Lung Cancer Diagnosis: Role of Pathology and Genetic ......1. Increased PD-L1 expression is associated with higher ORR 2. Negative PD-L1 expression does not preclude response 3. ORR

ESMO PRECEPTORSHIP ONLung Cancer Diagnosis:

Role of Pathology and Genetic Analysis

Teh-Ying Chou, MD, PhD, MBA

November 20, 2019

DISCLOSURES

I have nothing to disclose.

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3

Lung Cancer: Pathological Classification

20041999 2015

Histology

Targeted Therapy

Immunotherapy

Molecular Pathology-based Precision Medicine of Lung Cancer

Precision Medicine

Molecular Biomarkers

4

Lung Cancer: Molecular Classification

Carcinoma (CA)

Non-small Cell CA (NSCC)

Small Cell CA

Squamous Cell CA

Non-squamous Cell NSCC

Large Cell CA

Adenocarcinoma

Other NSCC

Large Cell Neuroendocrine CA

Other Large Cell CA

Typical Carcinoid

Atypical Carcinoid

Neuroendocrine CA

Molecular Analysis

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Lung Cancer: Molecular Diagnostics

EGFRoma, ALKoma…

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Lung adenocarcinoma: Molecular profile

7

Tsao et al. J Thorac Oncol. 2016 May;11(5):613-38.

8

Recommended biomarker tests for patients

with newly diagnosed NSCLC

� EGFR

� ALK

� ROS1

� BRAF

� PD-L1

� RET

� MET exon 14

� HER2

� KRAS

� NTRK

Am Soc Clin Oncol Educ Book. 2019 Jan;39:531-542.

Minimum necessaryRecommended

(for NGS panels)

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EGFR mutation in lung cancer

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Test mutations in EGFR exon 18-21 with ≧1% prevalence(codons 709, 719, exon 19 del, 768, exon 20 insertions, 790, 858, and 861)

TechniqueSensitivity

(% t DNA)Mutations identified

Comprehensive detection

of deletions and insertions

PCR/Direct sequencing 25 Known and new Yes

PCR-SSCP

(ss conformation polymorphism)10 Known and new Yes

PCR-RFLP and length analysis 5 Known only No

MALDI-TOF MS-based genotyping 5 Known only No

Real-time PCR based technology

•PNA-LNA PCR clamp 1 Known only No

•Scorpion/Arms 1 Known only No

•TaqMan PCR 10 Known only No

Mutant-enriched PCR 0.2 Known only No

Next generation sequencing 1 Known and new Yes

Modified from Pao et al. Clin Cancer Res 200711

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Mechanisms of acquired resistance to

1st and 2nd generation EGFR-TKIs

Ann Oncol. 2018 Jan 1;29(suppl_1):i10-i19

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EGFR testing at the time of recurrence

Piotrowska Z, et al. Cancer J. 2015

1st biopsy and EGFR testing

2nd biopsy and EGFR testing

3rd biopsy and EGFR testing

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Mechanisms of acquired resistance to first-

line Osimertinib therapy

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16

ALK rearrangement in lung cancer

Chromosome 2

1. Expression of ALK fusion protein

2. Dimerization of ALK

3. Constitutive activation of ALK

Shaw AT at al. Clin Cancer Res. 2011Soda M et al. Nature. 2007

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Testing for ALK rearrangement

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ALK testing by break apart FISH

Thunnissen E. et al. Virchows Arch. 2012

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ALK break apart FISH

ALK (-) ALK (+)

Cheng L. et al. Modern Pathology 2012

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� Normal lung tissue does not express ALK

� ALK fusion (+) lung cancer express ALK fusion protein

ALK Fusion: Immunohistochemistry

Adapted from IASLC Atlas of ALK Testing, 2013

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� Recommended IHC assay: D5F3 or 5A4 clone

� Pooled estimates of IHC relative to FISH

� 97% sensitivity

� 99% specificity

� Discrepant FISH and IHC results

� No consistent pattern of clinical outcome

ALK rearrangement: IHC vs. FISH

Arch Pathol Lab Med.

