Contents

Introduction to Biomarkers MSI-H/dMMR

 Role of biomarkers, defining biomarker cut  What is dMMR/MSI-H?
points
 Etiology of dMMR/MSI-H in
 Companion vs Complementary Diagnostics
CRC
PD-L1
 Assessment and scoring of PD-L1 expression
 Testing methods
TPS  Role of PD-1 blockade in MSI-
 What is TPS? H/dMMR cancers
 Staining Pattern for TPS  Prevalence of MSI-H/dMMR
 Scientific Rationale for TPS in NSCLC  Response
Tumor to pembrolizumab
Mutational Burden (TMB)
 Staining Pattern for CPS  What is TMB?
 Scientific Rationale for CPS  Role of TMB in predicting response to PD-1
inhibitors
 Correlation between TMB and response to
pembrolizumab
 Potential application of TMB
 Role of Biomarkers
• A biomarker is “a characteristic that is objectively measured and evaluated as an indicator of normal
biological processes, pathogenic processes or pharmacological responses to a therapeutic intervention”1

Role of the biomarker2 Description of use

Predictive
Used to identify patients who will benefit from a particular drug/therapy (e.g., EGFR)
(Patient Selection)

Used to determine how aggressive the disease process is and/or how a patient may expect to fare regardless of
Prognostic
therapy (e.g., KRAS)

Diagnostic Used to diagnose a disease, potentially before it is detectable by conventional methods (e.g., PSA)

1. Strimbu K, Tavel JA. Curr. Opin. Hiv Aids. 2010;5:463–466.

2. FDA/NIH BEST Resource Nov 2017
 Defining Biomarker Cut-points: ROC Curve and
• the Youden Index
After the analytical validity of a biomarker assay is established, the
clinical validation and utility of the test must be evaluated.1 Receiver Operating Characteristic Curve (ROC)
• Cut-points are selected to enrich for the optimal benefit from
pembrolizumab2,3
• Receiver operator characteristic curves (ROC) and the Youden Index are
used to identify the optimal cut point by maximizing the true positive
rate (TPR) and minimizing the false positive rate (FPR) 1,4

Test Result Responder Non-Responder

Positive True Positive (TP) False Positive (FP)

Negative False Negative (FN) True Negative (TN)

All Responders (AR) All Non-Responders (AN)

• Cut-points are explored with a pre-determined patient population

assigned as the training set to assess the number of responders4
• Practical aspects of scoring are also considered in cut-point choice (i.e.,
ease of use of the cut-point)1
1. Dobbin et al. Journal for ImmunoTherapy of Cancer (2016) 4:77. 2. Dolled-Filhart et al. Arch Pathol Lab Med Vol 140 (1243-1249) Nov 2016 3. Garon et al NEJM N Engl J Med 2015; 372:2018-28
4. Youden WJ. Index for rating diagnostic tests. Cancer 1950; 3: 32–5.
 Companion vs Complementary Diagnostics
Companion Diagnostic:
• A companion diagnostic is a medical device, often an in vitro diagnostic, which
provides information that is required for the safe and effective use of a
corresponding drug or biological product.
• Identify patients who are most likely to benefit from a particular therapeutic product;

Complementary Diagnostic:
• A complementary diagnostic is a type of in vitro diagnostic that can lend
information about the risk/benefit of a drug but the biomarker is NOT a
prerequisite for receiving a drug.
• Therapeutic benefit has been shown in all populations but may inform on enhanced benefit in
a subpopulation of patients1

1. Scheerens H. Clin Transl Sci. 2017 Mar; 10(2): 84–92.

Programmed Death-Ligand 1(PD-L1)

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 Assessing PD-L1 Expression: PD-L1 IHC 22C3
pharmDx
• PD-L1 protein expression is measured using immunohistochemistry (IHC), as determined by an FDA-approved test
− PD-L1 IHC 22C3 pharmDx is the only companion diagnostic approved by the FDA for use in the detection of PD-L1 protein in formalin-fixed,
paraffin-embedded (FFPE) non-small cell lung cancer (NSCLC), gastric or gastroesophageal junction (GEJ) adenocarcinoma, urothelial carcinoma
and cervical cancer tissues

