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Dr. Mirka Allerding
Anna Sophie Skof
Longitudinal Plasma Samples:
Paving the Way for Precision Oncology
Compact Collections of Slides to Products and Services
Copyright 2023. www.indivumedservices.com
3
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
Copyright 2023. www.indivumedservices.com
Cancer Research - Solid vs. Liquid Biopsy
Background
4
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.
Copyright 2023. www.indivumedservices.com
Liquid Biopsy & ctDNA
Background
5
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
Copyright 2023. www.indivumedservices.com
Oncological Liquid Biopsy Biobank
Indivumed Services – Biobanking
6
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
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
Copyright 2023. www.indivumedservices.com
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.
8
Additional information: “Quality Analysis of Circulating Cell-Free DNA from Indivumed
Services’ Plasma Sample Collection”
cfDNA Quality
(qPCR measurement)
cfDNA Yield
(qPCR measurement)
Copyright 2023. www.indivumedservices.com
Blood Collection, Shipment & Plasma Extraction
Indivumed Services – Biobanking
9
• 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
Copyright 2023. www.indivumedservices.com
Longitudinal Cell-free Plasma
Indivumed Services – Biobanking
10
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.
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
Copyright 2023. www.indivumedservices.com
Clinical Data
Indivumed Services – Biobanking
12
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
Copyright 2023. www.indivumedservices.com
13
Liquid Biopsy Inventory
Indivumed Services – Biobanking
~ 27.000 patients
~ 700.000 plasma Aliquots
~ 3,000 cancer patients/year
with all types of tumors
Copyright 2023. www.indivumedservices.com
14
Liquid Biopsy Inventory
Indivumed Services – Biobanking
Copyright 2023. www.indivumedservices.com
Analysis of ctDNA
Indivumed Services – Biobanking
15
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
Copyright 2023. www.indivumedservices.com
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
16
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
Copyright 2023. www.indivumedservices.com
• 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
17
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
Thank you
Liquid Biopsy Biobank – Indivumed Services
Indivumedservices.com
liquidbiopsybiobank_cbge@crownbio.com
Indivumed Services GmbH
Falkenried 88, Bldg. D
20251 Hamburg, Germany

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Longitudinal Plasma Samples: Paving the Way for Precision Oncology

  • 1.
  • 2. Dr. Mirka Allerding Anna Sophie Skof Longitudinal Plasma Samples: Paving the Way for Precision Oncology Compact Collections of Slides to Products and Services
  • 3. Copyright 2023. www.indivumedservices.com 3 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
  • 4. Copyright 2023. www.indivumedservices.com Cancer Research - Solid vs. Liquid Biopsy Background 4 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.
  • 5. Copyright 2023. www.indivumedservices.com Liquid Biopsy & ctDNA Background 5 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
  • 6. Copyright 2023. www.indivumedservices.com Oncological Liquid Biopsy Biobank Indivumed Services – Biobanking 6 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
  • 8. Copyright 2023. www.indivumedservices.com 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. 8 Additional information: “Quality Analysis of Circulating Cell-Free DNA from Indivumed Services’ Plasma Sample Collection” cfDNA Quality (qPCR measurement) cfDNA Yield (qPCR measurement)
  • 9. Copyright 2023. www.indivumedservices.com Blood Collection, Shipment & Plasma Extraction Indivumed Services – Biobanking 9 • 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
  • 10. Copyright 2023. www.indivumedservices.com Longitudinal Cell-free Plasma Indivumed Services – Biobanking 10 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
  • 12. Copyright 2023. www.indivumedservices.com Clinical Data Indivumed Services – Biobanking 12 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
  • 13. Copyright 2023. www.indivumedservices.com 13 Liquid Biopsy Inventory Indivumed Services – Biobanking ~ 27.000 patients ~ 700.000 plasma Aliquots ~ 3,000 cancer patients/year with all types of tumors
  • 14. Copyright 2023. www.indivumedservices.com 14 Liquid Biopsy Inventory Indivumed Services – Biobanking
  • 15. Copyright 2023. www.indivumedservices.com Analysis of ctDNA Indivumed Services – Biobanking 15 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
  • 16. Copyright 2023. www.indivumedservices.com 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 16 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
  • 17. Copyright 2023. www.indivumedservices.com • 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 17 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