Experts present a cell-free plasma biobank and describe the role of longitudinal plasma samples for cancer research, disease monitoring, and biomarker development. Through liquid biopsies, it is now possible to repeatedly and non-invasively interrogate the molecular landscape of solid tumors via a blood draw over the whole treatment course. Until now, liquid biopsies can be used for screening, disease monitoring and prognosis. Circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) have been the most explored targets in this technology for commercial applications up to the present time. In collaboration with a continuously expanding oncology network, Indivumed Services has established a unique high-quality cell-free plasma biobank that is exclusively focused on collecting longitudinal whole blood samples from cancer patients. This allows molecular insight by providing quick access to longitudinal plasma from cancer patients that have undergone treatment. ctDNA can then be isolated from longitudinal cell-free plasma to allow for monitoring of disease progression by providing diagnostic and prognostic information, potentially in real time. Key Topics Include: - Gain insights into Indivumed Services’ longitudinal plasma collection process - Understand the advantages and benefits of utilizing longitudinal plasma samples for cancer research - Explore applications of longitudinal plasma samples for biomarker research and development of companion diagnostics

2. Dr. Mirka Allerding
Anna Sophie Skof
Longitudinal Plasma Samples:
Paving the Way for Precision Oncology
Compact Collections of Slides to Products and Services

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Agenda
I. Indivumed Services’ longitudinal plasma
collection
II. Advantages and benefits of longitudinal
plasma samples for cancer research
III. Explore applications of longitudinal
plasma samples for biomarker research
and development of companion
diagnostics

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Cancer Research - Solid vs. Liquid Biopsy
Background
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Solid Biopsy Liquid Biopsy
Tissue Blood
e.g., cerebrospinal fluid, bone marrow, saliva, and urine
Invasive (surgery) Minimally invasive (blood draw)
Single snapshot at one time point Monitoring over multiple time points
Tumor information from one part of the tumor
i.e., extraction site
Intra-tumor heterogeneity is detectable
Limitations:
e.g., access to the tumor, patient condition, clinical complications,
spreading to other sites
Limitations:
e.g., low specificity due to high cell-free DNA background, false
positive and negative results
Tumor DNA Circulating tumor DNA, circulating tumor cells etc.

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Liquid Biopsy & ctDNA
Background
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Tumor tissue
Healthy tissue
Blood vessel with RBCs and WBCs
ctDNA =
• Human blood contains cells, cell-free
DNA (cfDNA) & RNA, proteins, and
exosomes.
• Part of cfDNA in cancer patients is
circulating cell-free tumor DNA
(ctDNA).
• Detection of ctDNA in plasma samples
may lead to the discovery of
hidden/unknown tumor sites.
Modified
from
Crowley
E.,
et
al.
Nat
Rev
Clin
Oncol.
2013

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Oncological Liquid Biopsy Biobank
Indivumed Services – Biobanking
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Start:
March 2016
Patients:
Constantly growing
patient pool,
~27,000
Headquarter:
Hamburg, Germany
Reliable Clinical Partners:
46 oncology clinics located
across Germany, and
continuously expanding
Longitudinal Plasma
Sample Collection:
Blood draw T0
up to Blood draw T5
Relevant Clinical Data:
~32-45 data points,
includes clinical response
& therapy information

7. Longitudinal Blood Collection and Processing
Plasma
storage
Plasma is stored at
-80°C in 1 ml aliquots
Shipment of
samples
Blood samples are
shipped to Indivumed
Services
Blood
collection
Patient's blood is
collected by oncology
clinic staff
Plasma
extraction
Cell-free, double spun
extraction protocol
used in-house
Longitudinal
sampling
Blood from multiple
time points and
therapies is collected

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Plasma Quality & cfDNA Yield
Indivumed Services – Biobanking
• Only 0.1-10 % cfDNA is ctDNA.
• Levels of cf- and ctDNA depend on:
› Cancer properties
› Cancer treatment types
› Other non-cancer related conditions
• Wrong sample handling leads to an artificial increase in
cfDNA.
• The detection of ctDNA depends on a high cfDNA
yield and a low cfDNA/ctDNA ratio.
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Additional information: “Quality Analysis of Circulating Cell-Free DNA from Indivumed
Services’ Plasma Sample Collection”
cfDNA Quality
(qPCR measurement)
cfDNA Yield
(qPCR measurement)

