1. Circulating DNA – Large datasets and big opportunity
Iwijn De Vlaminck

2. Big Data and Molecular diagnostics
Driven by technological advances (hardware and data management tools), reduction in cost of high throughput sequencing and rapid growth of public databases.
Opportunities: precision medicine, one stop shop diagnostics.
Challenges: (1) Condensing big data elements into a therapeutic decision (2) Dealing with diverse data elements, genetic data, public, pharmacological, and clinical data

3. 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: 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).
Nucleosome: histone octamer wrapped 1.65 times by 147 bp of DNA
Liberation rate’
Liver, kidney, spleen involved in the removal of circulating DNA

4. Circulating fetal DNA

5. PNAS 2008

6. Circulating tumor DNA
C. Bettegowda, et al. Science Translational Medicine (2014)

7. The challenge of post transplant therapy
Post transplant therapy requires a immunesuppressive therapy that balances the risks of infection and rejection.
You can imagine that the probability of rejection is some function of immune strength. The weaker the immune system the lower the prob of the occurrence of rejection.
Immune suppression can mitigate ethe problem of rejection in theory.
Opens the door for a new set of problems in the face of infections

8. The challenge of post transplant therapy
The weaker the immune system

9. The challenge of post transplant therapy
The therapeutic window is small, in some cases non-existent.
Drug toxicity/intolerance further complicates the situation

10. Outline
Rejection: Cell-free donor derived DNA as a marker for rejection
Heart transplants
Lung transplants
Infection:
Shotgun sequencing detects a broad array of infectious agents:
Immunosuppression
Temporal response of the virome in plasma to immunosuppressants and antivirals

11. Outline
Rejection: Cell-free donor derived DNA as a marker for rejection
Heart transplants
Lung transplants
Infection:
Shotgun sequencing detects a broad array of infectious agents:
Immunosuppression
Temporal response of the virome in plasma to immunosuppressants and antivirals

12. Post-transplant transplant surveillance
Endomyocardial biopsy: currently the gold standard
0R R R R
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)

13. 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: 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).
Nucleosome: histone octamer wrapped 1.65 times by 147 bp of DNA
Liberation rate’
Liver, kidney, spleen involved in the removal of circulating DNA

14. 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)

15. Heart transplant: prospective study design and numbers

16. 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

17. 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

18. Signal at time of rejection
Elevated donor DNA at time of rejection

19. Signal at time of rejection
Elevated donor DNA at time of rejection

20. Graft loss and re-transplant

21. Comparison signal non-rejectors/rejectors
Significant increase in fraction of donor-derived DNA at rejection

22. 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)

23. Outline
Rejection: Cell-free donor derived DNA as a marker for rejection
Heart transplants
Lung transplants
Infection: Shotgun sequencing detects a broad array of infectious agents? Temporal response of the virome in plasma to immunosuppressants and antivirals
Immunocompetence: Antibody repertoire sequencing measures the overall state of the immune system

24. Patient survival rates in lung transplantation
Bilateral transplants perform better.
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)

25. Study design

26. Lung transplants – signal in absence of rejection
Very high levels of donor DNA immediately post transplant
High background level compared to heart transplants

27. Signal elevated at rejection (transbronchial biopsy)

28. Analysis of performance against biopsy
Exclude samples collected within first 60 days
Account for volume difference for single/bilateral transplants

29. 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

30. Relative genomic abundance of species
Viruses from the anelloviridae family are most abundantly represented among viruses
Cell 2013

31. Relative genomic abundance of species
The anelloviridae fraction is primarily composed of viruses from the alphatorque genus
Cell 2013

32. 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)

33. Chronic Viral infection
Herbert W. Virgin et al., Cell, 138 (2009)

34. Immunosuppressants and antivirals alter structure of the virome
47 patients; 380 samples

35. Virome temporal dynamics
96 patients; 656 samples

36. Virome temporal dynamics
96 patients; 656 samples

37. Virome temporal dynamics
96 patients; 656 samples

38. Rejection vs infection
Infectious burden different for non-rejecting vs. rejecting patients?
non-rejecting
rejecting

39. 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)

40. 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)

41. 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.

42. Shotgun sequencing for the screening of infections
Many oncoviruses and pathogens are not screened for frequently in current clinical practice.
Shotgun sequencing reveals high incidence.
Disconnect between the frequency of clinical testing and incidence of infection.
Submitted

43. Outline
Rejection: Cell-free donor derived DNA as a marker for rejection
Heart transplants
Lung transplants
Infection:
Shotgun sequencing detects a broad array of infectious agents:
Immunosuppression
Temporal response of the virome in plasma to immunosuppressants and antivirals

44. Acknowledgements Steve Quake Quake lab Jennifer Okamoto
Christopher Vollmers
Lance Martin
Thomas Snyder
Norma Neff
Mark Kowarsky
Mickey Kertesz
Steve Quake
Stanford hospital:
Calvin Strehl, Garrett Cohen, Bitika Kohli, Helen Luikart, Daniel Bernstein, David Weill, Kapil Patel, Mark Niccols, David Cornfield, Hannah Valentine,
Kiran Khush
NIH grant RC4AI092673

46. Extra slides

47. Potential for early diagnostics
Donor DNA levels significantly elevated up to 5 months prior to a rejection event

48. Response to rejection treatment
Donor DNA after detection and treatment of severe rejection.

49. Error rate – independent measurement
Median error 0.04%
Matched SNP positions allow estimating the frequency of erroneous donor calls

50. Evidence of graft injury exclusively for CMV infections
Bonferroni adjusted significance threshold

51. 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

52. Infection diagnosis via shotgun sequencing?
CMV detected in plasma for patients that test positive for CMV.