Christian Röder, PhDInstitute for Experimental Cancer Research and

Centralized Biomaterial Bank P2N of the Medical Faculty, CAU

Biomarker for personalized medicine

biobanking, biomarker, diganostics, ethics

Outline of Lecture / Seminar

Lecture Dec-16-2019

• Biobanking as a Tool for Research / Biobanks / Aims

• Standards in Biobanking: Quality management, Ethics

• Biomarkers: Types / Utilization / Diagnostics / Limitations

Seminar

• Paper by Andrew Woolston et.al., Cancer Cell 36, 2019

Genomic and Transcriptomic Determinants of Therapy Resistance and Immune Landscape Evolution during Anti-EGFR Treatment in Colorectal Cancer

Learning Objectives

• define / describe the term biobank• name the requirements of high quality biobanking• name the major topics of a patient informed consent• biomarkers: name the characteristics of goodbiomarkers / diagnostic tests

• describe the different types and use of biomarkers aswell as their limitations

Time Magazine March 23, 2009:

• Biobanks are mentioned as1 of 10 ideas changing theworld right now

Atmosphere of awakening in geneticsand genomics with human clinicalspecimen

• 1st human genome sequence (2001)after 10 yrs at costs of 3 billion $

• Next Gen. Sequencing (NGS) (2005)• 1000 genomes project (2008)• 100,000 genomes project (UK, 2012)• US Precision Medicine Initiative

(2015), 1 Million Genome• EU 1+Million Genomes Initiativesigned on 10.4.2018 by meanwhile20 EU member states

Biomaterial Banks

Human Genome Sequencing

https://www.genome.gov/sequencingcostsdata/Wetterstrand KA. DNA Sequencing Costs: Data from the NHGRI Genome Sequencing Program (GSP)

What is a Biobank ?

Biobanks [are] collections of samples of human body substances[...], which are linked or may be linked to personal data andinformation of the donors. Biobanks simultaneously share

characteristics of a collection of specimen and data.

German National Ethics Council Statement (Nationaler Ethikrat, 2006)

www.ukbiobank.ac.uk

What is a Biobank ?

• Any collection of biospecimens or genetic and othermaterial thereof with and without associated data

• Retained for sharing and/or future use

• with or without identifying (donor) information

• de facto – collectionsamples kept in lab freezers on occasion: accidental bank

• de jure – biorepositorydesigned for storage and sharing, ethics board-approved, scope, governance, consent forms, sample catalog, use & access policy (board)

What is a Biobank ?

collection on occasion biorepository

Profile and Requirements of a Biobank ?

• sufficient (large) scale of collections• multiple aliquots of samples for multiple use• long-term storage• flexible scientific questions and future analyses,

driven by future research insights / requirements• connection of samples and data (research-, medical-

and follow up-)• ethics („ELSI“) regulations: data protection and

„Informed Consent“ of donors• quality management (SOPs)• use and access regulations

Standard Operating ProcedureGovernance / Quality Management

Informed Consent

Informed Consent

Informed ConsentSynopsis

• Easy to understand outline of the project, aims andcollected materials; broad (general) scientific question

• Information about combined sample and data collectionwith pseudonymization

• No direct benefit for the donor (neither medical norfinancial)

• Information about any reporting of individual researchdata (not intended in Kiel/P2N)

• Right of donors to withdraw their consent (impossiblein case of anonymization)

• Long-term storage of specimen

Biobanks - Advantages

• large and well characterized pre-existing collectionsfor investigations of weak or moderate biologigal effects

• no recruiting phase for donorsallows faster processing of scientific questions

• availability of quality-controlled reference materialsfor validation of in-vitro or per-clinical results

• access for the broad scientific community tovaluable human materials for reseach

• establishment of scientific and ethical standards in patient-based research

Biobanks - Disadvantages

• long-term determinationwith regard to donor cohorts, materials, preparations

• unflexible planning for requirements and scientificconceptsdue to SOPs and clinical workflows

• long-term commitment for budgets and lab ressources(rooms, staff, equipment and consumables)

• low scientific impact / „broad effects“in terms of „scientific output parameters“(papers, grants, graduate projects)

Different types of biobanksHuman biobanks

• banks of organs and tissues for therapy / transplantation- blood bank- organ bank (whole organs, heart valves, cornea)

• research-related biobanks- ethnical cohorts, twin-biobanks, ....- disease-oriented / clinical / epidemiological biobanks- population (prospective) biobanks↳ study of common, complex, or rare diseases over long time

