The clinical timeline, treatments and main diagnostic assessments in this patient are summarized in  Figure 1A . In July 2017, a 55-year-old woman underwent quadrantectomy and axillary lymphadenectomy for a multifocal (G3) ductal infiltrating breast carcinoma of the luminal B subtype: estrogen receptor (ER) 90%, progesterone receptor (PgR) 5%, Ki67 50%, HER2-low (immunohistochemistry 2+; non-amplified by in situ hybridization). In December 2017, one month only after the end of adjuvant therapy, early metastatic dissemination to the lung, bones and parasternal soft tissue called for first-line therapy with CDK4/6i plus Letrozole. Unfortunately, further rapid progression occurred (July 2018) involving the lung and spine (L2/L3), which prompted to surgical removal of a thoracic subcutaneous metastasis for diagnostic re-assessment and targeted NGS (see below). Routine immunohistochemistry of the metastatic tissue confirmed the ER+/PgR+/HER2 2+ status detected in the primary, and in addition revealed a very slight HER2 copy number gain (HER2/CEP17 SISH ratio: 2.4). Since Trastuzumab deruxtecan was not available in year 2018, a combined regimen was attempted of Trastuzumab plus Pertuzumab and weekly Taxol for double HER2/hormone blockade (2nd line). However, novel pleural and left costal lesions developed after 5 cycles. This prompted us to initiate 3rd line therapy with Trastuzumab emtansine (T-DM1) plus Letrozole. This combination was administered in the context of a LB study, now published. LB detected progressive accumulation of 3 actionable ESR1 circulating alterations (see below) during treatment, despite apparently stable disease. Clinical progression (massive dissemination to the lung, pleura and liver) occurred in June 2019 (total body CT scan,  Figure 1B ). The patient was referred to the Regina Elena intramural Molecular Tumor Board (MTB) in very poor general conditions and dyspnoic. Off-label Fulvestrant was recommended (see below) exclusively based on LB data, in association with Capecitabine (4th line), but the latter had to be discontinued after a single cycle due to severe gastrointestinal intolerance. In contrast, Fulvestrant was well tolerated and, despite administration as single-agent, it resulted in a quick and dramatic improvement with disappearance of most lung lesions and associated pleural exudate, as well as drastic regression of the extensive liver invasion ( Figure 1C ). Response lasted 8 months until early February 2020, when the patient, who was otherwise in good general conditions and essentially asymptomatic, suddenly experienced dyspnea and chest pain due to a pleural exudate requiring thoracentesis. Salvage treatment with Trastuzumab plus Vinorelbine (5th line) resulted in partial response of the pleural effusion and apparently stable thoracic disease for about 7 additional months, until massive pleural/lung recurrence and death in November 2020.
Tumor DNAs (tDNAs) were obtained from the three sampled tumor lesions: the primary tumor, the subcutaneous metastasis, and neoplastic cells isolated from the pleural effusion following overnight adherence to plastic dishes. Circulating tumor DNAs (ctDNAs) were obtained from 14 blood drawings. All 3 tDNAs and 8/14 ctDNAs ( Figure 1A , asterisks) were tested by NGS (Life Technologies, see Supplementary for technical details) with two targeted panels of different complexity. The tDNA-grade Oncomine  Comprehensive Assay Plus detected 9 single nucleotide variants (SNVs) and 19 copy number alterations (CNA) in a total of 505 tested genes. These 27 alterations are listed along with variant allele frequencies (VAFs) and estimated copy numbers in  Figures 1D, E, J . Due to its smaller size (it detects SNVs, CNAs and fusions in 52, 12 and 92 genes, respectively), the ctDNA-grade Oncomine  PanCancer Cell-Free Assay could only assess 3 of 9 SNVs and 2 of 19 CNAs. Results are shown for 7 representative blood drawings ( Figures 1F-M ; n.a.: not assessed).
The distribution of the 9 SNVs among the three tDNAs readily revealed marked phylogenetic divergence during tumor evolution: 5 SNVs (BRCA2 p.C1820, MGA p.R1106*, PRDM1 p.A546T, REG3G p.Q69H and TP53 p.G245S) were exclusive of the primary, 3 (HRAS p.A146T, RUNDC3B p.R4W and ESR1 p.D538G) were exclusive of the subcutaneous metastasis, and only one (KMT2C p.T816) was shared between any two lesions, namely between the subcutaneous metastasis and the neoplastic pleural effusion ( Figures 1D, E, J , top section; SNVs color-coded by site of detection in panel d).
Particularly in light of rapid clinical progression, BRCA p.C1820 and TP53 p.G245S losses appeared counterintuitive. The former is a potentially inheritable, likely pathogenic variant (), and the latter is a loss-of-function cancer driver strongly associated with poor outcome (OncoKB level Px1; ). These considerations prompted to orthogonal testing by custom-designed digital PCR (dPCR) assays. However, NGS calling was confirmed, and dPCR-assessed VAFs differed marginally (within +/- 3%) from NGS-assessed VAFs (see representative TP53 testing in  Table S1 ). Likewise, dPCR confirmed that BRCA2 p.C1820 was undetectable at both tested metastatic sites and in leukocyte DNA (not shown), conclusively ruling out a germ line origin.
