TDNA 500 copies/mL of a second PIK3CA ULK2 custom synthesis mutation detectable in their baseline ctDNA samp sample, even though in lesser quantities than the the tumour mutation (FigureInterestingly, even though in lesser quantities than tumour mutation (Figure two). two). Interestingly, all all six sufferers with PIK3CA-wildtype archival tumour had detectable circulating PIK3CA individuals with PIK3CA-wildtype archival tumour had detectable circulating PIK3CA m mutations in their in their baseline plasma ctDNA. In all participants,number of copies of of PIK3 tations baseline plasma ctDNA. In all participants, the the number of copies PIK3CA mutations in ctDNA fluctuated over the the coursetreatment, with no clear trend mutations in ctDNA fluctuated more than course of of therapy, with no clear trend in re in relation to therapy response or duration. A larger peakpeak quantity of mutant PIK3CA alle tion to therapy response or duration. A greater quantity of mutant PIK3CA alleles in ctDNA didn’t necessarily appear to correlate with having a shorter survival (Figure two). in ctDNA did not necessarily appear to correlate a shorter survival (Figure two).3.4. Serial Tumour Biopsy three.4. Serial Tumour Biopsy Sequencing Sequencing Two participants had tumour 5-HT1 Receptor Modulator manufacturer biopsies biopsies post-clinical trial with Two participants had voluntary voluntary tumour pre- and pre- and post-clinical trial with s ficient tumour material exome sequencing (WES) as well as archival tumour adequate tumour material for wholefor entire exome sequencing (WES) in conjunction with archival tumo from initial from initial diagnosis. diagnosis. In both sufferers, 20 on the 20 of gene mutations mutations detected, In each sufferers, fewer than fewer than somatic the somatic genedetected, such as includ predicted functional (deleterious) and non-functional have been typical to all predicted functional (deleterious) and non-functional mutations, mutations, were popular to three timepoints Most mutations mutations had been exceptional to one or all but not all th 3 timepoints (Figure S1).(Figure S1). Mostwere unique to a single or two but nottwothree timepoints in each patients reflecting significant temporal genomic heterogeneity. timepoints in each patients reflecting significant temporal genomic heterogeneity. In 1 patient one patient (patient X), two tumour biopsies have been obtained at pre plus the pre a In (patient X), two tumour biopsies have been obtained at every single of the every of post copanlisib plus trastuzumab time points. Within the pre-trialthe pre-trial biopsies, 80/98 (81.six ) post copanlisib plus trastuzumab time points. In biopsies, 80/98 (81.6 ) somatic mutations had been shared even though shared though only of somatic gene mutations gene commatic mutations have been only 10/33 (30.3 ) 10/33 (30.three ) of somatic had been mutations w mon towards the frequent to the two tumour biopsies possibly reflecting extra intra-tumoural intratwo tumour biopsies taken post-trial, taken post-trial, possibly reflecting extra heterogeneity as the tumour evolves. tumour evolves. moural heterogeneity as theCancers 2021, 13, 1225 Cancers 2021, 13, x9 of 13 ten ofFigure two. (a) Serial circulating PIK3CA mutant alleles (ctDNA) in patients with PIK3CA mutation archival tumour (n = = Figure 2. (a) Serial circulating PIK3CA mutant alleles (ctDNA) in sufferers with PIK3CA mutation inin archival tumour (n6); (b)(b) Serial Circulating PIK3CA mutant alleles (ctDNA) in patients withno PIK3CA mutation in archival tumour (n = six); six); Serial Circulating PIK3CA mutant alleles (ctDNA) in patients with no PIK3.