Download DeVlaminck Presentation

Circulating DNA
–
Large datasets and big opportunity
Iwijn De Vlaminck
Big Data and Molecular diagnostics
o Driven by technological advances (hardware and data management
tools), reduction in cost of high throughput sequencing and rapid
growth of public databases.
o Opportunities: precision medicine, one stop shop diagnostics.
o Challenges: (1) Condensing big data elements into a therapeutic
decision (2) Dealing with diverse data elements, genetic data, public,
pharmacological, and clinical data
Circulating cell-free DNA
 Abundant: 1,000 – 10,000 genome copies per ml.
=> up to 100 billion fragments of DNA per ml.
P. Mandel, P. Metais, Biologie Compters Rendus, (1948)
 Mean half life in circulation: 16.3 minutes.
Y.M. Dennis Lo et al., Am. J. Hum. Genet. (1999).
 Turnover: ~ 10,000 cells per second or ~ 1 ml cell volume per hour
(primarily hematopoeitic in origin).
Circulating fetal DNA
PNAS 2008
Circulating tumor DNA
C. Bettegowda, et al. Science Translational Medicine (2014)
The challenge of post transplant therapy
The challenge of post transplant therapy
The challenge of post transplant therapy
The therapeutic window is small, in some cases non-existent.
Drug toxicity/intolerance further complicates the situation
Outline
o Rejection: Cell-free donor derived DNA as a marker for rejection
o Heart transplants
o Lung transplants
o Infection:
o Shotgun sequencing detects a broad array of infectious agents:
o Immunosuppression
o Temporal response of the virome in plasma to
immunosuppressants and antivirals
Outline
o Rejection: Cell-free donor derived DNA as a marker for rejection
o Heart transplants
o Lung transplants
o Infection:
o Shotgun sequencing detects a broad array of infectious agents:
o Immunosuppression
o Temporal response of the virome in plasma to
immunosuppressants and antivirals
Post-transplant transplant surveillance
Endomyocardial biopsy: currently the gold standard
0R
1R
2R
3R
Subjective: Overall, all-grade agreement between pathologists: 71 ± 3 %
Crespo-Leiro et al., Transplantation (2012).
Expensive: > $4000
Invasive: major complications in ~ 1% of cases
~ 30,000 surveillance biopsies performed on heart transplants each year
(3000 rejections diagnosed)
Circulating cell-free DNA
 Abundant: 1,000 – 10,000 genome copies per ml.
=> up to 100 billion fragments of DNA per ml.
 Mean half life in circulation: 16.3 minutes.
Y.M. Dennis Lo et al., Am. J. Hum. Genet. (1999).
 Turnover: ~ 10,000 cells per second or ~ 1 ml cell volume per hour
(primarily hematopoeitic in origin).
 Donor-derived DNA circulates in the blood stream of transplant recipients.
Y.M. Dennis Lo et al., the Lancet (1998).
Cell free donor DNA as a marker for rejection
Pre-transplant genotyping, 1 Million or 2.5 Million SNP markers
Post-transplant sequencing of cell-free DNA T. Snyder, K.K Khush, H.A. Valantine and S.R. Quake, PNAS (2011)
I. De Vlaminck, S.R. Quake et al., Science Translational Medicine (2014)
Heart transplant: prospective study design and
numbers
Read and Donor Assignment Statistics
<24 Million> 50 bp sequences
<53,423> informative SNPs per donor-recipient pair
<13,300> sequences align to informative SNPs
Signal in absence of rejection
Elevated signal immediately post transplant followed by a quick decay
(decay time 2.4 days) to a low baseline level
Signal at time of rejection
Elevated donor DNA at time of rejection
Signal at time of rejection
Elevated donor DNA at time of rejection
Graft loss and re-transplant
Comparison signal non-rejectors/rejectors
Significant increase in fraction of donor-derived DNA at rejection
Performance of cell-free donor DNA as a marker of rejection
AUC 0.83
Threshold 0.25%
sensitivity: 58%
selectivity: 93%
donor DNA outperforms commercially available non-invasive diagnostic (AUC = 0.72).