• PD-L1 expression is assessed differently across tumor types based on Merck’s biomarker analyses of PD-L1 in tumor and/or immune cells in the
context of pembrolizumab response, by tumor type:

• Tumor Proportion Score (TPS): The percentage of PD-L1 expressing viable tumor cells (partial or complete membrane staining at any
intensity) relative to total number of viable tumor cells. This scoring method is used for NSCLC.

• Combined Positive Score (CPS): Number of PD-L1 staining cells (tumor cells, lymphocytes, macrophages) relative to the total number of
viable tumor cells, multiplied by 100. This method is used for urothelial, gastric/GEJ and cervical cancer, as well as other solid tumor types
under investigation.1

1. PD-L1 IHC 22C3 pharmDx Interpretation Manual, Agilent

 Scoring PD-L1 Expression: TPS and CPS TPS CPS
Malignancy NSCLC Other Solid Tumors

Companion Diagnostic (CDx) 1L , mono: TPS ≥50% Gastric/GEJ 3L; Cervical 2L: CPS ≥1
status 2L, mono: TPS ≥1% Bladder 1L cisplatin-ineligible: CPS≥10

Tumor cells  
Tumor associated immune cells
_ 
(lymphocytes, macrophages)

Scale 0-100% 0-100

• PD-L1 expression in immune cells did not improve predictive • PD-L1 expression in both tumor and immune cells improved patient
value patient identification 1 identification and objective response to pembrolizumab in other solid
tumors 5-9
• Incorporation of immune cells increased the false positive rate
Rationale and did not improve the true positive rate 1

• PD-L1 expression in tumor cells alone demonstrated a

relationship with pembrolizumab clinical efficacy 2,3,4

(1) Dolled-Filhart et al. Arch Pathol Lab Med Vol 140 (1243-1249) Nov 2016 (2) Garon et al NEJM N Engl J Med 2015; 372:2018-28 (3) Herbst et al Lancet 2016; 387: 1540–50 (4) Reck et al N Engl J Med 2016;375:1823-33 (5) Plimack et al Lancet
Oncolo 2017: 18; 212-220 (6) Muro et al Lancet Oncol 2016; 17: 717–26 (7) Chow et al Journal of Clinical Oncology 34 (32) Nov 2016 (8) Bauml et al. J Clin Oncol 2016; 35 (14):1542-49 (9) Bellmunt et al NEJM Feb 2017;376:1015-26.
 Tumor Proportion Score (TPS)
NSCLC

Reactive Use Only.

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 PD-L1 IHC 22C3 pharmDx:
• Tumor Proportion
TPS is used to evaluate PD-L1 expression inScore (TPS)
metastatic NSCLC - NSCLC
specimens.
• TPS is defined as the percentage of viable tumor cells showing partial or complete membrane staining at any
intensity divided by total number of viable tumor cells.1
• The specimen should be considered to have PD-L1 expression if TPS ≥1% and high PD-L1 expression if TPS ≥50%.1
• TPS is expressed as a percentage.1
• TPS does not include PD-L1 expression on immune cells.1,2

# 𝐏𝐃–𝐋𝟏 𝐬𝐭𝐚𝐢𝐧𝐢𝐧𝐠 𝐭𝐮𝐦𝐨𝐫 𝐜𝐞𝐥𝐥𝐬

TPS = x 100%
𝐓𝐨𝐭𝐚𝐥 # 𝐯𝐢𝐚𝐛𝐥𝐞 𝐭𝐮𝐦𝐨𝐫 𝐜𝐞𝐥𝐥𝐬

NSCLC = non–small cell lung cancer; PD-L1 = programmed death ligand 1.

