAS_G13; A146 KRAS_A146 FOLFIRI + anti VEGF durante 8 meses

KRAS_G13 KRAS_G13 KRAS_G13 FOLFIRI + anti VEGF durante 10 meses

KRAS_G12 WT WT FOLFIRI + anti VEGF durante 18 meses

KRAS_G12 WT NRAS_Q61 FOLFIRI + anti VEGF durante 7 meses

WT KRAS_G12 WT FOLFOX + anti EGFR durante 4 meses

WT KRAS_Q61 WT 10 semanas de FOLFIRI passa a WT ainda sem progressão

KRAS_G12 KRAS_G12 WT 12 semanas de FOLFIRI passa a WT ainda sem progressão

KRAS_G12 WT WT 19 semanas de FOLFIRI, passa a WT ainda sem progressão

WT WT NRAS_G12 10 semanas após FOLFIRI passa a NRAS mut sem progressão

KRAS_A146 KRAS_A146 KRAS_A146 FOLFIRI durante 3 meses

KRAS_G12 KRAS_G12 WT 8 semanas após FOLFIRI passa a WT ainda sem progressão

KRASG12; NRASQ61 WT WT 8 semanas após FOLFIRI passa a WT ainda sem progressão

BRAF_V600E BRAF_V600E BRAF_V600E Fez 1 ciclo de folfiri com deterioração e faleceu 5 meses após
 Towards Clinical Validation of Circulating Tumor DNA
ID 1 2 3 4 5 6 7 8 9 10 11 12 13
KRAS

Valor basal
NRAS
BRAF
UÇÃO DO STATUS MUTACIONAL RAS E BRAF NO FOLLOW-UP POR BIÓPSIA LIQUIDA
EGFR
nês Rêgo)
KRAS 
1ª linha

NRAS
BRAF
EGFR
KRAS 
progressão

NRAS
  
BRAF
EGFR
KRAS
2ª linha

NRAS
BRAF
EGFR
KRAS
progressão

NRAS 

BRAF
EGFR
KRAS
3ª linha

NRAS 
BRAF
EGFR
Status     
OS (meses) 12 10 4 9 8 8 8 6 6