Examples of biomarker — Keytruda PD-L1 / MSI-H / dMMR

Non-Small-Cell Lung Cancer (NCT01295827) — PD-L1 BM Define / Validation

indication	stage	group / size	assay	analysis
Non-Small-Cell Lung Cancer important! NCT01295827.)	BM define	training group, n=182	prototype assay	Initial Correlation analysis (→ 2014 Gandhi: 50% (potential) cut point 써 보니까, RR 높고, & differentiated responders from nonresponders in ORR)

PD-L1 Tumor Expression	N for irRC	ORR by irRC, n (%), [95% CI]	N for RECIST	ORR by RECIST (%), [95% CI]	PFS rate* at 6 months (95% CI)	PFS Statistics	OS rate^ at 6 months (95% CI)	OS Statistics
High (score ≥ potential cut point)	9	6 (67%), [30%, 93%]	7	4 (57%), [18%, 90%]	67% (42%-100%)	Median: not reached	89% (71%-100%)	Median: not reached
Low (score < potential cut point)	22	0 (0%), [0%, 15%]	20	1 (5%),# [0%, 25%]	11% (3%-40%)	Median: 2.1 months	33% (18%-62%)	Median: 3.9 months
HR (95% CI) for high vs low PD-L1 expression	-	-	-	-	0.22 (0.07-0.67)		0.32 (0.10-0.99)
One-sided P value (PD-L1 association test)		<0.001 (Fisher's exact test)	-	-	0.009 (Fisher's exact test)	0.004 (Cox regression)	-	0.024 (Cox regression)
*Based on investigators' irRC assessments.

50%이상인 환자는 위 표 보면 9 명 즉 29% 이네.

BM cut point defined (protocol says Youden Index): PD-L1 expression in at least 50% of tumor cells was selected as the cutoff from the training group., using a ROC curve (sensitivy 와 specificity 가 max 되는 point 일 것임 & the ease of use), 아래 그림에서 대략 AUC 가 0.7 정도 되어보이네. 아래는 PD-L1 과 (irRC-based) ORR 과의 관계임

correlation with severity/progression?

• Preclinical validation?: (Guo 2020) a previous study showed that blockade of the PD-1/PD-L1 pathway using PD-L1 antibody could inhibit T-cell apoptosis (25). This study also showed that PD-L1 antibody affected the survival of tumor cells in vivo (25).
• Disease impact? (ie natural Hx without using keytruda) :

source	indication	Tx	PD-L1	Results
Keytruda label. P58	NSCLC	Docetaxel	50%이상 vs 1-100%	OS, PFS 다 두 군간 차이 X
Keytruda label. P62	NSCLC	ChemoTx	50%이상 vs 1-100%	OS, PFS 다 두 군간 차이 X
Keytruda label. P67	hnscc	Cetuximab platinum FU	CPS 1이상 VS 20이상	OS, PFS 다 두 군간 차이 X

• 약쓰면 % ↓?: {Lee, 2019 #1881} nivolumab → ↓serum PD-L1 (cf serum PD-L1 correlates with tissue (liver) PD-L1 with R2=0.43)

What is downstream?
In another study, the median sPD-L1 levels were 500 pg/mL (range, 30-6,040 pg/mL) in patients with HCC and 780 pg/mL (range, 168-1,521 pg/mL) in healthy controls.1

Protocol amendment to add a coprimary endpoint to evaluate the efficacy in patients who expressed a high level of PD-L1 (ie. 50% PD-L1 아님)
위에서 도출된 아래의 가설을 검증함:

• 가설 1: 이 군에서 PD-L1 >50% 의 prevalence 는 50%일것이다.
• 가설 2: 이 군에서 RR >30% 일 것이다 (→ 결과적으로 45.2% 보임, cf. all group: 19.4%)

indication	stage	group / size	analysis
	BM validation	validation group, n=313)	see Protocol amendment block above
	2014 Gandhi 중요		A separate analysis (: from KEYNOTE-001) of patients with NSCLC treated with MK-3475 showed that PD-L1 expression in at least 50% of tumor cells predicted response to MK-3475. These findings were based on analysis of a training set of 146 NSCLC patients from the ongoing KEYNOTE-001 phase I study and were reported by Leena Gandhi, MD, PhD, of Dana-Farber Cancer Center, Boston. Using the 50% cutoff point, about 25% of patients were strongly positive for PD-L1 expression. The overall response rate for both PD-L1–positive and –negative groups was 19%. However, strong positivity clearly differentiated responders from nonresponders, Dr. Gandhi said. Median progression-free survival was 14.1 weeks in the PD-L1–positive patients vs 9.3 weeks in the weakly positive (1%–49% cutoff) and negative patients. Overall survival was 9.3 vs 7.3 months, respectively, which was not statistically significant.
	임상아니고 Natural Hx study		NSCLC, {Sun, 2016 #1293} high PD-L1 expression was correlated with severity (stage 3b & 4, ie crosssectional) and ↓ overall survival (ie longitudinal).
			NSCLC, {Sorensen, 2016 #1294} HIGH PD-L1 expression was correlated only with severity , but not with OS

