NGS en oncología

Mauricio Lema Medina MD
Clínica de Oncología Astorga / Clínica SOMA
Medellín
Inspirado en: Michael Bierut, 2013, Logo para Mohawk Fine Papers

@onconerd
Conflictos de interés
Mauricio Lema, 2020-2021
• Abbie
• Eli-Lilly
• Pfizer
• AZ
• ROCHE
• BMS
• Boheringer Ingelheim
Los comentarios sobre indicaciones de los
diversos medicamentos en esta presentación se
basan en evidencia clínica. Para muchos de ellos
no existe la aprobación regultaria por el INVIMA.

La genotipificación es la tecnología que detecta
pequeñas diferencias genéticas que pueden dar lugar
a cambios importantes en el fenotipo.

Melindre:
Delicadeza afectada y excesiva en palabras, acciones y
ademanes.

Genotipificación
Gen A Gen B Gen C Gen X
Gen A
Gen B
Gen C
.
.
.
Gen X
Secuencial
Paralela

Genotipificación
Gen A Gen B Gen C Gen X
Secuencial
Apropiada para pocos
genes de interés
No detecta genes
potencialmente
importantes, aunque
raros
Agotamiento tisular

Genotipificación
Gen A
Gen B
Gen C
.
.
.
Gen X
Paralela
Idónea para muchos
genes de interés
Puede ser más costosa, si
los genes de interés son
pocos

Big Data
Bio-informatics
Se debe evaluar la significancia de cada
mutación

Genotipificación
Gen A
Gen B
Gen C
.
.
.
Gen X
Exhaustiva
(cientos de
genes de
interés)
CDx
Acompaña aprobación
regulatoria
Perfilación genómica exhaustiva

55 años
Mujer
No fumadora
Dolor de cadera izquierda y hombro izquierdo por 9 meses

Incremento en la
captación en varias
estructuras óseas,
altamente
sugestiva de
metástasis
01.09.2017

• Biopsia de ganglio cervical
• Adenocarcinoma
• TTF1+
• Napsina+
• CK20-
• Estrógeno-
• PD-L1: 1-49%
Creado por Mauricio Lema Medina MD

• Adenocarcinoma
• TTF1+
• Napsina+
• CK20-
• Estrógeno-
• PD-L1: 1-49%
• Genotipificación
• EGFR mutado L858R
• ALK no mutado
09.2017

Johnson B, et al. ASCO 2013. Abstract 8019.
Lung Cancer Mutation Consortium:
OS by Mutation and Treatment
No driver mutation (n = 361; median OS: 2.1 yrs)
100
80
60
40
20
0
OS
(%)
0 1 2 3 4 5
Yrs

Driver mutation + no targeted therapy (n = 265; median OS: 2.4 yrs)
100
80
60
40
20
0
OS
(%)
0 1 2 3 4 5
Yrs

Driver mutation + targeted therapy (n = 313; median OS: 3.5 yrs)
Driver mutation + no targeted therapy (n = 265; median OS: 2.4 yrs)
100
80
60
40
20
0
OS
(%)
0 1 2 3 4 5
Yrs
Targeted therapy vs no targeted
therapy; P < .0001

• Adenocarcinoma
• TTF1+
• Napsina+
• CK20-
• Estrógeno-
• PD-L1: 1-49%
• Genotipificación
• EGFR mutado L858R
• ALK no mutado
• Inicia Afatinib
09.2017

10.01.2018
11.09.2017
Desaparecen las
metastasis
hepáticas

10.01.2018
11.09.2017
Disminución en la
captación ósea

03.06.2018
Nuevas lesiones en el hígado

L858R
Adeno NSCLC
Etapa IV
Afatinib
T790M
9.2017 6.2018
PD
+8 mo
Se inicia Osimertinib

L858R
Adeno NSCLC
Etapa IV
Afatinib
T790M
9.2017 6.2018
PD
+8 mo
Afatinib Osimertinib
T790M
9.2017 6.2018 9.2018 3.2019
PD PD
+8 mo +9 mo
03.2019

L858R
Adeno NSCLC
Etapa IV
Afatinib
T790M
9.2017 6.2018
PD
+8 mo
T790M
9.2017 6.2018 9.2018 3.2019
PD PD
+8 mo +9 mo
Quimioterapia
paliativa u otra
terapia dirigida?

L858R
Adeno NSCLC
Stage IV
Afatinib
T790M
9.2017 6.2018
PD
+8 mo
T790M
9.2017 6.2018 9.2018 3.2019
PD PD
+8 mo +9 mo
T790M
9.2017 6.2018 9.2018 3.2019
MET Ampl
PD PD
+8 mo +9 mo
Se encuentra otra
mutación
accionable
(amplificación de
MET), se agrega
crizotinib

L858R
Adeno NSCLC
Stage IV
Afatinib
T790M
9.2017 6.2018
PD
+8 mo
T790M
9.2017 6.2018 9.2018 3.2019
PD PD
+8 mo +9 mo
T790M
Osimertinib + Crizotinib
9.2017 6.2018 9.2018 3.2019
MET Ampl
PD PD
+8 mo +9 mo
Control de
enfermedad,
Paciente trabajando
Sin síntomas
+20 meses…

• 75 años
• Masa en region cervical izquierda
• En 02/10/2020 ecografía de cuello: nódulo solido complejo en cuello.
• En 14/10/2020 tomografía contrastada de cuello y tórax: en región
supraclavicular izquierda, en la estación ganglionar III, IV y VB, compromiso
ganglionar de aspecto tumoral. No hay evidencia de patología tumoral a
nivel del tórax.
• En 13/11/2020 biopsia de ganglio cervical izquierdo: tejido comprometido
por carcinoma, a favor de adenocarcinoma.
• En 18/11/2020 tomografía contrastada de abdomen y pelvis: crecimiento
prostático con cambios postquirúrgicos de RTU. Lesiones nodulares
suprarrenales bilaterales.

