Thread by @AshAlizadeh

Delighted to share our new study nature.com/articles/s4158… introducing PhasED-Seq out today @NatureBiotech.

A fantastic collaboration from @StanfordMedicine led by Dave_Kurtz & Joanne Soo with @max_diehn to help transform #cancer interception & monitoring by improving #LiquidBiopsy #ctDNA detection of #MRD.

For many cancers & nearly all currently available techniques, the impressive KM plots of #ctDNA #MRD immediately after definitive Rx w/ curative-intent unfortunately still miss ~50% of all events which occur in the MRD-negative subset, thus having modest NPV.

For example, moderate clinical sensitivity has been seen in large prospective MRD studies

https://twitter.com/aaleshin/status/1337064891254005760?s=20

We were motivated to improve this modest clinical sensitivity of existing #MRD methods & inefficiencies of techniques like #CAPP-Seq & #duplex #sequencing, which have reached a plateau even when using bespoke panels… sciencedirect.com/science/articl…

So, we set out to improve on these #MRD methods using a novel class of alterations we call Phased Variants (PVs). We consider PVs where >=2 alterations segregate in ‘cis’ on single DNA strands of individual short, nucleosomal #cfDNA fragments (i.e. mutations <170 bases apart)...

How common are PVs? We were struck by their high prevalence in #WGS across thousands of human #cancers in #PCAWG #TCGA #ICGC and elsewhere, including #lymphomas, #lungcancer, #breastcancer, #melanoma, #livercancer, #pancreaticcancer, and others...

What drives the genesis of PVs in tumors? We were also struck by PV’s ability to enrich signatures of specific mutagenic mechanisms in diverse cancer types, including AID, #APOBEC, #smoking, and others …

Where in the genome do PVs fall? For the vast majority of patients with #lymphoma, PVs are highly stereotyped across the genome & strongly cluster near canonical driver genes of individual tumor subtypes (eg #MYC #BCL2 #BCL6 #ID3).

This allows an off-the-shelf strategy for their efficient targeted PV capture directly from plasma #cfDNA, alleviating the need for tumor tissue and associated logistical hurdles… #lymsm

What makes PVs special for #MRD? In #cfDNA we found PVs to have strikingly low biological & technical background levels.

This means PVs have sublime sequencing error profiles, even lower than duplex, while having better efficiency of recovery & obviating the need for #UMIs & #barcoding! This allows ~40x improvement in analytical sensitivity allowing detection down to less than a part per million!

What does this 40 to 100+ fold improved analytical sensitivity mean clinically, and how is it relevant for #PrecisionMedicine? In profiling hundreds of samples, we observed >2x improved in clinical sensitivity of CAPP-Seq for monitoring #DLBCL, while maintaining high specificity

So, for MRD-driven trials, applying this more sensitive approach could mean capturing 2x the eligible patient population as being MRD+, yet doing the trial in 1/2 the time with 1/2 as many patients to achieve the same total number of events.

On the flip side, the high NPV should allow therapeutic abbreviations with less risk of false-negative results. That might allow us to do less harm by over treating fewer patients.

For example, this patient of mine discontinued therapy against medical advice for Stage IV DLBCL after only 1 rx cycle. This patient is nevertheless now alive without disease >6 years later, and was MRD-negative by PhasED-Seq just 25 days after start of therapy...

Does PhasED-Seq work for other tumor types beyond lymphomas? Yes! We were also able to translate the method to solid tumors including for monitoring localized #lungcancer and #breastcancer showing significant advantages of the approach #lcsm #bcsm

This study would not have been possible without the many contributions from the co-authors @StanfordCancer including @JakeChabon @mohamshah @co_keh @StefanAlig @everettmoding @emham @michael_c_jin @DrBinSquared @macaulay_cw @schroersmartin @schultzdre & others

We're also indebted to our international consortium & collaboration with @NCI @MDAndersonNews @iosilab @UniklinikEssen @UniFreiburg @LysaLymphoma including @RoschewskiMD @DrJasonWestin @WyndhamWilson @FSchererMD @WilsonWyndham @GianlucaGaidano @Lymphoma_Doc Davide Rossi & others.

We are so grateful for the patients and their families for participating in our studies. We’re working on bringing this assay to patients and providers thru @ForesightDx, as we envision this approach could be used in #PrecisionMedicine strategies across cancers.

Honored for the generous support of @NIH @NCI @lls @ASH_hematology @Ludwig_Cancer @DamonRunyon @TheVFoundation @ConquerCancerFd @EmCollective and others