Colorecat carcinoma tissue biomarkers

How tissue biomarker
data will help tailoring
treatment pathway?
By
Prof. Rasha Haggag
Head of Medical oncology Department
Zagazig Univerisity

 Colorectal cancer (CRC) is a disease in which
pathogenesis is influenced by genetic and epigenetic
events that occur with tumor initiation and progression.
 Precision oncology is becoming increasingly important
in the management and therapy of CRC since large
variation exists in individual patient prognosis and
response to chemotherapy that is due to molecular
heterogeneity.

Definition
A biomarker refers to a quantifiable biological parameter
that is measured and evaluated as an indicator of normal
biological, pathogenic, or pharmacologic responses to a
therapeutic intervention, as defined by the National
Institutes of Health.

Types of molecular markers:
 Diagnostic: used for risk stratification and early
detection.
 Prognostic: gives an indication of the likely progression
of the disease.
 Predictive: predicts treatment response.
 Surveillance: used to monitor disease recurrence.

Outline
• Extended RAS testing
• BRAF mutation
• Microsatellite instability testing
• Tumor Location: “Sidedness”
• Circulating tumor DNA for tumor profiling
• HER2 amplification
• Fusions
• Consensus Molecular Subtypes
• Circulating tumor DNA for minimal residual disease

KRAS and NRAS mutation
 KRAS and NRAS mutation status predict the efficacy of
anti-EGFR antibodies with clinical benefit restricted to
patient tumors with non-mutated KRAS or NRAS genes.
 More than one-third of CRCs carry mutations in exon 2
of KRAS, and an additional 15% of tumors were found to
carry mutations at exons 3 and 4 of KRAS and exons 2,
3 and 4 at NRAS that predict resistance to anti-EGFR
antibody therapy

Prevalence of Extended RAS Mutations
Exon 1 Exon 2 Exon 3 Exon 4
Exon 1 Exon 2 Exon 3 Exon 4
KRAS
NRAS
WT
12 13 61 146
12 13 61 146
4% 5%
4% 2% <1%
1 of 6 patients with codon 12/13 KRAS wildtype
tumors harbor extended RAS mutations
Atreya CE, …Kopetz S. J Clin Oncol. 2015;33(7):682-685.

 It is now recommended that expanded RAS mutation
testing be performed for all patients being considered
for anti-EGFR mAb treatment.
 In addition to RAS, mutations in the phosphoinositide 3-
kinase catalytic subunit alpha (PIK3CA; exon 20 vs exon
9), which is part of the EGFR signaling pathway, may
confer resistance to anti-EGFR therapies although
further study is needed.
 Stage III, colon cancer have shown that KRAS exon 2
mutations are associated with poor clinical outcome.

BRAF mutation
 The BRAF gene encodes B-RAF, a protein involved in cell signaling,
growth, and survival.
 The common BRAF V600E mutation leads to constitutive activation
of B-RAF monomers and subsequent activation of MEK1 and MEK2,
leading to increased cell proliferation and suppression of apoptosis.
 A subset (~8%) of CRCs carry a point mutation (V600E) in the BRAF
oncogene that is mutually exclusive with mutation in KRAS.
 BRAFV600E mutations have have a poor prognosis in the
metastatic setting AND also in stage III .

BRAF mutation
 The adverse prognostic impact of mutant
BRAFV600E is particularly evident for overall
survival.

Overall Survival: CALGB/SWOG 80405
Innocenti F, et al. J Clin Oncol. 2017;35(Suppl 4): Abstract 3504.
BRAF V600E Mutations Are Associated With Poor OS1
1Morris V, et al. Clin Colorectal Cancer. 2014;13(3):164-171.
Atypical Patterns of Metastases
IncreasedIncidenceComparedto
BRAFWildtype
CALGB/SWOG, The Cancer and Leukemia Group B/Southwest Oncology Group; L, left; OS, overall survival; R/T, right/transverse

BRAF mutation
 In contrast to patients with
BRAFV600E mutant melanoma,
CRCs that harbor BRAFV600E
mutations were found to be
resistant to inhibition of the
BRAF/MEK/ERK signaling pathway
by vemurafenib

BRAF mutation
 Resistance to vemurafenib was later
found to be due to feedback activation of
EGFR when BRAF is inhibited.
 In a recent report, the combination of a
BRAF inhibitor plus a MEK/ERK inhibitor
demonstrated modest activity in a subset
of patients with BRAFV600E mutant
metastatic CRC
14

Addressing BRAF/EGFR Resistance
1. Hong DS, et al. Cancer Discov. 2016;6(12):1352-1365. 2. Ahronian LG, et al. Cancer Discov. 2015;5(4):358-367.
Arm A - Triplet Therapy
Binimetinib* + Encorafenib +
Cetuximab
n = 205
Arm B - Doublet Therapy
Encorafenib + Cetuximab
n = 205
Arm C - Control Arm
FOLFIRI + Cetuximab or
irinotecan + Cetuximab
n = 205
Randomization
Patient
population
• BRAF V600E
mutant
• 1 to 2 prior
regimens in
metastatic
setting
Ongoing Phase III BEACON Study
Can response rate and PFS of BRAFi/EGFRi be augmented by MEKi? 1,2
48% response rate
PFS 8 months
Now available for enrollment in Japan (Dr Yoshino): NCT02928224
Huijberts S, et al. Ann Oncol. 2017;28(Suppl 5): Abstract 517P.