02/10/2020
Ecografía
TAC cuello y tórax
TAC abdomen y pelvis
11/2020
Día + 41: Adenocarcinoma NPI
Biopsia 13/10/2020
Es EPS (creo que PGP)

• En 19/11/2020 colonoscopia: hemorroides internas grado I-II. Enfermedad
diverticular no complicada en sigmoides.
• En 19/11/2020 endoscopia digestiva superior: gastropatía congestiva
erosiva antral.
• En 23/11/2020 inmunohistoquímica en biopsia de ganglio cervical
izquierdo: CK7, GATA 3: Positivo fuerte y difuso. CK20, TTF1, Napsin A,
CDX2, P63, S100: Negativos. Apoya el diagnóstico de tejido comprometido
por adenocarcinoma, como primera opción mama vs. glándula salival y
más remotamente urotelial.
• En 25/11/2020 biopsia de antro gástrico: gastritis crónica no atrófica con
actividad severa. H. pylori presente en escasa cantidad. Negativo para
metaplasia intestinal, displasia o malignidad.

02/10/2020
Ecografía
TAC cuello y tórax
11/2020
Biopsia 13/10/2020
Inmunohistoquímica:
puede ser cualquier
cosa, menos ovario

02/10/2020
Ecografía
TAC cuello y tórax
11/2020
Biopsia 13/10/2020

• En 26/11/2020 PET-CT: actividad tumoral de moderado a alto grado en:
adenopatías supradiafragmáticas, localizadas en la region supraclavicular
izquierda y retrocrurales bilaterales. El compromiso ganglionar de mayor
tamaño se encuentra en la region supraclavicular. Adenopatías
infradiafragmáticas; del hilio hepático y retroperitoneales, sin rango de
adenomegalia. Glándulas suprarrenales bilaterales, siendo de mayor
tamaño la derecha.
• En 2/12/2020: ACE 0.89 ng/mL, Ca 19-9: 98.27 U/mL, AFP 2.13 ng/mL,
tiroglobulina 10.5 ng/mL.
• En 15/12/2020 evaluado por cirujano oncólogo, recomienda:
nasofibrolaringoscopia y valoración por otorrinolaringología y oncología
clínica.

02/10/2020
Ecografía
TAC cuello y tórax
11/2020
PET-CT

• En 18/12/2020 evaluado por oncólogo EPS, quien recomienda
descartar primario adrenal con estudios funcionales y discutir la
posibilidad de biopsia en staff.
• En 21/12/2020 resonancia contrastada de cráneo y cuello: múltiples
adenopatías en el hemicuello izquierdo en los niveles III, IV, VB y
región supraclavicular, sin cambios con respecto a la tomografía
previa. Lesión de apariencia quística en la supraglotis en el espacio
preepiglótico en la línea media, de tipo inespecífico. Cambios
involutivos del parénquima cerebral acompañados de cambios
microangiopáticos.

02/10/2020
Ecografía
TAC cuello y tórax
11/2020
PET-CT
Día + 77: Oncólogo
Recomienda staff
Biopsia 13/10/2020
Endoscopia arriba
Endoscopia abajo
Biopsia estómago
RM de cráneo
Inmunohistoquímica tumor: no informa
Marcadores tumorales: Ca 19.9 altico

Me van a dejar MORIR!
Por favor, hagan ALGO
Segunda opinión

Perfilación genómica exhaustiva
Hallazgos de Biomarcadores
Estado de inestabilidad Microsatelital: MS- Estable
Carga mutacional del tumor – TMB: 8 M/Mb
Hallazgos Genómicos
Para obtener una lista completa de los genes analizados, consulte el Apéndice.
EGFR deleción del exón 19 (L747_T751del)
AKT2 amplificación
ARID1A Y551*, Q1493*
RICTOR amplificación
RB1 K8fs*13, Q850*
TERT promotor -124C>T
TP53 E224*

