The molecular pathologist’s view: how to satisfy increasing demand for multiple biomarker testing from limited sample size? Luca Quagliata Institute for Medical Genetics and Pathology University Hospital of Basel, Switzerland

Disclosure The data presented here are the sole property of the author and his institution. The idea and concepts proposed are based on his experience and are not influenced by any means by Novartis or Thermo Fisher Scientific. Novartis covered conference registration, travel, accommodation, and honorarium expenses for the speaker.

Disclosure – Year 2017 (research support, travel sponsor, honorarium)

NSCLC molecular subtypes Extensive genomics studies resulted in clinically relevant findings

Tsao AS, et al. J Thorac Oncol. 2016;11:613-38.

Unknown oncogenic

driver detected

31%

KRAS 25%

EGFR-sensitizing

17%

ALK 7%

EGFR other 4%

MET 3%

HER2 2%

ROS1 2%

BRAF 2%

RET 2%

NTRK1 1%

PIK3CA 1%

MEK1 1%

ALK, anaplastic lymphoma kinase; BRAF, B-Raf proto-oncogene; EGFR, epidermal growth factor receptor; HER2, human epidermal growth factor receptor 2; MEK1, mitogen-activated protein kinase 1; MET, mesenchymal-epidermal transition; NTRK1, neurotrophic tyrosine kinase receptor 1; RET, rearranged during transfection; PIK3CA, phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha; ROS1, ROS1 proto-oncogene receptor tyrosine kinase.

Key 1 - Phase 1 2 - Phase 2 3 - Phase 3 4 - Approved

EGFR-sensitizing • Gefitinib4

• Erlotinib4

• Afatinib4

• Osimertinib4

ALK • Crizotinib4

• Alectinib4

• Ceritinib4

• Lorlatinib2

• Brigatinib2

MET • Crizotinib2

• Cabozantinib2

HER2 • Trastuzumab

• Afatinib2

• Dacomitinib2

ROS1 • Crizotinib4

• Cabozantinib2

• Ceritinib2

• Lorlatinib2

• DS-6051b1

BRAF • Vemurafenib2

• Dabrafenib2

RET • Cabozatinib2

• Alectinib2

• Apatinib2 • Vandetanib2

• Ponatinib2

• Lenvatinib2

NTRK1 • Entrectinib2

• LOXO-1012

• Cabozantinib2

PIK3CA • LY30234142

• PQR 3091

MEK1 • Trametinib2

• Selumetinib3

• Cobimetinib1

NCCN testing guidelines A pressing demand for comprehensive molecular diagnostics

National Comprehensive Cancer Network. Non-Small Cell Lung Cancer Guidelines (Version 8.2017). Available from. https://www.nccn.org/professionals/physician_gls/PDF/nscl.pdf. Accessed September 2017. NCCN, National Comprehensive Cancer Network.

Molecular pathology has faced a sustained growth Historical trend of analysed samples (Basel)

CAGR, compound annual growth rate.

0

500

1,000

1,500

2010 2011 2012 2013 2014 2015 2016

Sam

ples

“Investments made within the Molecular Unit turned into a constant and substantial growth over the past years with an overall CAGR of 10.42%”

n = 750 n = 887 n = 907

n = 980

n = 1,156 n = 1,255

n = 1,501

Property of the University Hospital of Basel.

Examples of analysed sample types Heterogeneous source of starting material

Property of the University Hospital of Basel.

Tissue punch

Biopsy Resected Cytology Biopsy

Laser microdissection Liquid Biopsy

How to proceed with molecular testing – 2014 Swiss Lung Pathology Group recommendations (2014)

AC, adenocarcinoma; Amp, amplification; FISH, fluorescence in situ hybridization; KRAS, Kirsten rat sarcoma viral oncogene homologue; PD-L1, programmed death ligand 1; TAT, turn-around time. Courtesy of Prof Lukas Bubendorf - University Hospital of Basel

If negative

ALK FISH

ROS FISH If negative

RET FISH If negative

If positive If positive

Desirable TAT 1 day

Desirable TAT 10 days

AC/AC phenotype Stage IIIB/IV

Immunohistochemistry

ALK ROS1

Mutation analysis covering at least: EGFR KRAS HER2 BRAF – e.g. Sanger combination

Additional tests • Resistance mutations • PD-L1 IHC • EGFR IHC • MET Amp • METex14 Effective but

too complex!