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Blood Collection, Shipment & Plasma Extraction
Indivumed Services – Biobanking
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• Collection in Streck Cell-Free DNA BCT®
• Stabilizes cells, prevents cell lysis, reduces cfDNA background
• Allow collection of blood samples from all over Germany
• Blood can be stored for up to 7 days without quality loss
• Blood samples are processed within 24-72 hours after collection following an ISO-certified
double-spun protocol
• All steps are performed at room temperature
• Collection, sample transport and plasma aliquot preparation
• Highly standardized, state of the art collection of plasma

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Longitudinal Cell-free Plasma
Indivumed Services – Biobanking
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Patient
*4-12 weeks between each blood draw
Oncologist
Systemic Therapy #1
e.g. Chemotherapy (Carboplatin)
Blood draw T0
Before Therapy
Blood draws T1 to T5*
During Therapy
Clinical
Data
Oncologist
Systemic Therapy #2
e.g. Immunotherapy (Cetuximab)
Blood draw T0
Before Therapy
Blood draws T1 to T5*
During Therapy
Clinical
Data
Serial samples from the
same patient with matched
clinical data.
Multiple therapy lines
possible.

11. Longitudinal Blood Collection and Processing
Plasma
storage
Plasma is stored at
-80°C in 1 ml aliquots
Shipment of
samples
Blood samples are
shipped to Indivumed
Services
Blood
collection
Patient's blood is
collected by oncology
clinic staff
Plasma
extraction
Cell-free, double spun
extraction protocol
used in-house
Data
collection
Corresponding data
points are linked to the
samples
Longitudinal
sampling
Blood from multiple
time points and
therapies is collected

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Clinical Data
Indivumed Services – Biobanking
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Biomarkers
179p-Deletion ER
ALK FISH
BCR-Abl HER2
B-RAF JAK2
BRCA1 KRAS
BRCA2 MSI
CD15 NRAS
CD20 PDL1
CD30 PR
EGFR ROS
TP53
• General patient information
› e.g., age, gender
• Disease/Tumor classification
› ICD10, Morph-Code
• Therapy/Treatment information
• Clinical response
› e.g., clinical impairment, clinical
improvement, increase of tumor markers
• Tumor response
› CR, PR, SD, PD
• Biomarker information
› Information on mutation
status (i.e., yes/no, established
scoring schemes)
• Upon request:
› Method of detection
› Radiology data
~32 – 45 data points

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Liquid Biopsy Inventory
Indivumed Services – Biobanking
~ 27.000 patients
~ 700.000 plasma Aliquots
~ 3,000 cancer patients/year
with all types of tumors

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Liquid Biopsy Inventory
Indivumed Services – Biobanking

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Analysis of ctDNA
Indivumed Services – Biobanking
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Methods:
• PCR
→ qPCR, dPCR, ddPCR, BEAMing digital PCR
→ Single-locus/multiplexed assays, point-mutation analysis
→ Sensitive, fast and inexpensive
→ Only for limited number of known molecular alterations
• NGS
→ Highly sensitive, high throughput
→ Single-locus/multiplexed assays, point-mutation analysis, targeted
sequencing, chromosomal copy-number changes
→ Known and unknown multiple molecular alterations
→ More expensive than PCR
• WGS
→ Non-targeted NGS technology, whole genomic profile of the tumor DNA
→ Require high concentration of ctDNA

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Challenges:
• Low percentage of ctDNA, highly fragmented
• Trade-off between sensitivity and costs
• Many different genomic aberrations
• High specificity needed
• Screening and early
detection
• Patient stratification
• Treatment selection
Application of Longitudinal Plasma
Indivumed Services – Biobanking
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modified from Caputo et al. 2023, Explor Target AntitumorTher 4,102-38
ctDNA
levels
time
ctDNA detection
limit
Diagnosis
of cancer
Therapy
response
Metastatic
cancer
Therapy Progression
Therapy
response
Therapy
response

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• Detection of minimal residual disease (MRD)
and molecular relapse
• Risk for metastatic relapse (prognostic)
• Analyze spatial/temporal tumor heterogeneity
• Monitoring of clonal evolution and progression
• Real-time monitoring of treatment
response
• Detect development of resistances
• Detect molecular remission
• Screening and early
detection
• Patient stratification
• Treatment selection
Application of Longitudinal Plasma
Indivumed Services – Biobanking
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modified from Caputo et al. 2023, Explor Target AntitumorTher 4,102-38
ctDNA
levels
time
ctDNA detection
limit
Diagnosis
of cancer
Therapy
response
Metastatic
cancer
Therapy Progression
Therapy
response
Therapy
response

18. Thank you
Liquid Biopsy Biobank – Indivumed Services
Indivumedservices.com
liquidbiopsybiobank_cbge@crownbio.com
Indivumed Services GmbH
Falkenried 88, Bldg. D
20251 Hamburg, Germany