Population-based BiobanksGerman National Cohort (NAKO Gesundheitsstudie)

https://nako.de

• 18 study centers• 200.000 randomly chosencitzens (age 20-69)

• detailed medical examinationand anamnesis: medical data

• 28 mill. biomaterial samples• follow-up examination andmultiple follow-up anamnesis

• time span: 2013 - 2042

Population-based BiobanksGerman National Cohort (NAKO Gesundheitsstudie)

https://nako.de

Year 2013 2014 2015 2016 2017 2018 2019 2020 2021 2022 2023 ...........................................2042

base examination

follow up examination

active and passive follow up(every 2-3 yrs: questionaires, registries)

pilotstudy

utilization for research studies

PopGen 2.0 Network

Governance

ELSI

Governance

Institute ofPathology

Clinic for Neuro-Pediatrics Univ. Lung Center North

Clinic for Neurosurgery

Clinic for General SurgeryInst. of Pathology

Inst for Exp. Cancer Research

Institute for Epidemiology

Institute ofPharmacology

PopGen 2.0 Network: 10 partner BMBsBMBF grant 2011-2016 (5 Mio. €)

BMBEnzephalitis

BMBIKMB

BMBGyn

PopGen 2.0 Network

• Central management of patient consents; implementation of a „broad

consent“ for the use for research of residual clinical materials

• Central use & access structures for the utilization of samples and data

• P2N-wide LIMS / data- and sample-management software with central

data base hosting, maintenance and IT support

• Adaption and/or implementation of harmonized data and privacy

protection concepts

• Central quality management, harmonized SOPs,

perspective: ISO 9001 certification

• P2N-wide DNA preparation and storage facility

• Perspective: Harmonization and Cooperation with the new UKSH-CAU

medicine-informatics department

Aims

Biomaterial Storage Unit PopGen / P2N

Center of Molecular Biosciences (CMB) Kiel

LiCONiC walk-in repository-80�sample storage in the CMB

Biomaterial Bank PopGen

Stroke

Parodontitis

Gall stonesSarcoidosis

Morbus Crohn

ParkinsonDepression

PsoriasisPemphigus

Colitis ulcerosa

Biomaterial Bank for different diseases

> 80.000 participants> 120.000 biosamples> 150 clinical partner> 250 collaborations>> 200 high-ranked papesColorectal Cancer

Epilepsy

TuberculosisCardio-Vascular Diseases

Patient

Information

writtenconsent

Tumor ResectionClinic for General Surgery, Visceral, Thoracic,

Transplantation and Pediatric Surgery

Data

LIMS / Database ProWebDBCentraXX

Biomaterial Bank CCC

Blood, Serum, Plasma Cryo-Bone Marrow, Lavage Tissue

Biobank LaboratoryInst. f. Experimental Cancer Research

tumor & peritumoral non-malignant, IBD

Institute of Pathology

Data•Sample Logistics•Patient•Clinic•Pathology•Therapy•Research•Follow up

LIMS / Database Inst. f. PathologyCentraXX

Biomaterial Bank Patho

FFPE-,Cryo-Tissue

Oncological Biobank – BMB-CCCStructure and Procedures

Liquid Tissue

Molecular Cancer DiagnosticsGoing to improve medicine

Cancerpredisposition

Cancer detection /Early detection

Therapy /Drug selection

Follow-upRecurrence monitoring

Individualized therapy Which diseaseIs a patient atrisk for?

Has a patient a disease?

Which drugs should be prescribed?

Has the disease returned?Diagnosis Confirmation

and Prognosis

What is the likely course of a disease?

Key Questions

Biobank Materials are needed to defineBiomarkers for Personalized Medicine

Blood: Serum / Plasma / CellsPancreatic JuiceUrine / StoolSalivaBiopsy / Tissue / Cytology

Protein (Glycoproteins)DNA (SNPs/point mutations, methylated DNA)RNA (mRNA, miRNA, ncRNA, circRNA)Metabolites

Circulating & Disseminated Tumor Cells (CTC / DTC)Platelets

A good detection system should have 4 qualities:

ü Sensitivityü Specificityü Simplicity and Cost-Effectivenessü Robustness

Sensitivity means that the test must be able to detect very small amounts of target in case of a disease even in the presence of other molecules. (“positivity in disease”)

Specificity: the test yields a positive result for the target molecule and in the case of the disease only. (“negativity in health”)

Simplicity / Cost Limitations / Robustness: the test must be able to run efficiently and inexpensively on a routine basis and should run under various laboratory conditions. Benchmarking, “Ringversuche”