2018 Mar;142(3):321-346.

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Kohno et al. Transl Lung Cancer Res. 2015 Apr;4(2):156-64.

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ROS1

• Receptor tyrosine kinase (RTK) of the insulin receptor family,

closely related to ALK

(a) Glycoprotein-rich extracellular domain

(b) Transmembrane domain

(c) Intracellular tyrosine kinase

ROS1 gene: Chromosome 6q22

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Leads to constitutive

kinase activity

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Detection of ROS1 rearrangement

� Fluorescence in-situ hybridization (FISH)

� Reverse transcription polymerase chain reaction (RT-PCR)

� Immunohistochemistry (IHC)

� Next generation sequencing (NGS)

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Detection by FISH

Negative for ROS1

rearrangement

Positive for ROS1

rearrangement

(split signal in

>15% of cells)

3’ 5’

Positive for ROS1

rearrangement

(isolated 3’ signal

in >15% of cells)

Detect known and unknown fusion variants

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� Anti-ROS1 clone D4D6 (Cell Signaling)

� Anti-ROS1 clone SP384 (Ventana)

Detection by IHC

ROS1 FISH (+), IHC (+) ROS1 FISH (-), IHC (-)

Mod Pathol. 2014 May;27(5):711-20

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ROS1 IHC staining pitfalls

Macrophages/Giant cells Reactive pneumocytes

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� Pooled estimates of IHC (≧2+) relative to FISH� 96% sensitivity

� 94% specificity

� Up to 1/3 ROS1 FISH (-) cases show focal or weak ROS1

expression by IHC

ROS1: IHC vs. FISH

Arch Pathol Lab Med. 2018 Mar;142(3):321-346.

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CAP/IASLC/AMP molecular test guideline:

ROS1 testing in lung cancer

� ROS1 IHC may be used as a screening test

� Positive ROS1 IHC results should be confirmed by a

molecular or cytogenetic method

� Each laboratory must validate its own interpretive cutoff

from known positive and negative samples

Arch Pathol Lab Med. 2018 Mar;142(3):321-346.

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ROS1: IHC vs. FISH (VGH-TPE data)

ROS1 IHC

(SP384)

ROS1 FISH/NGS

Negative Positive Total

0 125 0 125

1+ 41 0 41

2+ 5 1 6

3+ 3 6 9

Total 174 7 181

0: H-Score = 0

1+: H-Score = 1~100

2+: H-Score = 101~200

3+: H-Score = 201~300

ROS1 IHC

No stain or

Weak / Focal stain

Moderate to Strong

Diffuse stain

ROS1 FISH

Negative PositiveNegative

VGH-TPE

approach

33

34


Kohno et al. Transl Lung Cancer Res. 2015 Apr;4(2):156-64.

http://www.apmggroup.net/innovation/molecular_testing/Colon_Pathways/colon.html35

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BRAF mutation in lung cancer

By Sholl LM, from BWH/DFCI data (~2500 NSCLC sequenced)

37

Dabrafenib plus trametinib in

BRAF-V600E mutant NSCLC (1st line)

Lancet Oncol. 2017 Oct;18(10):1307-1316.

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Characteristics of patients with

BRAF-V600E mutated lung tumors

Lancet Oncol. 2017 Oct;18(10):1307-1316.

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BRAF V600E IHC testing (clone VE1)

BRAF-V600E

mutated

tumor

BRAF

non-V600E

mutated

tumor

Ann Oncol. 2013 Mar;24(3):742-8.

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BRAF V600E IHC testing (clone VE1)

Study Sensitivity Specificity

Ilie et al.

Ann Oncol 2013

90.5%

(19/21)

100%

(19/19)

Sasaki et al.

Lung Cancer 2013

100%

(5/5)

95.2%

(20/21)

Gow et al.

Cancers 2019

96.6%

(28/29)

98.6%

(69/70)

Total94.5%

(52/55)

98.2%

(108/110)

Cancers (Basel). 2019 Jun 21;11(6).

