Robert Anders, MD, PhD, Robert L. Ferris, MD, PhD, and Lauren L. Ritterhouse, MD, PhD, prepared useful Practice Aids pertaining to biomarker testing for this CME/MOC activity titled "The Central Role of Biomarker Testing in Piecing Together the Immuno-Oncology Puzzle: Essential Guidance for Pathologists to Maximize the Potential of Cancer Immunotherapies." For the full presentation, monograph, complete CME/MOC information, and to apply for credit, please visit us at http://bit.ly/2QAudYX. CME/MOC credit will be available until December 26, 2019.

1. CRC: colorectal cancer; CSCC: cutaneous squamous cell carcinoma; dMMR: mismatch repair deficient; HCC: hepatocellular carcinoma; HNSCC: head and neck squamous cell carcinoma; MCC: merkel cell carcinoma;
MSI-H: microsatellite instability high; NS: nonsquamous; NSCLC: non–small-cell lung cancer; PD-L1: programmed death-ligand 1; pembro: pembrolizumab; RCC: renal cell carcinoma; S: squamous; SCLC: small-cell lung cancer.
1. https://www.fda.gov/drugs/informationondrugs/approveddrugs/ucm279174.htm. Accessed August 23, 2018.
IMMUNE CHECKPOINT INHIBITOR LANDSCAPE: SOLID TUMORS1
PRACTICE AID
Access the activity, “The Central Role of Biomarker Testing in Piecing Together the Immuno-Oncology Puzzle: Essential Guidance
for Pathologists to Maximize the Potential of Cancer Immunotherapies,” at www.peerview.com/UZM40.
This Practice Aid has been provided as a quick reference to help learners apply the information to their daily practice.
Melanoma
RCC
Bladder
HNSCC
CRC
Mar. 2011 Sept. 2014 Dec. 2014 Oct. 2015 Dec. 2015 Dec. 2017
Ipilimumab Pembrolizumab Nivolumab
Nivolumab + ipilimumab (1st line)
Ipilimumab (adjuvant)
Pembrolizumab
(1st line)
Nivolumab
(adjuvant)
May 2016 Feb. 2017 May 2017
Atezolizumab Nivolumab
Durvalumab
Avelumab
Pembrolizumab
Mar. 2017 May 2017 Sept. 2017
Avelumab
(MCC)
Pembrolizumab
(dMMR/
MSI-H cancers)
Pembrolizumab (gastric)
Nivolumab (HCC, 2nd line)
Jun. 2018
Pembrolizumab
(PD-L1+ cervical)Other
Aug. 2016 Nov. 2016
Pembrolizumab Nivolumab
Aug. 2017 Jul. 2018
Nivolumab
(dMMR/MSI-H)
Nivolumab + ipilimumab
(dMMR/MSI-H)
Nov. 2015 Apr. 2018
Nivolumab
Nivolumab + ipilimumab
(1st line)
Lung
Mar. 2015 Oct. 2015 Oct. 2016 May 2017 Feb. 2018 Aug. 2018
Nivolumab (S)
Nivolumab (NS)
Pembrolizumab
(PD-L1+ NSCLC)
Pembrolizumab
(PD-L1+ NSCLC, 1st line)
Atezolizumab (NSCLC)
Pembrolizumab +
chemo
(NS-NSCLC, 1st line)
Durvalumab
(stage III
NSCLC)
Nivolumab (SCLC, 3rd line)
Pembrolizumab + pemetrexed
+ platinum (NS-NSCLC)
May 2017
Pembrolizumab
(dMMR/MSI-H)
Sept. 2018
Cemiplimab
(CSCC)
Oct. 2018
Pembrolizumab + carboplatin +
paclitaxel or
nab-paclitaxel (S-NSCLC)
Atezolizumab +
bevacizumab +
chemo (NS-NSCLC)
Dec. 2018
Nov. 2018
Pembrolizumab
(HCC, 2nd line)