4
Del19 L858R
Afatinib
n=112
Cis/Pem
n=57
Afatinib
n=91
Cis/Pem
n=47
Mediana, mo 13.8 5.6 10.8 8.1
HR (95% CI), P-valor 0.26 (0.17-0.42), P<0.0001 0.75 (0.48-1.19), P=0.2191
LUX-Lung 3: SLP en mutación de subgrupos
L858R
Del19
No. en riesgo:
Afatinib 112 95 82 67 53 41 26 22 19 16 14 12 8 5 2 0
Cis/Pem 57 30 15 7 2 1 0 0 0 0 0 0 0 0 0 0
Tiempo de supervivencia libre de progresión (Meses)
Probabilidad
SLP
estimada
No. en riesgo:
Afatinib 91 75 61 48 37 29 24 15 12 8 6 5 2 2 1 0
Cis/Pem 47 32 20 10 7 5 2 2 2 2 2 2 2 2 1 0
0.0
0.2
0.4
0 3 6 9 12 15 18
18 21 24 27 30 33 36 39 42 45
0.6
0.8
1.0
0.0
0
Probabilidad
SLP
estimada
0.2
0.4
3 6 9 12 15 18
18 21 24 27 30 33 36 39 42 45
0.6
0.8
1.0
Afatinib
Cis/Pem
Afatinib
Cis/Pem

4
LUX-Lung 6: SLP en mutación de subgrupos
Del19 L858R
Afatinib
n=124
Cis/Gem
n=62
Afatinib
n=92
Cis/Gem
n=46
Mediana, mo 14.3 5.5 12.1 5.6
HR (95% CI), P-valor 0.17 (0.11-0.28), P<0.0001 0.32 (0.18-0.46), P<0.0001
L858R
Del19
Afatinib
Cis/Gem
No. en riesgo:
Afatinib 92 79 65 53 43 29 21 16 14 13 11 4 0 0
Cis/Gem 46 28 15 5 1 0 0 0 0 0 0 0 0 0
No. en riesgo:
Afatinib 124 110 98 92 77 58 44 37 29 25 18 14 3 0
Cis/Gem 62 39 11 6 1 1 0 0 0 0 0 0 0 0
0 3 6 12 21 33
0.0
0.2
0.4
0.6
0.8
1.0
Probabilidad
SLP
estimada
9 15 27 39
18 30
24 36
Afatinib
Cis/Gem
0 3 6 12 21 33
0.0
0.2
0.4
0.6
0.8
1.0
Probabilidad
SLP
estimada
9 15 27 39
18 30
24 36

Caso 3
Cortesía de Andrés Yepes

• Varón 36 años
• No fumador
• Adeno NSCLC - IV
• Adenocarcinoma pulmón
izquierdo
• Derrame pleural izquierdo
• Compromiso: SNC, supraclavicular,
mediastina, crura diafragmática
• --PD-L1 >50%
• --EGFR, ALK negativos
• Cis + Pem + Bevacizumab - 07/2018 - 02/2019
• Nivolumab: 22/03/2019 - 13/02/2020
• Docetaxel: 01/04/2020 - 16/09/2020
• Gemc + Carbo: 12/11/2020 - 13/01/2021
• 02/12/2020: FOUNDATION ONE LIQUID: ROS 1
POSITIVO (EZR-ROS 1 FUSIÓN), RAS51C,
CHEK2

Non-squamousNSCLC
2020
Mutation Prevalence ESCAT level Active drug
EGFR del19, L858R 15% (10-60%) IA Afa, Osimer…
T790M (exon 20) 60% (acquired) IA Osimer
G719X in exon 18, L861Q in
exon 21, S768I in exon 20
10% IB Afa, Osimert
Exon 20 insertion 2% IC Pozio
ALK fusion 5% IA Alec, crizo
METex14 3% IB Capma, tepo, crizo
MET ampl (in mEGFR) 3% IIA Crizo, capma, tepo
BRAF(V600E) 2% IB Dabra/Trame
ROS1 1-2% IB Crizo
NTRK fusions 0.23-3% IC Entrec, Larotrec
RET 1% IC Selperca
RAS (G12C) 12% IIB AMG-510
ERBB2 (mutations) 2-5% IIC T-Duroxtecan
BRCA1/2 1.2% IIIA iPARP
PIK3Ca 1.2-7% IIIA Alpelisib
NRG fusions 1.2% IIIC
Mosele F, et al. Ann Oncol, 2020

Rearreglos del ROS1
Frecuencia: 1-2% de NSCLC
No escamosos
Mujeres, no fumadores, histologías mixtas
Metástasis cerebrales: comunes (36%)
Diagnóstico
Cribado con
inmunohistoquímica,
confirmar con
estudios moleculares

Activity of Crizotinib in Pts With ROS1
Fusions: Best Overall Response
Shaw AT, et al. N Engl J Med. 2014;371:1963-1971.
 ORR: 72%
100
80
60
40
20
0
-20
-40
-60
-80
-100
Change
From
Baseline
(%)
PD
SD
PR
CR

Crizotinib in ROS1 Rearrangement–Positive NSCLC
 Median follow-up for OS: 62.6 mos
 14 patients (26%) remain in follow-up
ROS1-Rearranged NSCLC (N = 53)
Deaths, n (%) 26 (49.1)
Median OS, mos
(95% CI)
51.4 (29.3-NR)
40
OS
(%)
Mos
60
80
100
20
0
0 40
20 60 80
53
Pts at Risk, n 48 42 37 31 27 23 20 18 17 9 5 4 0
20
33 13 3
1-yr OS rate:
79%
4-yr OS rate:
51%
Censored
Shaw. Ann Oncol. 2019. [Epub]