1. Agilent Technologies, Inc. Instructions for Use: PD-L1 IHC 22C3 pharmDx. 2. Agilent Technologies, Inc. PD-L1 IHC 22C3 pharmDx Interpretation Manual.
 PD-L1 Expression and Staining Pattern for TPS
The TPS determines the PD-L1 expression of the specimen

Expression Level TPS Staining Pattern

No PD-L1 Expression < 1% Partial or complete cell membrane
staining (> 1+) in < 1% of viable tumor
cells
PD-L1 Expression > 1% Partial or complete cell membrane
staining (> 1+) in 1-49% of viable tumor
cells
High PD-L1 Expression > 50% Partial or complete cell membrane
staining (> 1+) in > 50% of viable tumor
cells
1. PD-L1 IHC 22C3 pharmDx Interpretation Manual, Agilent

1. PD-L1 IHC 22C3 pharmDx Interpretation Manual, Agilent

 Scientific Rationale for TPS in NSCLC
• PD-L1 can be expressed differently across cancer types; therefore, the
clinical utility of a scoring algorithm system must be appropriately
matched to that tumor type.1–5
• In NSCLC, incorporation of inflammatory cells into the PD-L1 scoring
algorithm system did not add any selection value.3
• IHC 22C3 pharmDx assay was prospectively validated in KEYNOTE-001,
KEYNOTE-010 and KEYNOTE-024:
− KEYNOTE-001 (1L and 2L+): validation of PD-L1 at the 50% cutoff 3
− KEYNOTE-010 (2L+): biomarker enriched with clinical validation of PD-L1 at the 1% cutoff 4
− KEYNOTE-024 (1L): validation of PD-L1 at 50% for 1st line 5

1. Kluger HM et al. Clin Cancer Res. 2017;23(15):4270–4280. 2. Dolled-Filhart M et al. Arch Pathol Lab Med. 2016;140(11):1243–1249. 3. Garon EB et al. N Engl J Med.
2015;372(21):2018–2128. 4. Herbst R, et al. Lancet 2016;387:1540-50. 5. Reck M et al. N Engl J Med. 2016;375(19):1823–1833.
Determination of PD-L1 TPS Cut Points for
Pembrolizumab in NSCLC

Reactive Use Only.

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 Combined Positive Score (CPS)
Gastric cancer/GEJ adenocarcinoma, Cervical cancer,
Urothelial carcinoma and Head and Neck Squamous Cell
Carcinoma

Reactive Use Only.

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 PD-L1 IHC 22C3 pharmDx: Combined Positive
Score
• CPS is defined as(CPS)
the number of PD-L1 staining cells (tumor cells, lymphocytes, macrophages) divided by the total
number of viable tumor cells, multiplied by 100. 1

• In gastric and cervical cancer, the specimen should be considered to have PD-L1 expression if CPS ≥1.1
• CPS is expressed as a numeric value.1
• Although the result of the CPS calculation can exceed 100, the maximum score is defined as CPS 100.1

# 𝐏𝐃–𝐋𝟏 𝐬𝐭𝐚𝐢𝐧𝐢𝐧𝐠 𝐜𝐞𝐥𝐥𝐬

𝐭𝐮𝐦𝐨𝐫 𝐜𝐞𝐥𝐥𝐬, 𝐥𝐲𝐦𝐩𝐡𝐨𝐜𝐲𝐭𝐞𝐬, 𝐦𝐚𝐜𝐫𝐨𝐩𝐡𝐚𝐠𝐞𝐬
CPS = x 100
𝐓𝐨𝐭𝐚𝐥 # 𝐯𝐢𝐚𝐛𝐥𝐞 𝐭𝐮𝐦𝐨𝐫 𝐜𝐞𝐥𝐥𝐬

1. Agilent Technologies, Inc. Instructions for Use: PD-L1 IHC 22C3 pharmDx.
 CPS
PD-L1 Expression and
Expression Level
Staining Pattern
Image (20x)
for CPS

