Papadopoulos:Ovary, CRC, Bladder, Gastro-esophageal at 100% sens for Stage IV. Other types not as well. #NGDx15
11:03am August 20th 2015 via Hootsuite
Papadopoulos: Widely applicable. Survey ctDNA, plasma from many pts. Looked in tumor, SafeSeqS or BEAMing (dPCR method) in parallel #NGDx15
11:02am August 20th 2015 via Hootsuite
Papadopoulos: Interpret the UID (barcode): present in sample 'legitimate' errors ass'd with same barcode. Error decrease 70x #NGDx15
11:00am August 20th 2015 via Hootsuite
Papadopoulos: Describes the Safe-SeqS method Kinde et al 2013 http://t.co/wNN4jdMGhA Each molecule has its own barcode #NGDx15
10:59am August 20th 2015 via Hootsuite
Papadopoulos: NGS is digital, high TP and automatable - but the error rate is too high for early detection #NGDx15
10:57am August 20th 2015 via Hootsuite
Papadopoulos: Refers to 1999 original PNAS digital PCR paper http://t.co/c5PoCcszo9 #NGDx15
10:56am August 20th 2015 via Hootsuite
Papadopoulos: Technical challenge of needle in haystack: amount is small. 1-5 mutant molecules in the wild-type ocean. #NGDx15
10:55am August 20th 2015 via Hootsuite
Papadopoulos: Tumor DNA as a marker can be in not only blood but stool (and urine, sputum, others) #NGDx15
10:54am August 20th 2015 via Hootsuite
Papadopoulos: Why:specific markers, window for early stage, non-invasive. Applications include risk, screen, Dx, monitor, pred resp #NGDx15
10:52am August 20th 2015 via Hootsuite
Papadopoulos: Markers for tumor DNA are point mutations, indels, translocations, methylation #NGDx15
10:51am August 20th 2015 via Hootsuite
Nick Papadopoulos (Johns Hopkins MD) "Detection of somatic mutation in biological fluids in the management of cancer" #NGDx15
10:50am August 20th 2015 via Hootsuite
Legendre: Q:How high to multiplex? A:Lots of optimization (also not much to say here). #NGDx15
9:55am August 20th 2015 via Hootsuite
Legendre: Q:W/multiple PCRs, what about error? A:Hi-fidelity polymerase (not much more to say) #NGDx15
9:54am August 20th 2015 via Hootsuite
.@MaceyLHenderson Feel free to send me a note with your question(s) about tranplantation (or anything else!) to dale@yuzuki.org
9:51am August 20th 2015 via Hootsuite in reply to MaceyLHenderson
Legendre: Work with MD Anderson: monitoring BRAF V600E increasing w/o therapy, and sharp drop after targeted therapy. #NGDx15
9:49am August 20th 2015 via Hootsuite
Legendre:Showed data down to 0.01% on RainDance T790M. Also on BioRad. >400x increase in sens. for both Sanger/NGS. dPCR >100x #NGDx15
9:48am August 20th 2015 via Hootsuite
Legendre: Did dilution of 0.04%, went through MX-ICP (resulted in 15%), re-calculating original amount and derived 0.05% #NGDx15
9:44am August 20th 2015 via Hootsuite
Legendre: Showed data 3% down to 0.03% (calculation before MX-ICP) which ended up at 10% after MX-ICP #NGDx15
9:42am August 20th 2015 via Hootsuite
Legendre: Products can then be Sanger seq or whatever assay downstream. 330ng is 10 copies at 0.1%. 4mL plasma needed #NGDx15
9:41am August 20th 2015 via Hootsuite
Legendre: 2nd round is a 5-step PCR; a reference-strand oligo that blocks WT from amplifying #NGDx15
9:37am August 20th 2015 via Hootsuite
Legendre: First round-multiplex generates population of WT and mutant. Second round: specialized nested PCR, preferential to mutant #NGDx15
9:36am August 20th 2015 via Hootsuite
Legendre: Called MX-ICP (multiplexed ice cold PCR) - can be coupled to Sanger, NGS, digital PCR, Pyrosequencing, even HRM #NGDx15
9:35am August 20th 2015 via Hootsuite
Legendre: NB: The 2011 original Nucl Acids Res paper with the method: http://t.co/hKlFkYc59r Assay improvement for better sens. #NGDx15
9:33am August 20th 2015 via Hootsuite
Ben Legendre (Transgenomic NE) "The use of multiplexed IC COLD-PCR w/analysis platforms for low level sequence alterations" #NGDx15
9:31am August 20th 2015 via Hootsuite
De Vlaminck:Q: Total cfDNA abundance of donor? A: Turnover and clearance vary by time of day and activity. #NGDx15
9:28am August 20th 2015 via Hootsuite