Biomarker DiagnosticsCharacteristics of a diagnostic test system

Diagnostic Biomarkers specifically indicate a particular disease

Prognostic Biomarkers predict the course of a diseasein untreated patients („high vs. low risk“)

Biomarker-Definitions

Molecular Cancer Diagnostics

• Classical cancer pathology: investigation of tissue biopsies, surgery resectates, lymph node pathology: TNM classification

Molecular Cancer Diagnostics

• Classical cancer pathology: investigation of tissue biopsies, surgery resectates, lymph node pathology: TNM classification

• „The TNM classification of the UICC gives a solid basis fortumor staging. It‘s still the most important instrument fortherapy decisions.• Up to now, the TNM classification was never

outperformed by any other prognosticbiomarker“

(C. Röcken, FORUM 2016 · 31:406–411)

Molecular Cancer Diagnostics

www.mindsofmalady.com

• Classical cancer pathology: investigation of tissue biopsies, surgery resectates, lymph node pathology: TNM classification

• Molecular pathology: genetic and phenotypic markers

• Liquid biopsy:• Cytological investigation of blood

- cytogenetics (karyotypes, FISH)- molecular markers / circulating tumor cells• Identification of virus infections• Analysis of cerebrospinal fluid• Analysis of cancer biomarkers in blood

(serum/plasma)

Tumor&Marker& Elevated&in:& Comment&CA1979& Pancreas(,*Gastric(,*

Cholangio(Ca.*carbohydrate(epitope*on*ganglioside*or*mucin*

CEA& Colon(,*Rectum(,*Gastric(,*Lung(,*Breast(Ca.*

membran*glycoprotein,*=CEACam*

Colorectal Cancer

Diagnostic Biomarkers for systemic Cancer Detection in Liquid Biopsies

Tumor-associated Antigens / Serum Markerin gastrointestinal Cancer

• some are long established

• not very sensitive nor specific

provide information on the likely prognosis or course ofthe disease in an untreated individual

• studies problematic in oncology, since most patientsreceive (besides surgery) some adjuvant therapy

• however, clinically important ’prognostic‘ studiescomprise early-stage tumors that are not treated byadjuvant therapy (e.g. stage I/II colorectal cancer)...

Prognostic Biomarkers

Prognostic Biomarkers

Kaplan-Meier-survival analysis

Log-Rank testP< 0.05 = significant

differencein survival

Colorectal carcinoma (CRC)

• many patients suffer from recurent disease- cancer patients mostly die from metastases or relapse

• prognostic markers are needed to selectotherwise untreated patients for adjuvant therapy

post-surgicalchemo-therapy

5-yr survival rate

Prognostic Biomarkers for Cancer Cell Detection in Liquid Biopsies

Schroeder et al.Nature Reviews Cancer 2012

Keratin20 (CK20)-nested RT-PCR

Circulating Tumor Cells (CTC)

„Rare Event Detection�

CK20 qRT-PCR

Detection of CTC / DTC of solid Tumors

MNC fraction+ tumor cells

APAAP complex

„Rare Event Detection� 10ml blood:100 million leukocytes

Enrichment: 50 billion erythrocytes• Ficoll cushion or• positive selection• negative selection

BiobankLab

2 x 106 MNC

• 10 ml venous blood

cytokeratin stainingwith mAB A45-B/B3

Ficoll

plasma, platelets

erythrocytes, granulocytes

Detection

biological properties ?

Ficoll

40 min, 400 xg

blood diluted 1:3 (RPMI1640)

Pan-keratin cytology

Patients: Liquid Biopsy

CK-20 RT-PCR

Kaplan-Meier-Analysis

Stage I+IIP<0.001 (log Rank)n=209

CK20 –

CK20 +

[months][cummulativesurvival]

• pioneering registry: www.genetests.org• Information resource e.g. for healthcare providers• Located at the University of Washington, Seattle• was funded by the National Institutes of Health• phased out 2013 from NIH that now runs own registry

Biomarker Registries

Biomarker (Genetic Testing) Registries

Prognostic Genetic Test Systems

Genes:BMPR1A, BRCA1, BRCA2, CDH1, CDKN2A, MEN1, MLH1, MSH2, MSH6, MUTYH, PMS2, PTEN, SMAD4, STK11, TP53, VHL, NTRK1, RET, APC

Method:Next Generation Sequencing

Material:6-9 ml blood or 5 µg DNA

Prognostic Genetic Test Systems

Prognostic Genetic Test Systems

Mar 27, 2013

• 5-10% of hereditary breast cancerwith autosomal dominant inheritance

• BRCA1 is affected with very high frequency

• woman with inherited germ line mutationstatistically develop breast cancer- 20 yrs earlier- 80-90% incidence