DNA Mismatch Repair/Microsatellite Instability
(MMR/MSI)
 Microsatellite instability (MSI)–high cancers are caused by mutations
and/or promoter hypermethylation in DNA mismatch repair (MMR)
genes.
 MMR genes encode proteins that “police” base-pair mismatches
during DNA replication to correct any errors.
 MMR deficiency results in 10-100 times more genetic mutations,
resulting in MSI-high hypermutant tumors.

DNA Mismatch Repair/Microsatellite
Instability (MMR/MSI)
 Microsatellite instability status (MSI) (high or low) is the primary
molecular marker for stratification of stage II CRC.
 In node negative CRC, patients that are MSI-high have better
outcomes than MSI-low tumors; therefore, adjuvant chemotherapy is
usually not indicated in MSI-high tumors.
 Although approximately 15% of early colorectal cancers display a
high degree of MSI-H, indicating dMMR, MSI-H status is found in
approximately 4% of metastatic CRC

Although several proteins have been implicated in
DNA repair, abnormalities in MSH2, MSH6, PMS2 and
MLH1 are the most commonly described.
MSI-high tumors may be the result of an inherited
mutation of the DNA repair genes (Lynch syndrome)
or, more commonly, the abnormal epigenetic
methylation of the MLH1 promoter gene (sporadic MSI-
high CRC).
Analysis of the DNA repair system may be directly
investigated by the tissue expression of MSH2, MSH6,
PMS2 and MLH1 by immunohistochemistry, or
alternatively by by PCR.

Microsatellite Instability High (MSI-H) Is Associated
With High Mutation Rates and Immune Activation
MSI-H Tumor: Anti-CD3 Ab
Vogelstein B, et al. Science. 2103;339(6127):1546-1558.
HLA, human leukocyte antigen

Immune Microenvironment of MSI Tumors
Xiao Y, et al. Cancer Discovery 2015, Llosa N, et al. Cancer Discov 2015, Dung TL, et al. ASCO 2015
The high mutational load in MSI tumors creates many tumor-specific neoantigens
Some of these neoantigens will be processed, presented on MHC, and
recognized as foreign by T cells
High level of tumor-infiltrating lymphocytes (TIL) and lymphocytic reaction
in MSI tumors

Le DT, et al. N Engl J Med. 2015;372(26):2509-2520.
Responses to Pembrolizumab in
Mismatch Repair-Deficient (dMMR) mCRC
RECIST, Response Evaluation Criteria in Solid Tumors

FDA News. www.fda.gov/newsevents/newsroom/pressannouncements/ucm560167.htm. Accessed May 16, 2018.

Overman MJ, et al. Lancet Oncol 18:
1182-1191, 2017
Overman MJ, et al. J Clin Oncol 36:
773-779, 2018

MAP Kinase Pathway
Growth factor
receptor
Cobimetinib
Bendell J, et al. #3502 ASCO 2016
Single agent MEK inhibition has shown little activity in mCRC
Cobimetinib Atezolizumab
Binding of PD-L1 to its receptors PD-L1 and B7.1
• This inhibition can enhance T-cell priming and
restore anti-tumor T-cell activity

The colorectal tract is highly heterogeneous
 Developmental, genetic and biological differences in the proximal (right-side) and distal
(left-side) segments of the colon have been documented for over 20 years, and may account
for differences in left- vs right-sided CRC tumours1-5
1. Bufill. 1990; 2. Missiaglia. 2013; 3. Brule. 2013;
4. The Cancer Genome Atlas Network. 2012; 5. Bendardaf. 2008
Right-sided tumours
(cecum, ascending colon, or transverse colon)
Left-sided tumours
(descending colon, sigmoid colon, rectum)
• Older patients
• Higher incidence in female patients
• Mucinous, signet ring histology
• Poorly differentiated
• Microsatellite instability
• Hypermethylation, higher mutation rates
• PI3KCA mutation
• KRAS mutations
• BRAF mutations
• Chromosomal aberrations; 18q loss and 20q gain
• Aneuploidy
• p53 mutation
• COX2 expression
• EGFR gain
• HER2 gain
• High EGFR ligand expression (epiregulin [EREG] and
amphiregulin [AREG] expression)
• High VEGF-1 mRNA expression
Right hepatic
flexure
Ascending
colon
Cecum
Appendix
Rectum
Sigmoid colon
Descending colon
Left splenic
flexure
Transverse
colon