<1 No PD-L1 Expression

>1 PD-L1 Expression

1. PD-L1 IHC 22C3 pharmDx Interpretation Manual – Gastric or Gastroesophageal Junction (GEJ) Adenocarcinoma Agilent
 Scientific Rationale for Scoring Algorithms:
Combined Positive Score (CPS)
• PD-L1 can be expressed differently across cancer types; therefore, the clinical utility of a scoring algorithm system must be
appropriately matched to that tumor type.1–4

• In NSCLC, incorporation of inflammatory cells into the PD-L1 scoring algorithm system did not add any predictive value.3

• In KEYNOTE-012 and KEYNOTE-028, the CPS scoring algorithm was evaluated against TPS retrospectively, to assess the impact of
including inflammatory cells in identifying responders to pembrolizumab. 4

• KEYNOTE-012 was an open-label, basket study that enrolled PD-L1 positive patients with recurrent, metastatic, or persistent gastric or gastroesophageal cancer,
urothelial cancer, triple-negative breast cancer, or squamous carcinoma of the head and neck, with any number of prior therapies. 4

• KEYNOTE-028 was an open-label, basket study that enrolled PD-L1 positive patients with 20 tumor types. 4

• CPS was shown to be highly reproducible scoring algorithm and was implemented prospectively in Gastric/GEJ Cancer with
KEYNOTE-059, Cervical Cancer with KEYNOTE-158 and 1L Urothelial Carcinoma in KEYNOTE-052.4

• In gastric/GEJ adenocarcinoma, cervical and urothelial cancer, PD-L1 staining of tumor, lymphocytes and macrophages combined
showed to be associated with patient response to pembrolizumab5,6,7
1. Kluger HM et al. Clin Cancer Res. 2017;23(15):4270–4280. 2. Dolled-Filhart M et al. Arch Pathol Lab Med. 2016;140(11):1243–1249. 3. Garon EB et al. N Engl J Med. 2015;372(21):2018–2128. 4. Kulangara et al, Arch Pathol Lab Med, 2018 . 5. Kulangara et al. Presented
at ASCO 2018 June 1-5 2018 6. Böger C et al. Oncotarget. 2016;7(17):24269–24283. 7. Agilent Technologies, Inc. Instructions for Use: PD-L1 IHC 22C3 pharmDx. 8. Balar et al. Lancet Oncol. 2017 Nov; 18(11):1483-1492.
 MSI-H/dMMR

Reactive Use Only.

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 What Are DNA Mismatch Repair (MMR) and
Microsatellite Instability (MSI)? MSI is a genetic condition caused by a dMMR system.1,2

DNA MMR MSI

MMR is a process in normal cells that permits the recognition and repair MSI is the expansion or reduction in
of genetic mismatches generated during replication. MMR deficiencies the length of repetitive DNA sequences (known as microsatellites) in
(dMMR) can result in the persistence of DNA mismatches that may then tumor DNA, compared with normal DNA2-3
be incorporated into the genetic code as mutations2

dMMR: pMMR: MSI-H: MSI-L: MSS:

When mutations are detected in When a tumors does not ≥2 mutated 1 mutated No mutated
MSH2, MLH1, MSH6, and/or demonstrate MMR deficiency by microsatellite microsatellite microsatellite
PMS2 genes via PCR2 IHC sequences3 sequence3 sequences3

1. Lee JK, Chan AT. Curr Colorectal Cancer Rep. 2011;7(2):136–44. 2. Boland CR, Goel A. Gastroenterology. 2010;138(6):2073–2087. 3. Boland CR, et al. Cancer Res. 1998;58:5248–5257.
 Genetic or Epigenetic Pathways Lead to dMMR Pathways to mismatch repair deficiency in colorectal cancer

Germline mutation Biallelic MLH1 methylation CIMP+

(MLH1, MSH2, MSH6, PMS2) Loss of functional MMR protein
Loss of functional MMR protein
via germline mutation (Lynch via
Lynch syndrome (~3%) Sporadic (~12%) somatic mutation or silencing
Syndrome)

