PMC9874630_01
Clinical timeline and tumor profiling. (A) Treatment history: adjuvant chemotherapy with Epirubicine plus Cyclofosphamide (EC: 90/600 mg/mq, q14), followed by Taxol (175 mg/mq, q14) plus Pegfilgrastim, and Aromatase Inhibitor (Ai: Letrozole 2.5 mg/day). First line therapy with Palbociclib (CDK/6i:125 mg/die for 21 days, every 28 days) plus Aromatase Inhibitor (Ai: Letrozole 2.5 mg/day). Surgical removal of a thoracic skin metastasis. Second line with Trastuzumab (TTZ: 8 mg dose loading   6 mg) plus Pertuzumab (PTZ: 840 mg dose loading   420 mg) and weekly Taxol. Third line with T-DM1 (3.6 mg/kg, every 21 days) and Letrozole (2.5 mg/day), beginning of LB monitoring. Fourth line, off-label Fulvestrant (500 mg day 1, 15, 28, then every 28 days) in association with Capecitabine (Cap: 2000 mg/m2 daily discontinued after a single cycle). Fifth line with Vinorelbine (VNR: 25 mg/mq at day 1 and 8) plus Trastuzumab (8 mg/kg -> 6 mg/kg) every 21 days. Asterisks: tissue and blood samples tested by both NGS and dPCR. All the other samples: testing by dPCR only. (B, C) CT scans before and following treatment with Fulvestrant; dates of clinical imaging are noted. Major sites of response to Fulvestrant are indicated by white arrows. Cap*, Capecitabine (D-M) Key time points at which tumor tissue/cells and blood drawings were obtained. Single nucleotide variants (SNV) are color-coded by tissue of origin in (D). Palettes rank alterations by variant allele frequency (different %VAFs) and copy numbers (CNA). Copy numbers are expressed per diploid genome: copy number-neutral n=2. tDNA, tumor DNA; ctDNA, circulating tumor DNA. (-) undetectable. n.a., not assessed because SNV/CNA was not included in the LB-grade NGS panel.
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The clinical timeline, treatments and main diagnostic assessments in this patient are summarized in  Figure 1A . Clinical progression (massive dissemination to the lung, pleura and liver) occurred in June 2019 (total body CT scan,  Figure 1B ). 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 ). 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 . Results are shown for 7 representative blood drawings ( Figures 1F-M ; n.a.: not assessed). 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. None of the three SNVs could be detected in the pleural effusion ( Figure 1J ). 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). 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 ). 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 ).
Fishplots and tumor phylogenesis. Fishplots displaying alterations in blood (A) and tumor lesions (B). Areas are proportional to relative SNV abundances, whereas CNAs are depicted in background, not to scale. (C) Tumor phylogenetic tree: boxes surround evolution modes. Connecting lines: parental relationship proven (continuous) and inferred (dotted). ESR1 mutations: dimension of dots (blood variants) is roughly proportional to clonal representation. Tumor clones are consistently color-coded between fishplot areas and the dots of the phylogenetic tree. Abbreviations as in 
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Figures 2A, B ). For simplified CNA rendering, we focussed on FGFR1 and MYC copy numbers, that are available for both blood and tissues. Specifically, the fishplot in  Figure 2A  clearly shows relative abundance and kinetics of ESR1 p.D538G p.Y537S and p.Y537N. 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 ). 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. 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. The inferred tumor phylogenetic tree is shown in  Figure 2C .