2. NSCLC: non–small-cell lung cancer; PD-L1: Programmed death-ligand 1; TMB: tumor mutational burden.
1. Hellmann MD et al. N Engl J Med. 2018. doi:10.1056/NEJMoa1801946. 2. Carbone DP et al. N Engl J Med. 2017:376;2415-2426. 3. Hellmann MD et al. Cancer Cell. 2018. doi: https://doi.org/10.1016/j.ccell.2018.03.018. Epub. 4. Ramalingam S et al. American Association
for Cancer Research Annual Meeting 2018 (AACR 2018). Abstract CT078. 5. Snyder A et al. N Engl J Med. 2014;371:2189-2199. 6. Rizvi NA et al. Science. 2015;348:124-128. 7. Le DT et al. N Engl J Med. 2015;372:2509-2520. 8. Van Allen EM et al. Science. 2015;350:207-211.
9. Hugo W et al. Cell. 2016;165:35-44. 10. Yarchoan M et al. N Engl J Med. 2017;377:2500-2501.
TUMOR MUTATIONAL BURDEN (TMB)
An Emerging Immuno-Oncology Biomarker 1-10
PRACTICE AID
Access the activity, “The Central Role of Biomarker Testing in Piecing Together the Immuno-Oncology Puzzle: Essential Guidance
for Pathologists to Maximize the Potential of Cancer Immunotherapies,” at www.peerview.com/UZM40.
This Practice Aid has been provided as a quick reference to help learners apply the information to their daily practice.
WHY
TEST
FOR TMB
HOW TO
TEST
FOR TMB
Mutations
Neoantigens
Immune
system
recognition
of tumor
TMB levels
vary in different
types of tumors
High TMB cancers
Lung Skin Bladder
But a large % of many other cancers
found to be TMB-high also →
opportunity to expand indications
for immunotherapies
Represents an emerging
immuno-oncology biomarker
Responses to immunotherapies
correlate with TMB across many tumors
Validated in CheckMate -227 as
independent biomarker in 1st-line
setting of metastatic NSCLC
Predictive, not prognostic
Complementary to PD-L1 expression—
identifies a distinct and independent
population of patients
TMB = Collective number of mutations within the coding region
of a tumor’s genome (exome)
Usually thought of as the burden of non synonymous
mutations in an exome
Type of mutations counted usually is missense
Often measured/reported as mutations per megabase (mut/Mb)
Thresholds for high vs low TMB are still in flux and depend on assay
and histology (various labs/groups may calculate it differently and
include different kinds of mutations)
TMB-high cut-point in CheckMate -227: ≥10 mut/Mb
Testing can be carried out via:
Tissue biopsy-based assessments
Whole exome sequencing
Targeted comprehensive genomic profiling
(more feasible option for
routine use in practice)
Liquid biopsy-based assessments

3. CRC: colorectal cancer; dMMR: deficient mismatch repair; IHC: immunohistochemistry; MMR: mismatch repair; MSI: microsatellite instability; MSI-H: microsatellite instability high; MSI-L: microsatellite
instability low; MSS: microsatellite stable; NGS: next-generation sequencing; PCR: polymerase chain reaction.
1. Umar A et al. J Natl Cancer Inst. 2004;96:261-268. 2. Hematology/Oncology (Cancer) Approvals and Safety Notifications. Available at: https://www.fda.gov/drugs/informationondrugs/approveddrugs/
ucm279174.htm.
This Practice Aid has been provided as a quick reference to help learners apply the information to their daily practice.
Access the activity, “The Central Role of Biomarker Testing in Piecing Together the Immuno-Oncology
Puzzle: Essential Guidance for Pathologists to Maximize the Potential of Cancer Immunotherapies,”
at www.peerview.com/UZM40.