Crizotinib in ROS1 Rearrangement–Positive NSCLC
ROS1-Rearranged
NSCLC (N = 53)
Shaw et al
2014 (N = 50)
Events, n (%) 36 (67.9) 23 (46)
Median PFS, mos (95% CI) 19.3 (15.2-39.1) 19.2 (14.4-NR)
PFS
(%)
53 41 35 31 19 17 16 11 10 9 4 2 2 0
14
22 7 2
Patients at Risk, n
40
60
80
100
20
0
0 40
20 60 80
Mos
Shaw. Ann Oncol. 2019. [Epub]

• Varón 36 años
• No fumador
• Adeno NSCLC - IV
• Adenocarcinoma pulmón
izquierdo
• Derrame pleural izquierdo
• Compromiso: SNC, supraclavicular,
mediastina, crura diafragmática
• --PD-L1 >50%
• --EGFR, ALK negativos
• Cis + Pem + Bevacizumab - 07/2018 - 02/2019
• Nivolumab: 22/03/2019 - 13/02/2020
• Docetaxel: 01/04/2020 - 16/09/2020
• Gemc + Carbo: 12/11/2020 - 13/01/2021
• 02/12/2020: FOUNDATION ONE LIQUID: ROS 1
POSITIVO (EZR-ROS 1 FUSIÓN), RAS51C,
CHEK2
• 02/2021: CRIZOTINIB

Entrectinib: Early Data in Patients With Solid Tumors
 2 phase I trials (N = 119)
‒ Age: 18-80 yrs
‒ Female: 54%
 ORR
‒ 100% for NTRK fusions
‒ 86% for ROS1 fusions
‒ 57% in ALK fusions
 Median DoR
‒ 2.6-15.1 mos for NTRK fusions
‒ 17.4 mos for ROS1 fusions
‒ 7.4 mos for ALK fusions
 Median PFS
‒ NR for NTRK fusions
‒ 19.0 mos for ROS1 fusions
‒ 8.3 mos for ALK fusions
Drilon. Cancer Discov. 2017;7:400.
Tumor Type, n (%)
ALKA-
372-001
STARTRK-1 Total
NSCLC 35 (65) 36 (56) 71 (60)
GI tract 9 (17) 9 (14) 18 (15)
CNS 4 (7) 1 (2) 5 (4)
Head and neck 1 (2) 4 (6) 5 (4)
Other 5 (9) 15 (23) 20 (17)

Entrectinib: Tumor Response by Gene Alteration
Maximum
%
CHANGE
FROM
BASELINE
in
Sum
of
Longest
Diameters 20
0
-20
-40
-60
-80
-100
NTRK ROS1 ALK
* 0% Change
LMNA-NTRK1
SQSTM1-NTRK1
ETV6-NTRK3
a
EZR-ROS1
ROS1+
(FISH)
ROS1+
(FISH)
ROS1+
(FISH)
GOPC-ROS1
ROS1+
(FISH)
ROS1+
(FISH)
ROS1+
(FISH)
ROS1+
(FISH)
ROS1+
(FISH)
ROS1+
(FISH)
CD74-ROS1
ALK+
(FISH)
D5F3-ALK
ALK+
(FISH)
CAD-ALK
VCL-ALK
ALK+
(FISH)
ALK+
(FISH)
*
b
aSDC4-ROS1
bROS1+ (FISH)

Entrectinib: Duration of Treatment
SQSTM1-NTRK1 NSCLC
BCAN-NTRK1 glioneuronal
ETV6-NTRK3 MASC
LMNA-NTRK1 mCRC
ROS1+ (FISH) NSCLC
ROS1+ (FISH) NSCLC
CD74-ROS1 NSCLC
ROS1+ (FISH) NSCLC
ROS1+ (FISH) NSCLC
EZR-ROS1 NSCLC
GOPC-ROS1 melanoma
ROS1+ (FISH) NSCLC
ROS1+ (FISH) NSCLC
ROS1+ (FISH) NSCLC
ROS1+ (FISH) NSCLC
ROS1+ (FISH) NSCLC
ROS1+ (FISH) NSCLC
SDC4-ROS1 NSCLC
ALK+ (FISH) NSCLC
ALK+ (FISH) NSLCLC
VCL-ALK RCC
ALK+ (FISH) NSCLC
CAD-ALK mCRC
ALK+ (FISH) NSCLC
D5F3-ALK unknown primary
NTRK
ROS1
ALK
0 3 6 9 12 15 18 21 24 27 30 33 36
Mos

Lorlatinib: Active Against ROS1 Resistance Mutations
(in vitro Data)
Zhou. AACR 2013. Abstr A277.
CD74-ROS1(s)
Mutation
Cellular ROS1 Phosphorylation Mean IC50
(nM)
Lorlatinib Crizotinib Ceritinib
NIH3T3
BaF3
0.23
0.11
11
3.9
51
G2032R
BaF3
186 2033 2666
IC50 < 100 nM
IC50 ≥ 100 < 200 nM
IC50 ≥ 200 nM

Rearreglo
ROS1
Crizotinib
Entrectinib
Lorlatinib
Primera línea Segunda línea
o Cabozantinib
o Ceritinib

Caso 4
Los Miamis – Mike Cusnir

• En 01/2020
• Masa curvatura mayor y cuerpo gástrico.
• Endoscopia: Masa no circunferencial, de tamaño medio, mide 4 cm.
• Adenocarcinoma pobremente diferenciado.
• Inmunohistoquímica: MLH1: negative, MSH2: positive. MSH6: positive. PMS2: negative.
• Omentectomía parcial: carcinoma metastásico.
• PET-CT: Positivo, estómago, peritoneo.
• PDL-1: baja expression (1-49%).
• En 15/02/2020 Perfilación genómica exhaustiva: mismatch repair status: deficient. Inestabilidad microsatelital: alta. PD-
L1: positive, CPS: 1. ERBB2 (Her2/Neu): no amplificado por FISH, negativo (1+) por IMH. BRCA2: variante patogénica en el
exón 11|p.k1108fs (somática, no germinal).

