Q: How quantitative? Tost: Report back 0.1% - 0.5%, 1-5%, and >5% #MDxE16
11:19am April 6th 2016 via Hootsuite
Tost: Shows multiplexing to 3-plex, BRAF V600E, KRAS G12/13, and NRAS Q61 #MDxE16
11:13am April 6th 2016 via Hootsuite
Tost: n=72, treated melanoma pts w/BRAF mutations. detected at 1,2 and 3mos. #MDxE16
11:09am April 6th 2016 via Hootsuite
Tost: QIAamp gave a bit more event; Norgen higher yield. Results published here '15 https://t.co/3UQeG7y3sp #MDxE16
11:07am April 6th 2016 via Hootsuite
Tost: Chemagic - good at 5mL but poor at lower volume. 3 good kits - Norgen, QIAamp, Nucleospin. #MDxE16
11:06am April 6th 2016 via Hootsuite
Tost: Tested 11 extraction methods; published, home-brew, most phenol chloroform. Results: "Don't try home-made products" #MDxE16
11:05am April 6th 2016 via Hootsuite
Tost: Shows sensitivity down to 0.1% for KRAS in cell lines. Showed two patient examples #MDxE16
11:04am April 6th 2016 via Hootsuite
Tost: They also use pyrosequencing; quantitative genotyping and allele ID. They've developed VBA scripts to ID KRAS muts #MDxE16
11:01am April 6th 2016 via Hootsuite
Tost: W/LNAs, critical temp becomes not so critical for ICE-COLD PCR. 0.5% gets brought up to 80% #MDxE16
10:57am April 6th 2016 via Hootsuite
Tost: Then miRNAs using Exiqon with LNAs that give better qPCR results. LNA sticks very well; thus enhanced ICE-COLD; w/o preamp #MDxE16
10:56am April 6th 2016 via Hootsuite
Tost: Shows chart of sensitivity, need to know a priori the mutation. Showed diagram of how COLD PCR works, ICE-COLD blocker mod #MDxE16
10:54am April 6th 2016 via Hootsuite
Jorg Tost (CNG Evry France): ID of rare and subclonal mut's and impact on personalized cancer treatment using enhanced ICE-COLD PCR #MDxE16
10:51am April 6th 2016 via Hootsuite
Heitzer: A modified FASt-SeqS method here '13 Clin Chem https://t.co/4egpgCim8I shows time-course in br ca, CEA, CA15-3, z-score #MDxE16
10:43am April 6th 2016 via Hootsuite
Heitzer: Her method Plasma-Seq for CNAs and SNVs, for prostate ca. '13 ref https://t.co/sbbXFhR4s1 #MDxE16
10:41am April 6th 2016 via Hootsuite
Heitzer: Shows Lo's Clin Chem WGS from plasma, and then onto methylation '15 PNAS https://t.co/rpCvP6QEXg #MDxE16
10:38am April 6th 2016 via Hootsuite
Heitzer: Her group looked at CNAs from plasma '15 ref https://t.co/DHlBgB4CEr in Stg IV CRC #MDxE16
10:36am April 6th 2016 via Hootsuite
Heitzer: Murtaza '13 Nature (again) WES and looking at treatment-assoc'd mutational changes. How mutations change w/treatment #MDxE16
10:35am April 6th 2016 via Hootsuite
Heitzer: This '15 EMBO https://t.co/EQrtMSGP9L looked at primary br ca and occult metastatic disease monitoring of rearrangements #MDxE16
10:34am April 6th 2016 via Hootsuite
Heitzer: Showed PARE paper from '12 STM Leary et al for chromosomal rearrangements; sens / spec >99% at 0.5% AF #MDxE16
10:32am April 6th 2016 via Hootsuite
Heitzer: Diehn's CAPP-Seq optimized low-input library, used TCGA data for tumor-specific selectors. #MDxE16
10:31am April 6th 2016 via Hootsuite
Heitzer: Safe-Seq method, improves accuracy 70-fold via barcoding. Reviewed TAm-Seq as well; paper got to 2% but improved since #MDxE16
10:28am April 6th 2016 via Hootsuite
Heitzer: Analysis of EGFR for lung ca - eligibility for tyr kinase inh; development of resistance and 2nd, 3rd line treatment #MDxE16
10:25am April 6th 2016 via Hootsuite
Heitzer: Input number, recovery rate: 10ng is 1500 diploid GE; 0.01% is 0.15 positive events from 10ng; 0.1% is 1.5 positives #MDxE16
10:22am April 6th 2016 via Hootsuite
Heitzer: Calls Bettegowda et al '14 'landmark paper' https://t.co/8O9jubZBXD High-res <1% include many methods. Detection LOD / LOQ #MDxE
10:21am April 6th 2016 via Hootsuite
Heitzer: Shows figure from this '11 review https://t.co/CYHNwBXm3u Challenges of fragmentation, low AF, but surrogate marker #MDxE16
10:19am April 6th 2016 via Hootsuite
Ellen Heitzer (Med Univ Graz, Austria): Recent technological advances in the analysis of ctDNA - are we ready for clinical use? #MDxE16