De Vlaminck:Q:Look at SNPs? A:They use genotyping of both donor / recipient; look at cfDNA at particular position. 0.5-1x WGS #NGDx15
9:26am August 20th 2015 via Hootsuite
De Vlaminck: 'Strong interaction between immune strength and virome observed' #NGDx15
9:24am August 20th 2015 via Hootsuite
De Vlaminck: Chart of isotype abundance - not much change. Abundant sequences change though - chart of repertoire of time #NGDx15
9:22am August 20th 2015 via Hootsuite
De Vlaminck: Error correction using barcoding of cDNA in first PCR step: push down the error 1/100, also absolute counting #NGDx15
9:21am August 20th 2015 via Hootsuite
De Vlaminck:Anellovirus lead them to antibody repertoire sequencing, to look at T-cell mediated B-cell activation #NGDx15
9:20am August 20th 2015 via Hootsuite
De Vlaminck: Can do shotgun sequencing (manuscript submitted) screening for infection (pathogens, oncoviruses, commensal) #NGDx15
De Vlaminck: Bone marrow transplant - Enterocytozoon bieneusi found via plasma DNA sequencing. Pt suffered from diarrhea. #NGDx15
9:19am August 20th 2015 via Hootsuite
De Vlaminck: Anellovirus load data compared rejecting vs non-rejecting patients. #NGDx15
9:17am August 20th 2015 via Hootsuite
De Vlaminck: Infection is looking at non-human DNA - 'more virus than bacteria' Anellovirus prominent #NGDx15
9:16am August 20th 2015 via Hootsuite
De Vlaminck: Bone marrow: 18K performed/y. Often due to leukemia. GVH disease: up to 30% in allogeneic. Can fail, be lost, infection #NGDx15
9:11am August 20th 2015 via Hootsuite
De Vlaminck:For lung, signal persists after transplant (unlike heart, where clearing occurs quickly). Applicable cross types #NGDx15
9:10am August 20th 2015 via Hootsuite
De Vlaminck: 10K cells / second. #NGDx15 One 2014 paper: http://t.co/pzpUwrOfXe AUC 0.72 of cfDNA as a rejection marker for heart xplants
9:09am August 20th 2015 via Hootsuite
De Vlaminck: Narrow window for monitoring, endomyocardial biopsy - only 71% agreement. cfDNA: half-life only 16.3 minutes! #NGDx15
9:07am August 20th 2015 via Hootsuite
De Vlaminck: ~180K people alive today with solid organ transplants (mainly kidney). Dual risk: rejection and infection #NGDx15
9:06am August 20th 2015 via Hootsuite
Iwin De Vlaminck (Cornell NY) "Diagnostic applications of cell-free DNA in solid organ and bone marrow transplantation" #NGDx15
9:02am August 20th 2015 via Hootsuite
Speicher: One of several Plasma-Seq papers 2014 http://t.co/3UY61vLQGr #NGDx15 Turnaround time is ~2d. Close collaborator with Klaus Pantel
9:01am August 20th 2015 via Hootsuite
Speicher: And yet no CNV observed. Work published 2014: http://t.co/trZOIpJubq #NGDx15
8:59am August 20th 2015 via Hootsuite
Speicher: Seq of 500 CTCs (singly or in pools) established a lineage between CTCs and lymph node cells. Non-responder, progressive .#NGDx15
8:58am August 20th 2015 via Hootsuite
Speicher: Looking now at CTCs, case with >100K CTCs from three different draws. (Mentioned yesterday.) DDR2, MLL3 high VAF #NGDx15
8:56am August 20th 2015 via Hootsuite
Speicher: Tyr Kinases 2009 GRAIL paper: http://t.co/FM5TJ0mO0n Loking at translocations. Showed example of prostate ca changing type NGDx15
8:54am August 20th 2015 via Hootsuite
Speicher: Prostate ca - much higher in ctDNA. Not reported in literature in pr ca - 'probably due to not looking at advanced cases' #NGDx15
8:51am August 20th 2015 via Hootsuite
Speicher: Cp to ctDNA (88 br ca smpls) very similar types of focal events. For4 prostate, large increase in 108 ctDNA vs 492 #TCGA #NGDx15
8:49am August 20th 2015 via Hootsuite
Speicher:Focal somatic CNVs in prostate, breast and other cancers. Looking at #TCGA data, 1058 smpls focal events by stage increases #NGDx15
8:47am August 20th 2015 via Hootsuite
Michael Speicher (Medical Univ Graz) "Whole genome sequencing of plasma DNA in patients with cancer" #NGDx15
8:45am August 20th 2015 via Hootsuite
.howitts_done Thanks for the kind word! Late to this AM's cfDNA talk and figuring out how I can manage at #NGDx with a broken laptop...
8:40am August 20th 2015 via Hootsuite