• En 01/2020
• Masa curvatura mayor y cuerpo gástrico.
• Endoscopia: Masa no circunferencial, de tamaño medio, mide 4 cm.
• Adenocarcinoma pobremente diferenciado.
• Inmunohistoquímica: MLH1: negative, MSH2: positive. MSH6: positive. PMS2: negative.
• Omentectomía parcial: carcinoma metastásico.
• PET-CT: Positivo, estómago, peritoneo.
• PDL-1: baja expression (1-49%).
• En 15/02/2020 Perfilación genómica exhaustiva: mismatch repair status: deficient. Inestabilidad microsatelital: alta. PD-
L1: positive, CPS: 1. ERBB2 (Her2/Neu): no amplificado por FISH, negativo (1+) por IMH. BRCA2: variante patogénica en el
exón 11|p.k1108fs (somática, no germinal).
• -- Pembrolizumab más FOLFOX
• En 06/2020 biopsia de gastrectomía y omentectomía: negativa para malignidad.

Mecanismos de reparación de ADN
Lord. Nature. 2012; 481:287. Martin. Clin Cancer Res. 2010;16:5107.
Single-strand
break
Double-strand
break
Bulky
adducts
Base mismatches,
insertions and
deletions Base alkylation
CH3
A
Double-strand
break repair
BER
PARP1
XRCC1
LIGASE 3
Proteins
Homologous
recombination
NHEJ
KU70/80
DNA-PK
BRCA1
BRCA2
PALB2
ATM
CHEK1
CHEK2
RAD51
Tumor types Breast, ovarian, pancreatic
NER
ERCC4
ERCC1
Xeroderma
pegmentosa
Mismatch
repair
MSH2
MSH1
Colorectal
G
Direct
reversal
MGMT
Glioma
MLH1
MSH2
PMS2/1
MSH6
Small IDLs (1-2 nucleotides)

Microsatellite Instability
 Durante la replicación las hebras de ADN pueden
mal-alinearse con facilidad
 Si los genes de reparación de mal-alineamiento de
ADN (MMR) mutan, la reparación es defectuosa
‒ Se denomina dMMR
 El ADN tiene regions llamados microsatélites
compuestos de “repeticiones”
‒ Estos microsatélites son únicos en que los
errores de replicación varían en longitud, en
vez de secuencia
‒ Se denomina inestabilidad microsatelidal, o
MSI-High
Kim. Cell. 2013;155:858.
Exomewide or
genomewide
microsatellite
instability
screening
Tumor
Normal
Tumor-specific DNA
slippage events
MS lengths
AAAAAAAA,
CTCTCTCT,
CAGCAGCAG,
…..

MSI and Immunotherapy
Slide credit: clinicaloptions.com
Sharabi. Oncologist. 2017;22:631.
MHC
PD-L1
PD-1
FAS-L
T-Cell
Tumor
Cell
Low Mutational Burden High Mutational Burden
PD-1
FAS-L
PD-L1
Anti–PD-L1
Antibodies
Tumor Cell
Flagged for
Attack
FAS MHC
TCR
PD-1
Anti–PD-1
Antibodies

Importancia de la carga mutacional tumoral
A mayor número de
mutaciones en el ADN
Mayor cantidad de
neoantígenos
Neoantígeno: antígeno nuevo
A más neoantígenos,
más oportunidad para
respuesta inmune
Inmunoterapia (anti
PD(L)1

MSI-H/dMMR in 39 Cancer Types; 11,139 Tumors
Uterine endometrial carcinoma
Colon adenocarcinoma
Stomach adenocarcinoma
Rectal adenocarcinoma
Adrenocortical carcinoma
Uterine carcinosarcoma
Cervical
Wilms tumor
Mesothelioma
Esophageal carcinoma
Breast carcinoma
Renal, clear cell
Ovarian
Cholangiocarcinoma
Thymoma
TOP 15
Bonneville. JCO Precis Oncol. 2017;2017. Slide credit: clinicaloptions.com