Treatment effects within subgroups defined by primary tumor
location in patients with RAS WT mCRC
Study Treatment n
OS,
months
HR OS (95% CI)
p value
PFS,
months
HR PFS (95% CI)
p value
ORR,
%
OR ORR (95% CI)
p value
Left-sided colorectal cancer
CRYSTAL
FOLFIRI 138 21.7 0.65 (0.50–0.86)
p=0.02
8.9 0.50 (0.34–0.72)
p<0.001
40.6 3.99 (2.40–6.62)
p<0.001FOLFIRI + cetux 142 28.7 12.0 72.5
PRIME
FOLFOX 159 23.6 0.73 (0.57–0.93)
p=NR
9.2 0.72 (0.57–0.90)
p=NR
53 1.9 (1.3–2.7)
p=NRFOLFOX + pani 169 30.3 12.9 68
CALGB
80405
FOLFOX/FOLFIRI
+ Avastin
152 32.6
0.77 (0.59–0.99)
p=0.04
11.2
0.84 (0.66–1.06)
p=0.15
–
1.6 (1.2–2.3)
p=NRFOLFOX/FOLFIRI
+ cetux
173 39.3 12.7 –
FIRE-3
FOLFIRI + Avastin 149 28.0 0.63 (0.48–0.85)
0.002
10.7 0.90 (0.71–1.14)
p=0.38
61.7 1.37 (0.85–2.19)
p=0.23FOLFIRI + cetux 157 38.3 10.7 68.8
PEAK
FOLFOX + pani 53 43.4 0.84 (0.22–3.27)
p=NR
14.6 0.65 (0.21–2.0)
p=NR
64 1.3 (0.7–2.5)
p=NRFOLFOX + Avastin 54 32.0 11.5 57
Holch. 2016

Treatment effects within subgroups defined by primary tumour location in
patients with RAS WT mCRC (cont’d)
Study Treatment n
OS,
months
HR OS (95% CI)
p value
PFS,
months
HR PFS (95% CI)
p Value
ORR,
%
OR ORR (95% CI)
p value
Right-sided colorectal cancer
CRYSTAL
FOLFIRI 51 15.0 1.08 (0.65–1.81)
p=0.76
7.1 0.87 (0.47–1.62)
p=0.66
33.3 1.45 (0.58–3.46)
p=0.43FOLFIRI + cetux 33 18.5 8.1 42.4
PRIME
FOLFOX 49 15.4 0.87 (0.55–1.37)
p=NR
7.0 0.80 (0.50–1.26)
p=NR
35 1.4 (0.6–3.1)
p=NRFOLFOX + pani 39 11.1 7.5 42
CALGB
80405
FOLFOX/FOLFIRI
+ Avastin
78 29.2
1.36 (0.93–1.99)
p=0.10
10.2
1.64 (1.15–2.36)
p=0.006
NR
1.1 (0.6–2.0)
p=NRFOLFOX/FOLFIRI
+ cetux
71 13.7 7.5 NR
FIRE-3
FOLFIRI + Avastin 50 23.0 1.31 (0.81–2.11)
p=0.28
9.0 1.44 (0.92–2.26)
p=0.11
50.0 1.11 (0.48–2.59)
p=0.83FOLFIRI + cetux 38 18.3 7.6 52.6
PEAK
FOLFOX + pani 22 17.5 0.45 (0.08–2.49)
p=NR
8.7 0.84 (0.18–3.79)
p=NR
63 1.8 (0.6–5.4)
p=NRFOLFOX + Avastin 14 21.0 12.6 50
Holch. 2016

Current View of mCRC Treatment in US
FOLFOXIRI + Bev
Cetuximab
+ irinotecan
+ vemurafenib
FOLFOX + Bev PD-1 inhibition
FOLFOX + Bev FOLFIRI + Bev
Salvage Oral
Agents:
Regorafenib
TAS-102
(Trifluridine
/tipiracil)
FOLFOX + Cet/Pan
(or Bev)
FOLFOX + Bev FOLFIRI + Bev Irinotecan + Cet/Pan
FOLFIRI + Bev
(or Cet/Pan)
RAS mutated
BRAF mutated, MSS
MSI-High
RAS/BRAF wildtype
• “Left sided”
• “Right sided”
Bev, bevacizumab; Cet/Pan, cetuximab/panitumumab; mCRC, metastatic colorectal cancer; MSI, microsatellite instable; MSS, microsatellite stable