How to proceed with molecular testing – 2017 Internal guidelines at the USB

Desirable TAT 1 day

Desirable TAT 5 days

AC/AC phenotype Stage IIIB/IV

Immunohistochemistry

ALK ROS1 PD-L1 MET

Mutation analysis covering: Oncomine™ Solid Tumour DNA kit

(EGFR KRAS HER2 BRAF)

Oncomine™ Solid Tumour Fusion panel (ALK ROS1 RET NTKR1)

If negative Additional tests: MET Amp METex14

If negative

Courtesy of Prof Lukas Bubendorf - University Hospital of Basel USB: University Hospital of Basel

Unidirectional, simplified and very effective!

Technological breakthrough in molecular pathology NGS is changing routine practice in pathology

Next generation sequencing (NGS) evolution NGS is a kind of revolution

Sanger Few base pairs A few hotspots One exone

One gene Whole exome Entire genome NGS

NGS workflow overview: an established path Is that compatible with our work?

QC, quality control. Modified from Quagliata L, et al. 2017, Schweizer Krebsbulletin, Nr. 1/2017, ISSN 2297-0703 . From the initial sample to the final report within 5-7 working days

Sample sources

DNA/RNA extraction Sequencing Data

generation Report

Small tissue biopsy

Cytology specimen

Liquid biopsy

Tumour-specific panel of genes

Library preparation

DNA/RNA extraction

Sequencing platform preparation

0% No template 33,811,202 100%

Enrichment

10% Empty wells 33,811,833 90%

Loading

38% Polyclonal 21,115,607 62%

Clonal

Bioinformatic analysis Variant calling

and classification

Signal processing

Alignment QC

Read filter

Base caller

Classification

DAT processing

L858R DEL18

INS9 G719A G719S L861Q INS12 V600E G12V G12A G12D

E545K

EGFR

ERBB2 BRAF

KRAS

PIK3CAC

Fusion variants described in the Cosmic database as related to lung cancer plus some additional variants identified by the OncoNetwork Consortium.

ALK translocation/ceritinib/crizotinib: approved ROS1 translocation/crizotinib: approved RET translocation/vandetanib: phase 33 clinical trial NTKR1: early clinical evidence

EGFR approved BRAF approved KRAS highly clinical relevant ERBB2 highly clinical relevant

What currently matters is there

NGS for NSCLC: a robust solution Covering all clinically relevant variants

NA, not applicable. Modified from the web info of

Sequencing of NSLCL: 2015–2016 results Sanger vs NGS: any winner? No need to fight

Quagliata L, et al. Unpublished data.

Sanger vs NGS sample distribution

No major differences in the incidence of driver gene alterations, as detected by the two methods. However, NGS intercepts a slightly higher number of EGFR and BRAF mutations

40.24% biopsy 59.76% cytology

Sanger (n = 1,225) N = 1,805

(n = 732)

(n = 493)

(n = 259)

(n = 312)

44.65% biopsy 55.35% cytology

NGS (n = 580)

Independent of the methods used, about 50% of samples still do not have driver gene mutations

n = 1,225

52.4% Driver-negative 29.5% KRAS (+) 13.0% EGFR (+) 3.8% BRAF (+) 1.0% HER2 (+) 0.2% EGFR + KRAS 0.2% KRAS + BRAF 0.1% EGFR + BRAF 0% EGFR + HER2 0% KRAS + HER2

Driver mutations distribution with Sanger 48.6% Driver-negative 30.0% KRAS (+) 14.1% EGFR (+) 6.2% BRAF (+) 0.5% HER2 (+) 0.2% EGFR + KRAS 0.2% KRAS + BRAF 0.2% EGFR + BRAF 0% EGFR + HER2 0% KRAS + HER2

Driver mutations distribution with NGS

n = 580

Molecular diagnostics: no tissue is left behind Make the best out of the limited resources

Property of the University Hospital of Basel.

For DNA extraction

(If needed) for RNA extraction

(If needed) for confirmation FISH

NSCLC samples overall rejection rate

Molecular diagnostics: no tissue is left behind It is a kind of Magic

Property of the University Hospital of Basel.

Molecular diagnostics: no tissue is left behind It is not just a matter of how much

Quagliata L, et al. Unpublished data.

Minimal additional time burden!