Next, we focused on the similarly puzzling observation of a simultaneous presence of three gain-of-function ESR1 SNVs (p.D538G, p.Y537S and p.Y537N), all known to be actionable and associated with resistance to hormone blockade. Interestingly, these three SNVs displayed very different origins and accumulation kinetics, as may be seen by comparing tDNA from the skin metastasis, and ctDNAs obtained before the T-DM1/Letrozole and Fulvestrant treatments, at progression, and at later time points ( Figures 1E-M ). Specifically, ESR1 p.D538G was the only ESR1 alteration to be detected in tissues. It displayed a very low VAF at onset both in tDNA and ctDNA, and then marked blood increases mostly coincident with clinical progression. In contrast, ESR1 p.Y537S and p.Y537N, initially undetectable, appeared de novo in blood, and their association with clinical outcome appeared to be less stringent. None of the three SNVs could be detected in the pleural effusion ( Figure 1J ). Also these changes were confirmed by orthogonal dPCR validation (representative results shown in  Table S1 ). In summary, BRCA2 p.C1820 and TP53 p.G245S were negatively selected, whereas ESR1 alterations were positively selected by successive events taking place for the most part at undetermined tumor sites.
Strikingly in contrast to the heterogeneous SNV distribution, the primary site and the skin lesion did share a large set of 19 collinear CNAs ( Figure 1D, E , bottom section). Most CNAs appeared to be inherited en bloc, since copy number gains and gene-to-gene ratios were roughly conserved between the two lesions. Only FAM135B displayed a discordant trend (increase instead of decrease/stability in copy numbers). Unfortunately, not all CNAs were testable by the ctDNA-grade NGS panel. Despite this limitation, we were able to confirm that both FGFR1 and MYC (the only two CNAs testable in blood) were amplified and, similar to tissues, copy number gains persisted in serial blood drawings, undergoing limited absolute and relative variations ( Figures 1F-L , bottom). Of special interest, FGFR1 and MYC amplifications were detected in peripheral blood collected at the time of thoracentesis despite tDNA from the pleural neoplastic cells had lost FGFR1, MYC, and all the other CNAs ( Figure 1J vs K ). The easiest interpretation of these results is that a clonal expansion containing at least FGFR1 and MYC CNAs was present throughout disease course, e.g. in the primary tumor, skin metastatic site and other unidentified tumor sites, as indirectly revealed by LB, but it was lost in the last lesion that had been sampled, e.g. tumor cells from the pleural cavity.
To display multiple SNV and CNA trajectories, longitudinal series of %VAF values and DNA copy numbers were elaborated by the fishplot package for RStudio. The 6 ctDNAs not tested by NGS were included by incorporating dPCR data in the model. To portray tumor evolution in its entirety we chose to represent SNVs and CNAs in the same graph, and generated separate fishplots for blood and tumor lesions ( Figures 2A, B ). For simplified CNA rendering, we focussed on FGFR1 and MYC copy numbers, that are available for both blood and tissues. These were averaged and merged into a single fishplot. However, since %VAF and gene copy numbers are incommensurable, the latter were displayed in background and not to scale. It is acknowledged that no inference can be made about relative SNV/CNA subclonal representation, whereas quantitative relationships among multiple SNVs are faithfully graphed.
Specifically, the fishplot in  Figure 2A  clearly shows relative abundance and kinetics of ESR1 p.D538G p.Y537S and p.Y537N. Quite strikingly, the two most abundant variants displayed a roughly reciprocal relationship whereby expansion of either is mirrored by contraction of the other. Unsurprisingly, circulating ESR1 alterations were prodromic to life-threatening clinical progression (see case description), which led to seek MTB multidisciplinary advice.
In elaborating therapeutic recommendations, the MTB considered the following points: (a) the 3 actionable (OncoKB level 3A) ESR1 SNVs had disabled two consecutive codons in the catalytic ESR1 site; (b) they had occurred in rapid succession within 7 months from the first T-DM1/Letrozole administration; (c) their distinct kinetics and tissue/blood distribution were suggestive of convergent adaptive events in distinct founders; (d) their timing of appearance and subsequent clinical progression at lung and liver foci was compatible with an elective topographical distribution of at least some ESR1 variants at these sites.
Preliminary evidence was also considered from the plasmaMATCH trial, now published, that subclonal ESR1 alterations when detected in blood do not confer sensitivity to targeted treatment. Nevertheless, in light of arguments (a-d), and in the absence of alternative therapeutic options, off-label Fulvestrant was recommended exclusively based on LB data, and in association with Capecitabine.
As outlined in case description, only Fulvestrant was tolerated, and dramatic clinical improvement (CT scans in  Figure 1C , performed in November) was mirrored, in December 2019, by nearly complete blood clearance of the newly arisen ESR1 p.Y537S and p.Y537N variants, although the ESR1 p.D538G clone (chronologically the first to appear) re-expanded, and FGFR1/MYC CNAs kept expanding steadily in blood ( Figure 2A ). These results formally prove direct variant (particularly ESR1 p.Y537S) targeting by Fulvestrant, and provide evidence for a dissociated response, both molecular and clinical.
At the time of further pleural relapse in early February 2020, the ESR1 p.D538G variant and the FGFR1 and MYC copy number gains were all abundant in blood ( Figure 2A ). However, none was detectable in tumor cells obtained on the same day from the neoplastic pleural effusion, that only retained a single 'undruggable' KMT2C SNV ( Figure 2B ) first seen in the 2018 cutaneous metastasis. While further supporting clearance of ESR1 variants from the pleural space, these results prevented target FGFR1 treatment, and prompted instead to Trastuzumab plus Vinorelbine as a possible salvage therapy. Partial response of the pleural effusion was accompanied by decreases in FGFR1, MYC and ESR1 p.D538G variants, and (re)-expansion of ESR1 p.Y537S ( Figure 2A ). Subsequent increases of all variants in October 2020 ( Figure 2A , last blood drawing) heralded massive pleural/lung recurrence and death in November 2020.
For synoptic view, the CNA trunk and the 11 SNVs detected in tDNAs and ctDNAs were assigned to specific tumor lesions and blood drawings by applying the pigeonhole principle and logical deduction. The inferred tumor phylogenetic tree is shown in  Figure 2C .