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� EGFR

� ALK

� ROS1

� BRAF

� PD-L1

� RET

� MET exon 14

� HER2

� KRAS

� NTRK



(for NGS panels)

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Key elements in biomarker

development for checkpoint inhibition

Nishino M. et al. Nat Rev Clin Oncol. 2017 Nov;14(11):655-668.

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Cells with PD-L1 expression

� Tumor cells

� Immune cells

� Lymphocytes (T cells, B cells)

� Dendritic cells

� Macrophages

� Stromal cells

� Certain types of normal epithelial cells

Analyzed by

Immuno-

histochemistry

(IHC)

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PD-L1 expression in tumor cells (TC)

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PD-L1 expression in immune cells (IC)

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Key points:

1. Increased PD-L1 expression is associated with higher ORR

2. Negative PD-L1 expression does not preclude response

3. ORR in high PD-L1 expression patients still below 50%Cancer Biol Med. 2016 Jun;13(2):157-70

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Confounders in PD-L1 expression IHC testing

� Multiple different assays and cut-off values

� Inter-observer variability

� Heterogeneous expression of PD-L1

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NivolumabPembro-

lizumabAtezolizumab

Durva-

lumabAvelumab

Detection

antibody28-8 22C3 SP142 SP263 73-10

IHC platform Dako Dako Ventana Ventana Dako

Epitope

locationExtra-cellular Extracellular Intracellular

Intra-

cellular

Intra-

cellular

Cell types

evaluatedTC TC TC + IC TC TC

Cut-offs in

clinical trials1, 5, 50%

1%

50% (High)

TC: 1, 5, 50%

IC: 1, 5, 10%25% 1%

FDA

diagnostic

status

Complementary

(Not required)

Companion

(Required)

Complementary

(Not required)

PD-L1 testing: at least 5 different assays

TC: tumor cells; IC: immune cells

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Blueprint project (phase 2):

PD-L1 IHC comparability study

Tsao MS. et al. J Thorac Oncol. 2018 May 22. pii: S1556-0864(18)30626-9.

Tumor staining:

SP142: Lower

73-10: Higher

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Interobserver variability (for TC scoring)

in Blueprint phase 2

Presented by Tsao MS at WCLC 2017

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Interobserver variability (for IC scoring)

in Blueprint phase 2


Presented by Tsao MS at WCLC 2017

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Tumor heterogeneity

� Intra-tumoral heterogeneity

� PD-L1 expression is frequently heterogeneous within a tumor

� Sampling error

� Inter-tumoral heterogeneity

� Differences between primary and metastasis?

Fusi A et al. Lancet Oncology 2015; 16(13),1285-1287

PD-L1 Tumor Heterogeneity

McLaughlin et al, JAMA Oncol., 2016.

H&E PD-L1 (SP142)

PD-L1 Tumor Heterogeneity

57PD-L1 (SP142)

PD-L1 TPS > 50%

PD-L1 TPS < 1%

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Discordance of PD-L1 expression between paired

biopsy and resection specimens

Ilie M et al. Ann Oncol. 2016 Jan;27(1):147-53.

Overall concordance rate = 52%

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Primary tumor vs. Metastasis

Concordance: 80.1% (1% cutoff), 90.7% (50% cutoff)

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PD-L1 expression as a biomarker

� Imperfect & Weak predictive power

� Multiple different assays and cut-off values

� 22C3/28-8/SP263 are highly analytically comparable

� Interobserver agreement

� Good for tumor cells, Bad for immune cells

� Challenges

� Heterogeneous expression

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� EGFR

� ALK

� ROS1

� BRAF

� PD-L1

� RET

� MET exon 14

� HER2

� KRAS

� NTRK



(for NGS panels)

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Cocco E, Scaltriti M & Drilon A, Nature Reviews Clinical Oncol 201864

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Journal of Thoracic Oncology 2019 14, DOI: (10.1016/j.jtho.2019.08.051) 70

Documents

Lung Cancer Diagnosis: Role of Pathology and Genetic ......1. Increased PD-L1 expression is associated with higher ORR 2. Negative PD-L1 expression does not preclude response 3. ORR