PMC4970582_01
Assembly of a distributed network. Components in place for all patients enrolled in ITOMIC-001 are shown in black. Components added specifically for the patient presented here are shown in red. Arrow indicates timeline of the study from day 0 to the time of the patient's death on day 276.. Abbreviation: CTC, circulating tumor cell.
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F1
Over the course of the patient's participation in the study, we sought the expertise of 32 researchers from 14 different institutions (Figure 1).
Clinical status at time of enrollment, bone marrow biopsies, and mutation profiles. (A) Extensive tumor involvement in a prestudy bone marrow biopsy (box). (B) Progressive decline in platelet counts in the weeks before study enrollment. (C) Nucleated red blood cells (closed arrowheads) and tear drop cells (open arrowheads), consistent with a myelophthisic process. (D) Presence of tumor cells in touch preparations from bone marrow cores obtained at study entry. (E) Representative flow cytometry profile of hematopoietic cells (green) versus tumor cells (red) in a baseline bone marrow sample. (F) Schematic depiction of biopsy sites (circles) from the left (L) and right (R) posterior iliac crest. Filled circles (designated B1, B2, and B3) indicate samples that were analyzed. (G) Schematic depiction of the mutational profile of bone marrow samples B1-B3 as determined by MuTect. Select genes are noted. Colors denote variant allele frequencies (VAF).
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Prestudy bone marrow biopsy results (Figure 2A), declining platelet count (Figure 2B), normal coagulation profile (data not shown), and leukoerythroblastic blood smear results (Figure 2C) were compatible with tumor replacement of the marrow. Following a platelet transfusion for thrombocytopenia, 5 core bone marrow biopsies were performed under conscious sedation (Figure 2D-F). Core samples were minced to generate cell suspensions and the 3 most abundant (B1-B3) (Figure 2F) were analyzed. To encompass all potentially actionable variants, we leveraged multiple samples, capture platforms, and computational algorithms (Figure 2G). A total of 67 single nucleotide variants and indels (including a TP53 nonsense mutation) were found in 2 of 3 samples, of which 45 (67%) were detected in the spatially proximate B1 and B2 samples (Figure 2F, G), consistent with regional spread of tumor sub-clones within the marrow. A total of 125 variants were present in all 3 bone marrow samples, and 44 missense, splice-site, and nonsense mutations were estimated to be present in most tumor cells based on their relatively high frequencies across marrow samples (Figure 2).
Dynamic changes in circulating tumor cells (CTCs) in response to therapeutic interventions. (A) CTCs; (B) CTC fragments, and (C) CTC clusters over the study period in the context of CA15-3 (D), platelet counts (E), and ECOG performance status (F). Solid arrows denote weekly cisplatin treatment. Open arrows denote cisplatin treatment every 3 weeks. L refers to leukapheresis procedures. Crizotinib treatment is noted by an open rectangle. Eribulin treatments are denoted by check marks.
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Cisplatin was discontinued after 21 weeks because of neuropathy (Figure 3). CTCs increased sharply following the initiation of crizotinib, accompanied by a decline in platelet count and worsening performance status, and the drug was stopped after 3.5 weeks (Figure 3A). CTCs decreased sharply but only transiently in response to eribulin (Figure 3A). Notably, although CTC levels increased moderately in concert with her clinical deterioration, a marked increase in CTC clusters was observed in the days preceding her death (Figures 6A, 3C).
PMC4970582_02
Dynamic changes in circulating tumor cells (CTCs) in response to therapeutic interventions. (A) CTCs; (B) CTC fragments, and (C) CTC clusters over the study period in the context of CA15-3 (D), platelet counts (E), and ECOG performance status (F). Solid arrows denote weekly cisplatin treatment. Open arrows denote cisplatin treatment every 3 weeks. L refers to leukapheresis procedures. Crizotinib treatment is noted by an open rectangle. Eribulin treatments are denoted by check marks.