dMMR in Patients With Advanced/Metastatic Cancers
1. Le. Science. 2017;357:409. 2. Conley. AACR 2017. Abstr A053. 3. Hall. ASCO 2016. Abstr 1523. Slide credit: clinicaloptions.com
CARIS[1]
(N = 12,019)
MATCH[2]
(N = 4901)
Foundation Medicine[3]
(N = 11,573)
Tumor Type[2,3] MSI High,
n/N (%)
Uterine 39/277 (14.1)
Small bowel 6/70 (8.6)
Prostate 11/178 (6.2)
CRC 42/1185 (3.5)
CUP 22/815 (2.7)
Hepatobiliary 9/389 (2.3)
Gastroesophageal 6/400 (1.5)
Pancreatic 1/459 (0.2)
NSCLC 5/2112 (0.2)
Breast 2/1459 (0.1)
Tumor Type
Lack MLH1 and/or
MSH2 Expression,
n/N (%)
GI cancer 33/1325 (2.5)
GYN cancer 31/301 (10.3)
Brain cancer 2/52 (3.8)
Sarcoma 6/106 (5.7)
Thyroid/
Parathyroid
2/3 (66.7)
Prostate 7/122 (5.7)
Breast 8/566 (1.4)
Neuroendocrine 5/192 (2.6)
Other 3/326 (0.9)
18%
16%
14%
12%
10%
8%
6%
4%
2%
0%
Late stage
Early stage

Response to Pembrolizumab in MSI-H/dMMR
Cancers
1. Pembrolizumab PI. 2. Le. Science.
2017;357:409.
Tumor Type[1] N ORR, % (95% CI)
CRC 90 36 (26-46)
Non-CRC 59 46 (33-59)
Endometrial 14 36 (13-65)
Biliary 11 27 (6-61)
Gastric/GEJ 9 56 (21-86)
Pancreatic 6 83 (36-100)
Small Intestine 8 38 (9-76)
Breast 2 PR, PR
Prostate 2 PR, SD
Bladder/esophageal 1/1 NE/PE
Sarcoma 1 PD
Thyroid 1 NE
Retroperitoneal
adenocarcinoma
1 PR
SCLC 1 CR
Renal 1 PD
Tumor Type and Response[2]
Slide credit: clinicaloptions.com

KEYNOTE-177: Study Design
Andre. ASCO 2020. Abstr LBA4. Slide credit: clinicaloptions.com
 Randomized, open-label phase III trial
Patients with
treatment-naive MSI-H
(PCR)/dMMR (IHC)
stage IV CRC;
ECOG PS 0/1;
measurable disease
(N = 307)
Investigator-choice of chemotherapy*
(n = 154)
Pembrolizumab 200 mg Q3W for up to 35 cycles
(n = 153)
 Dual primary endpoints: PFS,† OS
‒ Trial positive if pembrolizumab superior to
chemotherapy for either primary endpoint
 Secondary endpoints: ORR,† safety
 Data cutoff: February 29, 2020
 Median follow-up: 28.4 mos in
pembrolizumab arm, 27.2 mos in
comparator arm
*Chemotherapy options included mFOLFOX6 or FOLFIRI ± bevacizumab or cetuximab.
†Blinded independent central review per RECIST v1.1.
Crossover permitted at
disease progression

KEYNOTE-177: PFS (Primary Endpoint; ITT)
Andre. ASCO 2020. Abstr LBA4. Reproduced with permission. Slide credit: clinicaloptions.com
PFS
(%)
Mos
Patients at Risk, n
153 96 77 72 64 60 55 37 20 7 5 0 0
154 100 68 43 33 22 18 11 4 3 0 0 0
12-mo rate
55%
37%
24-mo rate
48%
19% Median, Mos (95% CI)
16.5 (5.4-32.4)
8.2 (6.1-10.2)
Pembrolizumab
Chemotherapy
0
20
40
60
80
100
0 4 8 12 16 20 24 28 32 36 40 44 48
Events, % HR (95% CI) P Value
54 0.60 .0002
73 (0.45-0.80)

KEYNOTE-177: Duration of Response
Andre. ASCO 2020. Abstr LBA4. Reproduced with permission. Slide credit: clinicaloptions.com
Patients
in
Response
(%)
Median DoR, Mos (95% CI)
NR (2.3+ to 41.4+)
10.6 (2.8 to 37.5+)
≥ 24-mo response duration
83%
35%
Mos
Patients at Risk, n
67 64 57 50 48 41 29 13 6 4 2 0 0
51 48 35 19 13 11 9 5 2 1 0 0 0
0
20
40
60
80
100
0 4 8 12 16 20 24 28 32 36 40 44 48
Pembrolizumab
Chemotherapy

A recent report on immunotherapy in metastatic colorectal cancer has
shown that an unacceptable percentage of patients (almost 10%) had
been enrolled in immunotherapy trials and experienced failure due to
false positive dMMR or MSI-PCR results assessed by local laboratories.
Thus, the consensus panel recommends the use of both MMR-IHC and
MSI-PCR to assess the eligibility to treatment with immune checkpoint
inhibitors of metastatic colorectal cancer and other cancers of the Lynch
syndrome spectrum
Luchini C, ESMO recommendations on microsatellite instability testing for immunotherapy in
cancer, and its relationship with PD-1/PD-L1 expression and tumour mutational burden: a
systematic review-based approach. Ann Oncol. 2019

An NGS represents an alternative molecular
test to assess MSI. One main advantage of
this, is the opportunity to couple MSI
analysis with the determination of TMB.
Luchini C, ESMO recommendations on microsatellite instability testing for immunotherapy in
cancer, and its relationship with PD-1/PD-L1 expression and tumour mutational burden: a
systematic review-based approach. Ann Oncol. 2019

