Reversion and non-reversion mechanisms of resistance to PARP inhibitor or platinum chemotherapy in BRCA1/2-mutant metastatic breast cancer.

BACKGROUND: Little is known about mechanisms of resistance to poly(adenosine diphosphate-ribose) polymerase inhibitors (PARPi) and platinum chemotherapy in patients with metastatic breast cancer and BRCA1/2 mutations. Further investigation of resistance in clinical cohorts may point to strategies to prevent or overcome treatment failure. PATIENTS AND METHODS: We obtained tumor biopsies from metastatic breast cancer patients with BRCA1/2 deficiency before and after acquired resistance to PARPi or platinum chemotherapy. Whole exome sequencing was carried out on each tumor, germline DNA, and circulating tumor DNA. Tumors underwent RNA sequencing, and immunohistochemical staining for RAD51 foci on tumor sections was carried out for functional assessment of intact homologous recombination (HR). RESULTS: Pre- and post-resistance tumor samples were sequenced from eight patients (four with BRCA1 and four with BRCA2 mutation; four treated with PARPi and four with platinum). Following disease progression on DNA-damaging therapy, four patients (50%) acquired at least one somatic reversion alteration likely to result in functional BRCA1/2 protein detected by tumor or circulating tumor DNA sequencing. Two patients with germline BRCA1 deficiency acquired genomic alterations anticipated to restore HR through increased DNA end resection: loss of TP53BP1 in one patient and amplification of MRE11A in another. RAD51 foci were acquired post-resistance in all patients with genomic reversion, consistent with reconstitution of HR. All patients whose tumors demonstrated RAD51 foci post-resistance were intrinsically resistant to subsequent lines of DNA-damaging therapy. CONCLUSIONS: Genomic reversion in BRCA1/2 was the most commonly observed mechanism of resistance, occurring in four of eight patients. Novel sequence alterations leading to increased DNA end resection were seen in two patients, and may be targetable for therapeutic benefit. The presence of RAD51 foci by immunohistochemistry was consistent with BRCA1/2 protein functional status from genomic data and predicted response to later DNA-damaging therapy, supporting RAD51 focus formation as a clinically useful biomarker.

Whole-exome sequencing of cell-free DNA and circulating tumor cells in multiple myeloma.

Liquid biopsies including circulating tumor cells (CTCs) and cell-free DNA (cfDNA) have enabled minimally invasive characterization of many cancers, but are rarely analyzed together. Understanding the detectability and genomic concordance of CTCs and cfDNA may inform their use in guiding cancer precision medicine. Here, we report the detectability of cfDNA and CTCs in blood samples from 107 and 56 patients with multiple myeloma (MM), respectively. Using ultra-low pass whole-genome sequencing, we find both tumor fractions correlate with disease progression. Applying whole-exome sequencing (WES) to cfDNA, CTCs, and matched tumor biopsies, we find concordance in clonal somatic mutations (~99%) and copy number alterations (~81%) between liquid and tumor biopsies. Importantly, analyzing CTCs and cfDNA together enables cross-validation of mutations, uncovers mutations exclusive to either CTCs or cfDNA, and allows blood-based tumor profiling in a greater fraction of patients. Our study demonstrates the utility of analyzing both CTCs and cfDNA in MM.

Computer-assisted analysis of particulate axoplasmic flow in organized CNS tissue cultures.

Using organized nervous system tissue cultures of embryonic mouse spinal cord, time-lapse motion pictures of intra-axonal particle movements were made using Nomarski optics. The paths of 272 particles were followed and analyzed using digital computer methods. Particles had a bidirectional saltatory motility with net anterograde and retrograde velocities which were shown to be the same (about 1 micron per second). When anterograde moving and retrograde moving particles were analyzed separately for anterograde and retrograde saltations, the velocities of saltation in the direction of net movement were found to be 1.5 times greater than saltations against the direction of net movement. These differences were statistically significant. In addition, it was shown that there were regions within the axon where variations in particle motion were similar from particle to particle as they passed, and that more disturbed motion was found to occur near intra-axonal objects which were judged to be mitochondria. No decrease of particle velocity was noted in regions away from mitochondria nor was in increase in velocity noted near them. Computer-drawn particle plots illustrate the various facets of particle motion.

Experimental acetylcholine blockade of the neuromuscular junction. Effects on end plate and muscle fiber ultrastructure.

To investigate the effect of acetylcholine on the formation and maintenance of the end plate, presynaptic (hemicholinium-3 and botulinum toxin) and postsynaptic (curare and alpha-bungarotoxin) blocking agents were injected into the amniotic sac or chorioallantoic circulation of chick embryos during different stages of their development (days 3 to 11, 3 to 18, and 12 to 18). Tntercostal or anterior and posterior latissimus dorsi muscles were examined by electron microscopy. Regardless of the duration of treatment, the type of agent, or the type of muscle, nerve ingrowth into muscle and end-plate formation were not arrested and the ultrastructure of nerve terminals and postsynaptic regions was normal. By contrast, muscle fiber ultrastructure was affected by acetylcholine blockade if continued beyond day 11. By day 11 both treated and control muscles were composed primarily of myotubes. Muscles treated beyond day 11 showed impaired fiber differentiation and persistence of many myotubes. In addition, many fiber regions contained unassembled fine filaments intermingled with other organelles. In hemicholinium-3 treated muscles ther were also mitochondrial abnormalities and an increased number of lipid droplets. The findings suggest that the altered structure of the neuromusclar junction in myasthenia gravis is not due to the lack of a trophic effect of acetylcholine. The impaired fiber maturation in the chick embryos may be related to the lack of acetylcholine mediated fiber activity.