Deficient MMR repair

Second hit (mutation, LOH,

methylation)

Microsatellite instability (MSI)

Frameshift mutations in genes with

Gain of other mutant proteins coding microsatellites BRAFV600E Gain of other mutant proteins
detected by T-cells2 mutation detected by T-cells2
Other mutations
Colorectal Cancer
BRAFV600E = amino acid substitution at position 600 in BRAF gene; CIMP = CpG island methylator phenotype; LOH = loss of heterozygosity; MMR = mismatch repair.
1. Sinicrope FA et al. Clin Cancer Res. 2012;18(6):1506–1512.
2. Overwijk WW. Nature Medicine. 2015;21(1):12-14.
 Testing Methods
• MSI polymerase chain reaction (PCR) and immunohistochemistry (IHC) are conventionally used for clinical MSI/MMR testing. 1-2
• PCR detects instability in the microsatellite repeats by comparing the lengths of specific microsatellites from the tumor
cells to those same regions in healthy cells.1
• IHC analyzes MMR protein expression to detect loss of one or more of these proteins in the tumor.2
• Both testing methods are considered equally sensitive and produce concordant results in CRC and endometrial cancer. 3-4
• Next-generation sequencing (NGS) is an alternative testing method for identification of patients with dMMR/MSI-H.5
• NGS uses massive parallel sequencing to detect altered microsatellite markers
• Different computational tools have been developed for the determination of MSI using NGS (e.g., Mantis, MSIsensor,
MSIseq, MOSAIC)
• Merck is working in a post-marketing commitment to deliver a diagnostic device that will identify tumors that are MSI-H or
dMMR.6
− Current testing methods are acceptable for determining MSI-H and dMMR for use with pembrolizumab

1. Vilar E et al. Nat Rev Clin Oncol. 2010;7(3):53–162. 2. Richman S. Int J Oncology. 2015;47(4):1189–1202. 3. Bedeir et al. Arch Path Lab Med 2011;135(5):578–587. 4. McConechy MK, et al. Gynegologic Oncology. 2015; 137(2):306-10. 5. Baretti M, Le DT.
DNA mismatch repair in cancer. Pharmacol Ther. 2018 Apr 15. pii: S0163-7258(18)30067-6. 6. Lemery S et al. N Engl J Med. 2017;377(15):1409–1412.
 Scientific Rationale for Immune-checkpoint
Blockade in MSI-H/dMMR Tumors 1

• MSI-H/dMMR tumors are characterized by:

− 10-50 times more tumor-specific neoantigens than MSS tumors1,2
− high level of TILs and an active Th1/CTL environment1,2
− high expression of checkpoint molecules such as PD-1, PD-L1, CTLA-4, LAG-3, and IDO1,2

1. Llosa NJ, et al. Cancer Discov. 2015;5(1):43–51. 2. Pardoll DM. Nat Rev Cancer. 2012;12(4):252–264.
MSI-H/dMMR Frequency Across Solid Tumors
Mismatch repair deficiency (dMMR) was assessed using NGS across 12,019 tumor samples. dMMR was identified in 24 of
32 tumor subtypes tested, more often in early-stage disease (defined as stage <4)

Le DT, et al. Science. 2017;357:409-413

 dMMR/MSI-H Response to Pembrolizumab
The efficacy of pembrolizumab 200 mg Q3W or 10 mg/kg Q2W was evaluated in 149 patients with MSI-H or dMMR
solid tumors, across 5 clinical trials
• Tumor status was evaluated prospectively in 135/149 tumor samples using laboratory-developed PCR for MSI-H or
IHC for dMMR.
• Fourteen of the 149 were retrospectively identified as MSI-H from 415 samples using a central laboratory-developed
PCR
• 47 patients had dMMR cancer identified by IHC, 60 had MSI-H identified by PCR, and 42 were identified using both
tests.
• 98% of patients had metastatic disease and 2% had locally advanced, unresectable disease
Endpoint N=149
Objective response rate
ORR (95% CI) 39.6% (31.7, 47.9)
Complete response rate 7.4%