10:17am April 6th 2016 via Hootsuite
Casadio: Onto prostate; '13 ref https://t.co/ZYh8dIuwfR But DNA integrity isn't better for prostate ca. #MDxE16
Casadio: Shows ROC curve for bladder ca: sens 0.73, spec 0.84. Combining cytology (spec 0.53) w/DNA integrity (spec 0.78) #MDxE16
9:23am April 6th 2016 via Hootsuite
Casadio: 2mL urine; quant via spectrophotometry; use qPCR to look at 125bp STOX1, HER2, BCAS1, C-MYC, AR genes #MDxE16
9:20am April 6th 2016 via Hootsuite
Casadio: Normal cells release fragmented DNA, bladder/prostate ca cells will be higher DNA integrity #MDxE16
9:19am April 6th 2016 via Hootsuite
Casadio: Current early-detection: bladder ca has urine cytology low sens; prostate ca has PSA w/low spec #MDxE16
Casadio: Urine DNA highly fragmented, passing through glomerulus. Shows list of references of urine cfDNA sens/spec 0.7/0.8 #MDxE16
9:18am April 6th 2016 via Hootsuite
Valentina Casadio (IRCCS Italy): Urine cell-free DNA integrity analysis for the early detection of bladder and prostate cancer #MDxE16
9:14am April 6th 2016 via Hootsuite
Q:How accurate does it have to be for clinical use? Massie: Diff bet. control and pt samples; have good sep between background #MDxE16
9:11am April 6th 2016 via Hootsuite
Massie: Plasma samples over time overlaid; stem mutations tracked (Murtaza '15 ref) shows progression of lung metastasis #MDxE16
9:07am April 6th 2016 via Hootsuite
Massie: One pt (who died) consented to multiple sampling of tumor sites; comparison of mutations across sites w/private muts #MDxE16
9:05am April 6th 2016 via Hootsuite
Massie: Shows unpublished data on P53 and PIK3CA mutations over time, looking at one may miss the emergence of the other over time #MDxE16
Massie: Showing melanoma timepoints, most genes mutated have low recurrence rates; within pts there is clonal diversity #MDxE16
9:03am April 6th 2016 via Hootsuite
Massie: Over time, when below 1% (54 mutations) 'det of any individ mutation is unreliable'. Multiple mutations increases sampling #MDxE16
9:02am April 6th 2016 via Hootsuite
Massie: Inivata applies to 0.1-0.2% sens, he's looking at pt-specific panels. Melanoma study: AF's shown above / below 1% threshold #MDxE16
9:00am April 6th 2016 via Hootsuite
Massie: 1:25K or 1:200K sens (NGS; dPCR). May be looking for things 'that aren't there'. 100 cfDNA molecules; unlikely to be present #MDxE16
8:59am April 6th 2016 via Hootsuite
Massie: Detection and monitoring: making sure 'there's only hay in the stack'. 10mL blood = 4mL plasma = 1mL used. 2K copies, 0.05% #MDxE16
8:58am April 6th 2016 via Hootsuite
Massie: The challenge - heterogeneity between tumors. Needle in haystack problem of larger volumes, more sensitive methods #MDxE16
8:57am April 6th 2016 via Hootsuite
Massie: Late-stage pts - multiple logs of diff in abundance. Showed data from others on prognostic indicator of risk of relapse #MDxE16
8:56am April 6th 2016 via Hootsuite
Massie: Rosenfeld's group: TAm-Seq https://t.co/YzfRqfKvWO Shows data that monitoring, relapse, data from Bettegowda STM '14 #MDxE16
8:54am April 6th 2016 via Hootsuite
Massie: Shows applications for cancer: stratification, treatment, monitor relapse; his focus is on early detection / screening #MDxE16
8:53am April 6th 2016 via Hootsuite
Massie: Most come from dying hematopoetic cells. About 10^3 cfDNA copies/mL in plasma; advantages of local sampling (saliva, urine) #MDxE16
8:52am April 6th 2016 via Hootsuite
Charlie Massie (CRUK UK): Enhanced sensitivity for cell-free tumour DNA analysis using multiple patient-specific assays #MDxE16
8:50am April 6th 2016 via Hootsuite
Makrigiorgos: Method can be upfront of any PCR or multiplex PCR/COLD-PCR, and whatever readout (MALDI, Sanger, NGS, dPCR etc) #MDxE16
8:48am April 6th 2016 via Hootsuite
Makrigiorgos: Reviews how NaME can be applied to bisulfite-treated ctDNA; showed from 50% C/T to 100% w/probes and DSN #MDxE16
8:47am April 6th 2016 via Hootsuite