Potential to complement or replace biopsies
Science Translational Medicine (2014)
Outline
o Rejection: Cell-free donor derived DNA as a marker for rejection
o Heart transplants
o Lung transplants
o Infection: Shotgun sequencing detects a broad array of infectious
agents? Temporal response of the virome in plasma to
immunosuppressants and antivirals
o Immunocompetence: Antibody repertoire sequencing measures the
overall state of the immune system
Patient survival rates in lung transplantation
Major pulmonary complications following transplantation:
- Bronchial Obliterans Syndrome: progressive loss of lung function ~ chronic rejection
- Acute rejection
- Infections of the lung
ISHLT report, 30,1104 (2011)
Study design
Lung transplants – signal in absence of rejection
Very high levels of donor DNA immediately post transplant
High background level compared to heart transplants
Signal elevated at rejection (transbronchial biopsy)
Analysis of performance against biopsy
Exclude samples collected within first 60 days
Account for volume difference for single/bilateral transplants
Non-human origin of a subset of sequences
Data from 656 heart and lung transplant samples
Viruses more abundantly represented than fungi and bacteria
Cell 2013
Relative genomic abundance of species
Viruses from the anelloviridae family are most abundantly represented among viruses
Cell 2013
Relative genomic abundance of species
The anelloviridae fraction is primarily composed of viruses from the alphatorque genus
Cell 2013
Relative genomic abundance of species
Bacteria at the phylum and order level of classification
I. De Vlaminck , S.R. Quake et al., Cell, 155, 1178 (2013)
Chronic Viral infection
Herbert W. Virgin et al., Cell, 138 (2009)
Immunosuppressants and antivirals alter structure of
the virome
47 patients; 380 samples
Virome temporal dynamics
96 patients; 656 samples
Virome temporal dynamics
96 patients; 656 samples
Virome temporal dynamics
96 patients; 656 samples
Rejection vs infection
Infectious burden different for non-rejecting vs. rejecting patients?
non-rejecting
rejecting
Anellovirus load for rejecting vs non-rejecting
recipients
Anellovirus load for rejecting patients (20 patients, 177 datapoints) and non-rejecting
recipients (40 patients, n = 285 datapoints)
Hypothesis independent screening of pathogens via
plasma DNA sequencing.
Enterocytozoon bieneusi load in a bone marrow transplant patient
confirms clinical positives (stool sample, red arrows)
Hypothesis independent screening of pathogens via
plasma DNA sequencing.
Similar parasite load observed in an untested lung transplant patient
that was never diagnosed but suffered from similar symptoms.
Shotgun sequencing for the screening of infections
o Many oncoviruses and pathogens are not screened for frequently in current clinical practice.
o Shotgun sequencing reveals high incidence.
o Disconnect between the frequency of clinical testing and incidence of infection.
Submitted
Outline
o Rejection: Cell-free donor derived DNA as a marker for rejection
o Heart transplants
o Lung transplants
o Infection:
o Shotgun sequencing detects a broad array of infectious agents:
o Immunosuppression
o Temporal response of the virome in plasma to
immunosuppressants and antivirals
Acknowledgements
Quake lab
Jennifer Okamoto
Christopher Vollmers
Lance Martin
Thomas Snyder
Norma Neff
Mark Kowarsky
Mickey Kertesz
Steve Quake
NIH grant RC4AI092673
Stanford hospital:
Calvin Strehl, Garrett Cohen, Bitika Kohli,
Helen Luikart, Daniel Bernstein, David
Weill, Kapil Patel, Mark Niccols, David
Cornfield, Hannah Valentine,
Kiran Khush
Extra slides
Potential for early diagnostics
Donor DNA levels significantly elevated up to 5 months prior to a rejection event
Response to rejection treatment
Donor DNA after detection and treatment of severe rejection.
Error rate – independent measurement
Median error 0.04%
Matched SNP positions allow estimating the frequency of erroneous donor calls
Evidence of graft injury exclusively for CMV infections
Bonferroni adjusted significance threshold
Infection-related tissue damage
653 CMV tests performed on 47 patients.
PCR tests of serum or Bronchial lavage samples (BAL)
This observation is unique to CMV, 73 infection types clinically reported
Observation of CMV-related graft injury
Infection diagnosis via shotgun sequencing?
CMV detected in plasma for patients that test positive for CMV.