TMB
1 MEGABASE 1 MEGABASE
1 MEGABASE 1 MEGABASE
Normal TMB-L
TMB-H
2
20

Gliomas
Tomado de León Darío Ortiz

Para llevar a casa
 El estado de codeleccion 1p/19q derivado de F1-CDx es altamente concordante
con FISH (96.7%)
 El perfilamiento genómico brinda otros biomarcadores moleculares como IDH 1 y
2
 Si bien en investigación se requieren paneles cada vez mas amplios
 En la practica clínica se requiere un panel especifico para cada tipo de tumor, que
se revalúe constantemente
 Genómica se debe complementar con el análisis epigenómico, transcriptómico,
proteómico, y metabolómica
 Compaginándolo con los métodos tradicionales, esto es clínica, imagenología,
patología convencional, etc.
LD Ortiz

Recommendations for the use of next-
generation sequencing (NGS) for patients
with metastatic cancers: a report from the
ESMO Precision Medicine Working Group
Mosele F, et al. Ann Oncol, 2020
https://doi.org/10.1016/j.annonc.2020.07.014

NGS for metastaticcancer:ESMO recommendations
Next-generation sequencing (NGS) allows sequencing of a high number
of nucleotides in a short time frame and at an affordable cost per
patient.
2020
ESMO recommendations
Public Health Research (hospital level) Individual patient

2020
The general strategy was to determine whether NGS can substitute complex or multiple testings.
All recurrent genomic alterations were identified in the eight cancers that are associated with highest
number of deaths
The ESMO Scale for Clinical Actionability of molecular Targets (ESCAT) ranking was then determined
for each alteration.
ESCAT is a framework that ranks a match between drug and genomic alterations, according to their
actionability.
ESCAT is a framework that ranks a match between drug and genomic alterations, according to their
actionability.

2020
ESCAT level I
means that the
match of an
alteration and a
drug has been
validated in clinical
trials, and should
drive treatment
decision in daily
practice.
ESCAT level II
means that a drug
that matches the
alteration has been
associated with
responses in
phase I/II or in
retrospective
analyses of
randomised trials
ESCAT level III
includes
alterations that are
validated in
another cancer, but
not in the disease-
to-treat.
ESCAT level IV
includes
hypothetically
targetable
alterations based
on preclinical data.
We calculated the number of patients to test with NGS, to identify one patient that
can be matched to an effective drug in daily practice (ESCAT level I), and KN158

Health economic evidence
• Comprehensive genomic panels may be moderately cost-effective in
NSCLC as compared to sequential single-gene evaluation
• From a public health perspective, it must also be considered that the
results of NGS panels could lead to recommend expensive drugs
outside of their approved indications.
• There is a need to regulate the volumes of NGS procedures at the
national level.

Non-squamousNSCLC
2020
EGFR del19, L858R 15% (10-60%) IA Afa, Osimer…
T790M (exon 20) 60% (acquired) IA Osimer
G719X in exon 18, L861Q in
exon 21, S768I in exon 20
10% IB Afa, Osimert
Exon 20 insertion 2% IC Pozio
ALK fusion 5% IA Alec, crizo
METex14 3% IB Capma, tepo, crizo
MET ampl (in mEGFR) 3% IIA Crizo, capma, tepo
BRAF(V600E) 2% IB Dabra/Trame
ROS1 1-2% IB Crizo
NTRK fusions 0.23-3% IC Entrec, Larotrec
RET 1% IC Selperca
RAS (G12C) 12% IIB AMG-510
ERBB2 (mutations) 2-5% IIC T-Duroxtecan
BRCA1/2 1.2% IIIA iPARP
PIK3Ca 1.2-7% IIIA Alpelisib
NRG fusions 1.2% IIIC

Non-squamousNSCLC
2020
Summary of recommendations. It is recommended that a tumour (or plasma) sample from a
patient with advanced non-squamous NSCLC is profiled using NGS technology, in order to
detect level I alterations. Considering the high frequency of fusions, RNA-based NGS, or
DNA-based NGS designed to capture such fusions, are the preferred options. There is no
evidence that panels detecting genes with a lower level of evidence brings additional value
from a public health perspective. They could be used only if the report ranks genomic
alterations according to valid ranking systems (e.g. ESCAT, OncoKB) and on the basis of
specific agreements with payers taking into account the overall cost of the strategy
(including offlabel use of drugs) as compared with small panels. Regarding this latter point,
ESMO does not recommend the use of off-label drugs matched to genomic alterations, except
if an access programme and a procedure of decision has been developed at the national or
regional level, as illustrated by the drug rediscovery protocol programme

Breast cancer
2020
ERBB2 amplification 15-20% IA Anti HER2 agents
ERBB2 mutation 4% IIB Nera
PIK3Ca 40-60% IA Alpelisib
BRCA1/2 germline 4% IA Olaparib (iPARP)
BRCA1/2 somatic 3% IIIA Olaparib (iPARP)
MSI-H 1% IC Anti PD1
NTRK fusions 1% IC Entrec, Larotrec
ESR1 10% IIA Fulvestrant
PTEN 7% IIA AKT inhibitors
AKT1 (E17K) 5% IIB AKT inhibitors
NF1 6% N/A Resistance to anti-estrogens
MDM2 amplification 1% IIIA N/A
ERBB3 mutations 1% IIIC N/A

Breast cancer
2020
Summary of recommendations. Considering that somatic sequencing
cannot fully substitute germline BRCA testing, that PIK3CA status can
be determined by PCR on the three hotspots and pending that HER2
testing is accurately done by immunohistochemistry (IHC) in the local
centre, there is currently no need to perform tumour multigene
NGS for patients with mBC in the context of daily practice. From
the perspective of clinical research centres, and considering the high
number of level II alterations, it is important to include mBC patients in
molecular screening programmes and include them in trials testing
targeted therapies matched to genomic alterations (AKT1E17K, PTEN,
ERBB2 mutations, ESR1 and NF1 mutations).