Circulating tumor DNA for tumor profiling
 The term “liquid biopsy” in cancer arose
when circulating tumor cells (CTC) were
proposed as alternatives to conventional
tissue biopsy for prognosis and evaluation
of treatment responses in breast cancer
and then CRC.
 The clinical applications of liquid
biopsy in CRC continue to grow,
including:
 detecting premalignant and early-stage
cancers,
 identification of aggressive phenotypes
and high-risk patients,
 assessing tumor heterogeneity,
residual, and recurrent disease, and
monitoring treatment response[
36

Circulating tumor DNA for tumor profiling
 Possible sources of liquid biopsies include blood,
urine, saliva, and stool, which contain cancer-
derived subcellular components, such as circulating
tumor DNA (ctDNA) and circulating miRNAs.
 Tumour-tissue remains the “gold standard”, but the
advent of ctDNA analysis from blood samples has
promise as a non-invasive biomarkers.

Concordance of Tissue and ctDNA in mCRC: 93% Accuracy
Prospective AGEO Study:
Bachet J-B, et al. J Clin Oncol. 2017;35(Suppl 4): Abstract 11509.
NGS:
A subset of patients did not have detectable
ctDNA:
Metachronous disease, peritoneal only, low
tumor markers
AGEO, Association des Gastro-Entérologues Oncologues; NGS, next generation sequencing

HER2 Amplification: 5-14% of CRC Tumors
Valtorta E, et al. Mod Pathol. 2015;28(11):1481-1491.

HER2 and Apparent Lack of Benefit from EGFR Inhibition
Median: 2.9 v 8.1 m
(P<.001)
Median: 2.9 v 9.3 m
(P<.001)
HER2amp
HER2NA
HER2amp
HER2NA
Cohort1Cohort2
Median: 9.7 v 10.1 m
(P=.848)
Median: 13.7 v 11.3 m
(P<.616)
HER2amp
HER2NA
HER2amp
HER2NA
A
A
B
B
EGFR-based regimen Non-EGFR-based regimen
Raghav K, et al. J Clin Oncol. 2016;34 (suppl): Abstract 3517.
V
a
l
i
d
a
t
i
o
n
o
f
a

Fusions: Rare But Actionable
• Relative prevalence of the key
actionable fusions in CRC
4,290 pt data
FGFR
2
ROS
NTRK1ALK FGFR3
RET
Unpublished

In total, these
fusions are
present in <1% of
mCRC
No detectable
fusion
Van Morris
In total, these
fusions are present
in <1% of mCRC
Absolute Prevalence
of Fusions
4,290 pt data
Unpublished

Amatu A, et al. Br J Cancer. 2015;113(12):1730-1734.
ALK Fusion
• Novel CAD-ALK gene rearrangement is
drugable by entrectinib in colorectal cancer

Guinney J, et al. Nat Med. 2015;21(11):1350-1356.
Consensus Molecular Subtypes (CMS)
•How do CRC differ by gene expression?
•
USING 6 different classification systems, they
developed 4 consensus molecular subtypes
(CMS) of CRC with distinguishing features
.
Grouping ~4,000 patient tumors by RNA profiling:

• Stage II (5% prevalence of ctDNA+)
SafeSeq/Roche Molecular assay
Assay with 197 genes; at least one mutation detected 99.3% of tumor tissue
57% sensitivity for recurrence; 100% specificity
• Stage III (16% prevalence of ctDNA+)
HR 54.4
95% CI: 9.5-311.7
P<.0001
HR 20.0
95% CI: 5.9-67.8
P<.0001
Diehn M, et al. J Clin Oncol. 2017;35(Suppl 4):Abstract 3591.
ctDNA Levels After Resection Predicts Recurrence
With Near 100% Specificity

NCCN-Recommended Tumor Biomarker Testing and
Impact on Treatment
52

Summary
 Colorectal cancer is a major cause of morbidity and mortality
 RAS WT Left sided tumors do well overall and on anti-EGFR
therapies
 BRAF mutation signifies aggressive disease and should be treated
with BRAF inhibitors (on and off trials)
 HER2 amplified tumors are resistant to anti-EGFR therapies and
should be treated on trials with anti-HER2 drugs (early trials show
promising benefit)
 The future of mCRC requires furthering of precision therapy

Summary
• Treatment decision-making based on latest evidences
– Biomarker status
– Sidedness of the primary tumor
– Patient characteristics
•
• Front-line therapy for patients with RAS MUT, BRAF MUT, and MSI-H
disease needs to be improved

Yoshino T…Yeh K-H. Ann Oncol. 2018;29(1):44-70.
BSC, best supportive care; CRC, colorectal cancer; CT, chemotherapy

Thank You
Doing now what patients need next

Colorecat carcinoma tissue biomarkers

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