Mutually exclusive variants

Top mutated genes

NGS of NSLCL: what can we learn? NGS provides a handful of information

Quagliata L, et al. Unpublished data.

Agents Status Agents Status Agents Status Agents Status AKT1 On trial FGFR3 On trial EGFR Approved PIK3CA On trial ALK On trial KRAS Clin relevant ERBB2 Approved PTEN Clin relevant

BRAF Approved MET Approved FBXW7 Clin relevant STK11 Clin relevant DDR2 On trial NRAS Clin relevant FGFR1 On trial TP53 Clin relevant

Data highlights that 52% of patients are eligible for targeted therapy (in/off label Data also highlights that 71% has some mutations that might be clinically relevant

Not all is bright and shining Criticisms to precision medicine – how bad can it be?

The causes and consequences of genetic heterogeneity in cancer evolution

NSCLC are indeed heterogeneous entities How can we sort out the problem of tumour heterogeneity? Listen to the next talk…

Burrell RA, et al. Nature. 2013;501:338-45.

Lars Grimm, MD, MHS | December 22, 2015, Medscape

Imaging suspect

Tumour biopsy

Traditional histology-based classification

Acknowledgements

Contact: luca.quagliata@usb.ch

Institute of Medical Genetics and Pathology

Molecular Team:

Alexandra, Anja, Barbara, Claudia, Linda, Tanja, Sybille I, Sybille II, Valeria

R&D Unit:

PD Dr C Ruiz, Dr I Bratic Hech, Dr C NG, Dr S Piscuoglio, Dr P Jermann, Dr S Nicolet

Molecular Pathologists:

Prof L Terracciano, PD Dr M Bihl, Dr M Matter, PD Dr S Holler

Lung Specialists:

Prof L Bubendorf, PD Dr S Savic

Lung tumour classification Large vs small specimens – an open issue

Modified from Gridelli C, et al. Nat Rev Dis Primers. 2015;1:15009.

Adenocarcinoma • Mixed subtypes • Lepidic (non-mucinous or mucinous) • Acinar • Papillary • Micropapillary • Solid

Large-cell carcinoma • Large-cell neuroendocrine

carcinoma

Squamous Non-squamous

Small-cell lung cancer Non-small-cell lung cancer

Lung cancer

15% 85%

70%

90%

30%

10%

Living in the personalized-medicine era The concept in a nutshell

Modified from Quagliata L, et al. 2017, Schweizer Krebsbulletin, Nr. 1/2017, ISSN 2297-0703 . Available from: www.aboutcancer.com/lung_CT_BMC_Feb_2007.jpg. Available from: business.inquirer.net/122469/first-endobronchial-ultrasound-procedures-in-ph.

Clinical suspect and investigation

DRUG TARGET MANUFACTURER Erlotinib (Tarceva) EGFR Roche

Dacomitinib/PF-00299804 EGFR Pfizer Gefitinib (Iressa) EGFR AstraZeneca Afatinib (Gilotrif) EGFR Boehringer Ingelheim

Rociletinib/CO-1686 EGFR T790M Clovis AZD9291 EGFR T790M AstraZeneca Icotinib EGFR Beta Pharma, Inc

Necitumumab/IMC-11F8 EGFR (Mab) Lilly Trastuzumab (Herceptin) ERBB2 Roche

T-DM1 (Kadcyla) ERBB2 Roche MM-121 ERBB3 Merrimack

Crizotinib (Xalkori) ALK, ROS Pfizer LDK378/ceritinib (Zykadia) ALK Novartis

Alectinib/RO5424802/ ALK Roche, Chugai PF-06463922 ALK, ROS Pfizer

RXDX-101 ALK, ROS, NTRK1 Ignyta Cabozantinib/XL184 MET, RET Exelixis

INC280 MET Novartis, Incyte Vandetanib/ZD6474 RET AstraZeneca

Living in the personalized-medicine era The concept in a nutshell

Modified from Quagliata L, et al. 2017, Schweizer Krebsbulletin, Nr. 1/2017, ISSN 2297-0703 . Available from: www.aboutcancer.com/lung_CT_BMC_Feb_2007.jpg. Available from: business.inquirer.net/122469/first-endobronchial-ultrasound-procedures-in-ph.

Clinical suspect and investigation

Sample extraction and molecular profiling

Data analysis and reporting

Sample collection and assessment

Treatment decision

Our working frame: precision medicine A shifting paradigm in medicine