Understanding the evolutionary pathways to metastasis and resistance to immune-checkpoint inhibitors (ICI) in melanoma is critical for improving outcomes. Here, we present the most comprehensive intrapatient metastatic melanoma dataset assembled to date as part of the Posthumous Evaluation of Advanced Cancer Environment (PEACE) research autopsy program, including 222 exome sequencing, 493 panel-sequenced, 161 RNA sequencing, and 22 single-cell whole-genome sequencing samples from 14 ICI-treated patients. We observed frequent whole-genome doubling and widespread loss of heterozygosity, often involving antigen-presentation machinery. We found KIT extrachromosomal DNA may have contributed to the lack of response to KIT inhibitors of a KIT-driven melanoma. At the lesion-level, MYC amplifications were enriched in ICI nonresponders. Single-cell sequencing revealed polyclonal seeding of metastases originating from clones with different ploidy in one patient. Finally, we observed that brain metastases that diverged early in molecular evolution emerge late in disease. Overall, our study illustrates the diverse evolutionary landscape of advanced melanoma. Significance: Despite treatment advances, melanoma remains a deadly disease at stage IV. Through research autopsy and dense sampling of metastases combined with extensive multiomic profiling, our study elucidates the many mechanisms that melanomas use to evade treatment and the immune system, whether through mutations, widespread copy-number alterations, or extrachromosomal DNA. See related commentary by Shain, p. 1294. This article is highlighted in the In This Issue feature, p. 1275

Circulating tumor cells (CTCs) detected by CellSearch are prognostic in non-small-cell lung cancer (NSCLC), but rarely found. CTCs can be extracted from the blood together with mononuclear cell populations by diagnostic leukapheresis (DLA), therefore concentrating them. However, CellSearch can only process limited DLA volumes (≈2 mL). Therefore, we established a protocol to enumerate CTCs in DLA products with Isolation by SizE of Tumor cells (ISET), and compared CTC counts between CellSearch^® and ISET. DLA was performed in NSCLC patients who started a new therapy. With an adapted protocol, ISET could process 10 mL of DLA. CellSearch detected CTCs in a volume equaling 2 × 10⁸ leukocytes (mean 2 mL). CTC counts per mL were compared. Furthermore, the live cell protocol of ISET was tested in eight patients. ISET successfully processed all DLA products—16 with the fixed cell protocol and 8 with the live cell protocol. In total, 10–20 mL of DLA was processed. ISET detected CTCs in 88% (14/16), compared to 69% (11/16, p < 0.05) with CellSearch. ISET also detected higher number of CTCs (ISET median CTC/mL = 4, interquartile range [IQR] = 2–6, CellSearch median CTC/mL = 0.9, IQR = 0–1.8, p < 0.01). Cells positive for the epithelial cell adhesion molecule (EpCAM+) per mL were detected in similar counts by both methods. Eight patients were processed with the live cell protocol. All had EpCAM+, CD45−, CD235- cells isolated by fluorescence-activated cell sorting (FACS). Overall, ISET processed larger volumes and detected higher CTC counts compared to CellSearch. EpCAM+ CTCs were detected in comparable rates.

Chemotherapy resistance and relapses are common in high-risk neuroblastoma (NB). Here, we developed a clinically relevant in vivo treatment protocol mimicking the first-line five-chemotherapy treatment regimen of high-risk NB and applied this protocol to mice with MYCN -amplified NB patient-derived xenografts (PDXs). Genomic and transcriptomic analyses were used to reveal NB chemoresistance mechanisms. Intrinsic resistance was associated with high genetic diversity and an embryonic phenotype. Relapsed NB with acquired resistance showed a decreased adrenergic phenotype and an enhanced immature mesenchymal–like phenotype, resembling multipotent Schwann cell precursors. NBs with a favorable treatment response presented a lineage-committed adrenergic phenotype similar to normal neuroblasts. Novel integrated phenotypic gene signatures reflected treatment response and patient prognosis. NB organoids established from relapsed PDX tumors retained drug resistance, tumorigenicity, and transcriptional cell states. This work sheds light on the mechanisms of NB chemotherapy response and emphasizes the importance of transcriptional cell states in chemoresistance.