Prognostic Genetic Test Systems

Mar 27, 2013Mar 10, 2016

Diagnostic Biomarkers specifically indicate a particular disease

Prognostic Biomarkers predict the course of a diseasein untreated patients („high vs. low risk“)

Predictive Biomarkers predict the probability of a therapy responsein a patient

Biomarker-Definitions

A ‘companion diagnostic’ is a test or assay that detects a predictivebiomarker, thereby allowing classification of patients (tumors) intoresponders and non-responders

Synonyms: ‘predictive diagnostic’, ‘precision diagnostic’, ‘theranostic’,‘advanced personalized diagnostic’

Targeted Tumor TherapyTumor-specific aberrations

• Overexpression ofoncogenes

• Overexpression of tyrosinekinases e.g. Her2

• Expression of fusion proteinse.g. Bcr-Abl

• Mutations / SNPse.g. ras-family, raf-family

• Loss of tumorsuppressor gene(function), e.g. TP53

Targeted Tumor Therapy

identify subgroups of patients who are most likelyto respond to a given therapy

• predictive markers are the basis of individualizedtreatment

Predictive Biomarkers

Examples (predictive markers in tumor tissue):• estrogen- / progesterone-receptor status

in breast cancer for hormone therapy

• HER2-status for Herceptin treatment

• KRAS mutation or wildtype for Anti-EGFR therapy↳ Molecular Pathology Precision Oncology

• Cetuximab („Erbitux“) approval in 2003 (US) and 2004 (EU)

• Approval for •CRC (EGFR+, KRAS-wt) in combination withchemotherapy and •squamous cell head & neck cancer in combination with radiation or chemotherapy

• Well known side effects (>20%) e.g. ulcerous skin inflammation

• Increased survival in responders by months

• Costs: 14-day cycle: 5000 €; standard: 12 cycles = 60.000 €

Anti-EGFR Tumor TherapyCetuximab / Erbitux

Gonzalez de Castro et al., Br J Cancer 107, 345 (2012):

• Patients with colorectal tumors containing activating KRAS mutations do not benefit from therapy with cetuximab and panitumumab.

• KRAS mutation status is a key determinant of response to anti-EGFR therapy.

• The European Medicines Agency states that anti-EGFR therapy is only indicated in patients with wild-type KRAS tumors.

• The US Food and Drug Administration states that such treatment is not recommended in tumors with mutations in codons 12 and 13 of KRAS.

• The European Society for Medical Oncology recommends that tumours are KRAS wild type, without naming specific mutations.

Anti-EGFR Tumor TherapyDetection of KRAS point mutations

K-Ras wildtype as predictivebiomarker for Anti-EGFR (e.g. Cetuximab) therapy

• Analysis of pointmutations in tumor tissue:

Cetuximab / Erbitux

Anti-EGFR Tumor TherapyDetection of KRAS point mutations

Anti-EGFR Tumor TherapyDetection of KRAS point mutations

K-Ras wildtype as predictivebiomarker for Anti-EGFR (e.g. Cetuximab) therapy

?

K-Ras wildtype as predictivebiomarker for Anti-EGFR (e.g. Cetuximab) therapy

• Analysis of pointmutations in tumor tissue:

- resected specimen maybe heterognous: e.g. stroma-rich:selection by dissection

- realtime PCR orsequencing

Cetuximab / Erbitux

Anti-EGFR Tumor TherapyDetection of KRAS point mutations

CUSA: Cavitron UltrasonicSurgical Aspirator

Cancers 2013, 5, 357-371

Antigen HeterogeneityIn tissue fragments after ultrasonic aspiration

Tsiatis et al., J Mol Diagn 12:425(2010)

Arrow = Detection Limit

Pyro-Sequencing: 5% mut.Sanger Seq.: 18% allelesMelting Curve: 18%(realtime PCR)

PS

SS

MC

Detection of KRAS point mutationsSensitivity of techniques

Detection of KRAS point mutationsCOBAS KRAS Mutation Analysis Test (Roche)

Cree et al., Biomol Detect Quantification 8:29 (2016)

Lee et al., Virchow‘s Arch 460:141(2012)

Calculateconcentrationfrom numberof droplets

FAM

am

plitu

de

VIC amplitude

Patient plasma Sample ispartitionedinto 20,000 droplets

Measurefluorescenceintensity in eachdroplet

Run PCR cycles in all dropletssimultaneously

1. Partition 2. Cycle 3. Read

Digital PCR