Colorectalcancer
2020
Since most level I alterations are hotspot mutations in KRAS, NRAS
and BRAF, and considering that MSI status is determined by IHC or
PCR, there is no need to test samples using multigene NGS in the
context of daily practice. Nevertheless, multigene NGS can be an
alternative to PCR tests only if it does not generate extra cost
compared with standard techniques already implemented in
routine. This would allow detection of ERBB2 amplifications, and, in
some panels, detect MSI status with high accuracy. If large panel
NGS is carried out, it should include detection of NTRK fusions. As
for mBC patients, patients with mCRC can present oncogenic
alterations for which drugs are being developed and it is therefore
recommended for clinical research centres to include patients in
molecular screening programmes to propose access to innovative
agents in clinical trials.

Prostatecancer
2020
In countries where PARPi are accessible for
patients with prostate cancer, it is
recommended to perform NGS on tumour
samples to assess the mutational status of,
at least, BRCA1/2. According to the preliminary
results of the phase III trial with AKT inhibitors
in patients with PTEN alterations, this gene
could be added to the panel. Given that they
are unlikely to be cost-effective in these cases,
larger panels can be used only on the basis of
specific agreements with payers taking into
account the overall cost of the strategy
(including off-label use of drugs) and pending a
ranking of additional alterations using a valid
ranking system. These panels should include
DNA repair genes and MSI signature.

Gastric cancer
2020
There is no current need to
perform tumour multigene NGS in
patients with mGC in daily
practice. Detection of MSI and
NTRK fusions should be done
using cheap standard methods.

Pancreaticcancer
2020
It is not currently recommended to perform
tumour multigene NGS in patients with advanced
PDAC in daily practice. Considering the unmet
medical needs and the high number of alterations
ranked as level IIeIV, ESMO considers it is the
mission of clinical research centres and their
networks to propose multigene sequencing to
patients with advanced PDAC in the context of
molecular screening programmes, in order for
patients to get access to innovative drugs. If
multigene sequencing is not carried out, detection
of MSI and NTRK fusions should be done using
cheaper standard methods, pending drugs are
approved and reimbursed.

Hepatocellular carcinoma
2020
It is not currently recommended to perform
tumour multigene NGS in patients with advanced
hepatocellular carcinoma (HCC) in daily practice.
Considering the unmet medical needs and the
number of alterations ranked as level IIeIV, ESMO
considers it is the mission of clinical research
centres to propose multigene sequencing to
patients with advanced HCC in the context of
molecular screening programmes. If multigene
sequencing is not carried out, detection of MSI and
NTRK fusions should be done using cheaper
standard methods, pending drugs are approved and
reimbursed.

Cholangiocarcinoma
2020
Tumour multigene NGS could be used
to detect level I actionable alterations in
cholangiocarcinoma. Given that they
are unlikely to be cost-effective in these
cases, larger panels can be used only
on the basis of specific agreements
with payers taking into account the
overall cost of the strategy (including
off-label use of drugs) and pending a
ranking of additional alterations using a
valid ranking system.
IDH1: Ivosedinib
FGFR2: Pemigatinib
NTRK fusions: Entrectinib, larotrectinib…

TMB
2020
TMB should be determined only in cervical
cancer, NET, salivary cancers, vulvar
cancers, thyroid cancers. Considering that
the study was not agnostic, but limited to
few cancers, the group thinks that
additional studies are needed before
implementing TMB in all cancers where
anti-PD(L)1 antibodies are not approved.
KN158 – TMB High:
ORR 27%

NTRKfusions
2020
Considering the very low incidence, the
group recommends using NGS to
detect NTRK fusions only in cancers
where this technology is recommended
otherwise. In cancers where there is no
need for multigene sequencing, it was
considered that the detection of NTRK
fusion is not an argument per se to
recommend NGS since alternative,
cheaper, diagnostic methods exist. Such
alternative, cheaper methods should be
prioritised to screen patients for NTRK
fusions, in countries where TRK inhibitors
are available..
NTRK fusions
Entrectinib
Larotrectinib
ORR: 80%

Conclusiones
• La perfilación genómica exhaustiva es una herramienta
potencialmente transformadora en escenarios cada vez más amplios
de la oncología clínica.
• Identifica mutaciones accionables susceptibles a terapia dirigida.
• Comunes: EGFR, ALK, BRCA1/2
• Menos comunes: ROS1, FGFR, IDH1/2
• Raras: NTRK
• Puede identificar pacientes candidatos a inmunoterapia
• MSI (alternativa a PCR).
• Tumores sólidos con TMB-H

NGS en oncología

In this document