Purpose: Tumor cells from patients with lung cancer are expelled from the primary tumor into the blood, but difficult to detect in the peripheral circulation. We studied the release of circulating tumor cells (CTCs) during surgery to test the hypothesis that CTC counts are influenced by hemodynamic changes (caused by surgical approach) and manipulation. Experimental Design: Patients undergoing video-assisted thoracic surgery (VATS) or open surgery for (suspected) primary lung cancer were included. Blood samples were taken before surgery (T0) from the radial artery (RA), from both the RA and pulmonary vein (PV) when the PV was located (T1) and when either the pulmonary artery (T2 open) or the PV (T2 VATS) was dissected. The CTCs were enumerated using the CellSearch system. Single-cell whole-genome sequencing was performed on isolated CTCs for aneuploidy. Results: CTCs were detected in 58 of 138 samples (42%) of 31 patients. CTCs were more often detected in the PV (70%) compared with the RA (22%, P < 0.01) and in higher counts (P < 0.01). After surgery, the RA but not the PV showed less often CTCs (P = 0.02). Type of surgery did not influence CTC release. Only six of 496 isolated CTCs showed aneuploidy, despite matched primary tumor tissue being aneuploid. Euploid so-called CTCs had a different morphology than aneuploid. Conclusions: CTCs defined by CellSearch were identified more often and in higher numbers in the PV compared with the RA, suggesting central clearance. The majority of cells in the PV were normal epithelial cells and outnumbered CTCs. Release of CTCs was not influenced by surgical approach.

While comprehensive molecular profiling of histone H3.3 mutant pediatric high-grade glioma has revealed extensive dysregulation of the chromatin landscape, the exact mechanisms driving tumor formation remain poorly understood. Since H3.3 mutant gliomas also exhibit high levels of copy number alterations, we set out to address if the H3.3K27M oncohistone leads to destabilization of the genome. Hereto, we established a cell culture model allowing inducible H3.3K27M expression and observed an increase in mitotic abnormalities. We also found enhanced interaction of DNA replication factors with H3.3K27M during mitosis, indicating replication defects. Further functional analyses revealed increased genomic instability upon replication stress, as represented by mitotic bulky and ultrafine DNA bridges. This co-occurred with suboptimal 53BP1 nuclear body formation after mitosis in vitro, and in human glioma. Finally, we observed a decrease in ultrafine DNA bridges following deletion of the K27M mutant H3F3A allele in primary high-grade glioma cells. Together, our data uncover a role for H3.3 in DNA replication under stress conditions that is altered by the K27M mutation, promoting genomic instability and potentially glioma development.

Chromosomal instability (CIN) and aneuploidy are hallmarks of cancer. As most cancers are aneuploid, targeting aneuploidy or CIN may be an effective way to target a broad spectrum of cancers. Here, we perform two small molecule compound screens to identify drugs that selectively target cells that are aneuploid or exhibit a CIN phenotype. We find that aneuploid cells are much more sensitive to the energy metabolism regulating drug ZLN005 than their euploid counterparts. Furthermore, cells with an ongoing CIN phenotype, induced by spindle assembly checkpoint (SAC) alleviation, are significantly more sensitive to the Src kinase inhibitor SKI606. We show that inhibiting Src kinase increases microtubule polymerization rates and, more generally, that deregulating microtubule polymerization rates is particularly toxic to cells with a defective SAC. Our findings, therefore, suggest that tumors with a dysfunctional SAC are particularly sensitive to microtubule poisons and, vice versa, that compounds alleviating the SAC provide a powerful means to treat tumors with deregulated microtubule dynamics.