MSI/MMR STATUS AS A KEY
IMMUNO-ONCOLOGY BIOMARKER
Testing and Treatment Options
PRACTICE AID
HOW
TO TEST
FOR
MSI-H/dMMR
STATUS
HOW
TO TREAT
PATIENTS WITH
MSI-H/dMMR
TUMORS
FDA-Approved Immune Checkpoint Inhibitors for Treatment
of Patients With MSI-H or dMMR Tumors2
Nivolumab:
Approved 07/31/17
Patients ≥12 years old with MSI-H or dMMR metastatic CRC that
have progressed following treatment with a fluoropyrimidine,
oxaliplatin, and irinotecan
Pembrolizumab:
Approved 05/23/17
Adult and pediatric patients with:
Unresectable/metastatic MSI-H or dMMR solid tumors that have
progressed following prior treatment and who have no satisfactory
alternative treatment options
MSI-H or dMMR CRC that has progressed following treatment with
a fluoropyrimidine, oxaliplatin, and irinotecan
Nivolumab +
ipilimumab:
Approved 07/10/18
Patients ≥12 years old with MSI-H or dMMR metastatic CRC that
have progressed following treatment with a fluoropyrimidine,
oxaliplatin, and irinotecan
Options:
MSI status: PCR
MMR status: IHC
Alternative method: NGS
MMR Testing by IHC
MLH1/PMS2/MSH2/MSH6
MSI Testing by PCR
Revised Bethesda Guidelines:1
5 microsatellites tested:
0 unstable à MSS
1 unstable à MSI-Low
≥2 unstable à MSI-High
Original NCI MSI panel included BAT25, BAT26, D2S123,
D5S346, D17S250:
Only dinucleotide repeats are mutated à test secondary
panel of microsatellite markers with mononucleotide repeats
(BAT40 and/or MYCL) to exclude MSI-L
Dinucleotide repeats less sensitive than mononucleotide
repeats for MSI-H, but provide an internal control for sample
mix-up prevention
Pentaplex panel of 5 quasimonomorphic mononucleotide
repeats may be more sensitive for MSI-H tumors than other
markers; requires ≥3 mutant alleles to indicate MSI-H
MSH2
MLH1
MSH6
PMS2
MSI-H à Lynch syndrome
MSI-L and MSS à
Clinicopathologically similar
MSI-L à Not associated with Lynch

4. a
Non–small-cell lung cancer and melanoma. b
Non–small-cell lung cancer, gastric cancer, cervical cancer, and bladder cancer. c
Bladder cancer.
IHC: immunohistochemistry; PD-1: programmed cell death protein 1; PD-L1: programmed death ligand 1.
TESTING FOR PD-L1 EXPRESSION AS A KEY IMMUNO-ONCOLOGY BIOMARKER
Complementary and Companion Diagnostics
PRACTICE AID
Access the activity, “The Central Role of Biomarker Testing in Piecing Together the Immuno-Oncology Puzzle: Essential Guidance
for Pathologists to Maximize the Potential of Cancer Immunotherapies,” at www.peerview.com/UZM40.
This Practice Aid has been provided as a quick reference to help learners apply the information to their daily practice.
COMPANION
vs
COMPLEMENTARY
DIAGNOSTICS
Companion
Result is required for prescription of drug
• Specified on drug label
• Category typically only required when the test is
among inclusion criteria for the trial (except gastric
and bladder)
IMMUNE
CHECKPOINT
INHIBITORS &
ASSOCIATED
PD-L1 ASSAYS
Complementary
Test result is predictive, but not required for
prescription of drug
• Nice to have but do not need to have
• Category mostly used when the assay is integrated
into the trial, but not used among inclusion criteria
Nivolumab Pembrolizumab Atezolizumab Durvalumab Avelumab
Target PD-1 PD-1 PD-L1 PD-L1 PD-L1
Antibody
clone/epitope
28-8 extracellular 22C3 extracellular
SP142
cytoplasmic
SP263
cytoplasmic
73-10
cytoplasmic
IVD class III
diagnostic
partner
Dako Dako Ventana Ventana Dako
Diagnostic
Complementarya
:
PD-L1 IHC 28-8
pharmDx test
Companionb
:
PD-L1 IHC 22C3
pharmDx test
Companionc
:
Ventana PD-L1
(SP142) assay
Complementaryc
:
Ventana PD-L1
(SP263) assay
Unknown
ü
X