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F3
Cisplatin was discontinued after 21 weeks because of neuropathy (Figure 3). CTCs increased sharply following the initiation of crizotinib, accompanied by a decline in platelet count and worsening performance status, and the drug was stopped after 3.5 weeks (Figure 3A). CTCs decreased sharply but only transiently in response to eribulin (Figure 3A). Notably, although CTC levels increased moderately in concert with her clinical deterioration, a marked increase in CTC clusters was observed in the days preceding her death (Figures 6A, 3C).
Experts contacted regarding the ROS1(Y2092C) variant observed in this patient. Solid lines denote primary contacts.
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F4
After a PubMed search, we contacted individuals with relevant expertise (Figure 4).
Development of a differential high throughput drug screen. (A) Following leukapheresis, circulating tumor cells (CTCs) were partially enriched using lineage depletion, and both the CTC-enriched and CTC-depleted cells were tested in a high-throughput screen of 160 investigational and approved oncology drugs. (B) Responses to BCL-2 inhibitors (ABT-263, ABT-737, obatoclax) and mitoxantrone (used as a positive control). Left columns show responses among CTC-enriched cells (blue lines) and CTC-depleted cells (red lines). Left middle column depicts summation of effects on CTC-enriched versus CTC-depleted cells. A declining slope indicates relatively greater potency against neoplastic cells, a rising slope indicates relatively greater potency against normal hematopoietic cells, whereas a flat line indicates similar potencies against neoplastic and hematopoietic cells. Right middle column depicts FITC-labeled, apoptotic cells detected using an antibody (M30), and the far right column depicts the corresponding bright field images. (C) Responses (indicated by area under the curve [AUC]) to ABT-263, ABT-737, and obatoclax across 20 triple-negative breast cancer (TNBC) cell lines (designated 1-20). (D) All correlations among all 160 drugs in the screen across the 20 TNBC cell lines. Open arrow shows that the correlation between ABT-263 and ABT-737 ranks in the top 3% approximately, whereas neither drug correlates with obatoclax (gray and black arrows).
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To interrogate a heterogeneous CTC-enriched mixture, we developed a differential drug screen, in which both cell pools obtained after magnetic bead separation:the CTC-enriched and CTC-depleted fractions:were tested and compared (Figure 5A). Among 160 drugs, only 2 exhibited preferential toxicity against the CTC-enriched population: ABT-263 and ABT-737, both BH3-mimetic inhibitors of BCL-2 (Figure 5B). ABT-263 and ABT-737 (Figure 5B), but not to obatoclax. ABT-263, ABT-737, and obatoclax were further compared across 20 TNBC lines, and although responses to ABT-263 and ABT-737 were highly correlated (r=0.89), there was no correlation between either drug versus obatoclax (Figure 5C, D). These findings are consistent with the similarities in structure and high-level affinity for BCL-2 family members for ABT-263 and ABT-737 relative to obatoclax, and with off-target effects associated with obatoclax.
Circulating tumor cell (CTC) cluster late in the study and extensive tumor occlusion of the pulmonary arteriolar vasculature. (A) A CTC cluster from study day 271. Left panel shows a merged image; middle panel shows cytokeratin staining; right panel shows EpCAM staining (original magnification x40). The blue color indicates DAPI (4',6-diamidino-2-phenylindole)-stained nuclei. (B) A low-power view of lung tissue in which multiple vessels contain clusters of dark-staining cells (arrowheads) (hematoxylin-eosin [H&E], original magnification x4). (C) Image shows no identified involvement of pulmonary lymphatics (H&E, original magnification x10). (D) Images shows a pulmonary infarct (H&E, original magnification x2). (E) Magnified view of Figure 6B (H&E, original magnification x10). (F) In many of these arteries, there is near occlusion of the lumen by breast carcinoma cells, and intimal expansion by smooth muscle cells, myofibroblasts, and extracellular material (H&E, original magnification x40). (G) A Movat stain revealing that the vessels are arteries (containing both internal and external elastic laminae) (H&E, original magnification x40).
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An autopsy revealed widely disseminated intravascular carcinoma involving the lungs, epicardium, and brain, with diffuse occlusion of the arteriolar vasculature (Figure 6). Notably, although CTC levels increased moderately in concert with her clinical deterioration, a marked increase in CTC clusters was observed in the days preceding her death (Figures 6A, 3C).