Partial response rate 32.2%

Response duration
Median in months (range) NR (1.6+, 22.7+)

% with duration >6 months 78%

Keytruda Prescribing Information

 Response by Tumor Type
Tumor type N Objective response rate DOR range (months)
n (%) 95% CI
CRC 90 32 (36%) (26%, 46%) (1.6+, 22.7+)
Non-CRC 59 27 (46%) (33%, 59%) (1.9+, 22.1+)

Other tumor types:

- Endometrial - Breast - Thyroid
- Biliary - Prostate - Retroperitoneal adenocarcinoma
- Gastric or GEJ - Bladder - Small cell lung
- Pancreatic - Esophageal - Renal cell
- Small intestinal - Sarcoma

Keytruda Prescribing Information

 MSI-H/dMMR Safety Results
• Most common adverse reactions (reported in ≥20% of patients)
were fatigue, musculoskeletal pain, decreased appetite, pruritus,
diarrhea, nausea, rash, pyrexia, cough, dyspnea, constipation, pain,
and abdominal pain.

1. Pembrolizumab Prescribing Information

 Tumor Mutation Burden (TMB)

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 Tumor Mutation Burden (TMB)
• TMB is a count of the total number of somatic, nonsynonymous mutations within a
tumor exome1-2
• Measured by high-throughput nucleic acid sequencing technologies known as next-
generation sequencing (NGS)
• Tumor DNA is extracted from tumor tissue or blood and processed for sequencing via a series of
molecular biology steps
• Bioinformatics analysis is then applied to align the raw sequencing data and identify specific genetic
alterations3
• TMB is reported as either a whole number, when measured by whole-exome
sequencing (WES), or as mutations/megabase (Mb), when measured by
comprehensive genomic profiling (CGP) on a targeted panel of genes3
• Retrospective analyses are investigating the role of TMB as a potential predictive
biomarker for checkpoint inhibitors4-7

1. Champiat S OncoImmunology 2. OncoImmunology. 2014;3(1). 3. Vanderwalde A Cancer Med. 2018;7(3):746-756. 4.Yarchoan M 2017 NEJM 5. Cristescu, R ASCO-SITC 2017 6. Ott PA ESMO 2017 7. Hellman 2018 NEJM
 TMB as a Potential Predictive Biomarker for PD-1/PD-L1 Inhibitors1-2

1. Rizvi Science 2015 2. Snyder NEJM 2014

 Correlation of TMB and ORR with Anti-PD-1/Anti-PD-L1 Across Tumors1
• After an extensive literature search, the ORR for anti–PD-1 or anti–PD-L1
therapies against the corresponding median TMB across 27 tumor types
was plotted

• For each tumor type, the response data was pooled from the largest
published studies that evaluated the ORR

• Only studies of anti–PD-1 or anti–PD-L1 monotherapy that enrolled at

least 10 patients regardless of PD-L1 expression

• The median TMB for each tumor type was obtained from a validated
comprehensive genomic profiling assay performed and provided by
Foundation Medicine.

1. Yarchoan et al. 2017 NEJM

Correlation of TMB and ORR with Anti-PD-1/Anti-PD-L1 Across Tumors1

1. Yarchoan et al. 2017 NEJM

Potential Application of TMB
• TMB is an emerging biomarker that has been shown to be correlated with
improved outcomes in patients treated with anti-PD-1 therapies, but has
yet to be clinically validated in a prospective study to show a correlation
with overall survival.

• In the NSCLC monotherapy setting, MSD continues to pursue our PD-L1-

based patient selection strategy while also investigating the relevance and
utility of TMB as a potential diagnostic for patient selection.

• In addition, MSD continues to explore TMB across other select

Pembrolizumab indications, evaluating TMB prospectively where it may be
believed to provide value