Keytruda with MSI-H or dMMR

Indication

• colorectal cancer.,or (any) solid tumors
• MSI-H () or
  • Determined by PCR
• mismatch repair deficient (dMMR)
  • Determined by IHC

MSI-H/dMMR can be sporadic or hereditary. Of the estimated 15% of patients with colorectal cancer who have MSI-H, about 83% have sporadic disease and only about 17% have Lynch syndrome.1
PCR for MSI and IHC for MMR are different assays measuring the same biological effect.

PCR = polymerase chain reaction; IHC = immunohistochemistry; MSS = microsatellite stable; MSI-L = mic rosatellite instability-low.

MSI / MMR Eligibility Diagram

MSS No microsatellite markers exhibit instability	MSI-L Instability shown in 1, or 1% to 29%, of the microsatellite markers	MSI-H Instability shown in ≥2, or ≥30%, of the microsatellite markers	MSI PCR test results16,17
↓ Normal PCR		↓ ELIGIBLE FOR KEYTRUDA

Proficient MMR Intact expression of all MMR proteins is detected	Loss of MLH1 or Loss of MSH2 or Loss of MSH6 or Loss of PMS2	MMR IHC test results16,17
↓ Normal IHC	↓ ELIGIBLE FOR KEYTRUDA

Definitions

Microsatellite instability (MSI)

• is the condition of genetic hypermutability (predisposition to mutation) that results from impaired DNA mismatch repair (MMR). The presence of MSI represents phenotypic evidence that MMR is not functioning normally.
• MSI occurs in about 15% of CRC

Mismatch repair (MMR)

• refers to deficiency in proteins responsible for DNA MMR: MSH2, MSH6, MLH1, PMS2.
• MMR usually occurs due to mutations that code for the above genes
• MMR deficiency leads to the MSI-H phenotype.

Microsatellite

• repeated sequences of DNA. These sequences can be made of repeating units of one to six base pairs in length. Although the length of these microsatellites is highly variable from person to person and contributes to the individual DNA “fingerprint”, each individual has microsatellites of a set length. The most common microsatellite in humans is a dinucleotide repeat of the nucleotides C and A, which occurs tens of thousands of times across the genome. Microsatellites are also known as simple sequence repeats (SSRs).

Mechanism

normally functioning MMR→ unable to correct errors that occur during DNA replication → accumulate errors in DNA microsatellites (short repetitive sequences in DNA; ref. 3)→ microsatellite instability (MSI) → the creation of novel microsatellite fragments → predisposition to mutation (high mutation) → ↑antigen presentation by tumor→ autologous immune recognition of cancer cells → potentially become more susceptible to immunotherapy than tumors with few mutations (4–6).

Clinical validation

KEYNOTE (KN) 016

MSI-H Tumor Phenotype Associated with Efficacy in Colorectal and Non-Colorectal Patients Treated with Pembrolizumab

Phase 2 Study of MK-3475 in Patients With Microsatellite Unstable (MSI) Tumors
Initiated in 2013, sponsored by Johns Hopkins- Sidney Kimmel Comprehensive Cancer Center in collaboration with MSD

Uncertain Spans

location	transcription	uncertainty
PD-L1 table Low row RECIST cell	1 (5%),# [0%, 25%]	The # between (5%) and [0%, 25%] is visible in the source; appears to be a footnote marker but the referenced footnote is not visible on this photo.
ROC plot legend	PS, P2S, P3S, HS	Legend text is small; reading is consistent across tile crops but the curve labels could correspond to alternative cohort acronyms.
Keytruda label P67 PD-L1 column	CPS 1이상 VS 20이상	The mid-cell separator VS is uppercase here while other rows use lowercase vs; transcribed as written.
MSI-L definition typo	mic rosatellite instability-low	The footnote line shows a visible space inside microsatellite (mic rosatellite), apparently a layout artifact; transcribed as written.