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Background: Cancer genomes provide a durable record of the genetic alterations that are acquired during normal cell development and carcinogenesis from DNA damage and DNA repair defects. As DNA repair-deficient tumors often become dependent on backup repair pathways, mutational signatures found in such tumors are thought to reflect the absence of a particular repair pathway as well as the activity of the backup repair mechanism responsible for maintaining genome integrity. While homologous recombination (HR) deficiency is primarily a disorder of double-strand break (DSB) repair, the mutation classes most specifically associated with HR-deficiency in cancer are paradoxically small variants, namely single nucleotide variants and short deletions. Mechanistic and cytogenetic studies, however, indicate that HR-deficiency should compromise structural genomic integrity and yield complex rearrangements. Here we elucidate complex structural variants that are specific for HR-deficient cancers and identify rearrangements that differentiate BRCA1 from BRCA2 loss and illuminate divergent backup DNA repair mechanisms. Methods: To investigate the role of complex SVs in HR-deficient cancers, we assembled a cohort of 2,367 WGS profiles from four tumor types (breast, ovarian, prostate, and pancreatic cancer) known to be associated with HR-deficiency. We identified 48 BRCA1-/- and 87 BRCA2-/- cases and called samples lacking (mono- or biallelic) mutations in BRCA1, BRCA2, or any other HR associated gene (e.g. PALB2, RAD51C) as wild-type. To validate observed structural variant patterns associated with homologous recombination deficiency from this larger dataset, a new cohort of 49 cases of invasive breast cancer with known BRCA1 (N = 29) or BRCA2 (N = 20) deficiency was collected as part of a prospective research study at Memorial Sloan Kettering Cancer Center and sequenced with 10X-linked read WGS in addition to standard Illumina short-read WGS. Results: Analysis of nearly 2,400 short-read whole genomes revealed distinct quasi-reciprocal structural variants (SVs) highly enriched in BRCA1-/- versus BRCA2-/- cancers. Applying high physical coverage (>150X) long molecule WGS to 49 tumor-normal pairs from breast cancer patients with inherited BRCA1 or BRCA2 loss-of-function mutations, we show that these SVs are associated with distinct cis or trans somatic allelic phases despite having nearly identical short read WGS footprints. Trans (crossover) outcomes were found to give rise to large-scale chromosomal variants that mediate the loss-of-heterozygosity patterns previously described as a cardinal feature of HR-deficient cancers. We find that these quasi-reciprocal SVs can be explained as distinct template switching outcomes from a shared intermediate arising after replication fork stalling in HR-deficient cancers. Furthermore, our WGS analyses reveal that BRCA2 genomes are highly enriched in deletions that harbor long (50-1000bp) tracts of inexact microhomology. These events, validated by long molecule WGS, indicate that single-stranded annealing serves as an active backup repair pathway in BRCA2-/- but not BRCA1-/- cancers. Conclusions: These results provide direct genomic evidence linking large-scale structural changes in HR-deficient tumors with specific backup repair pathways that suggest novel, therapeutically targetable dependencies. Our findings elucidate backup repair mechanisms responsible for generating structural variation in HR-deficient tumors, demonstrate the genotype-specific divergence of such compensatory DNA repair, and provide genomic features that improve the detection accuracy of HR-deficiency with utility for the optimal selection of treatment. Citation Format: Jeremy Setton, Kevin Hadi, Huasong Tian, Arnaud Da Cruz Paula, Joel Rosiene, Zi-Ning Choo, Julie Behr, Xiaotong Yao, Olivier Elemento, Britta Weigelt, Nadeem Riaz, Jorge S Reis-Filho, Marcin Imielinski, Simon N Powell. Pathognomonic long molecule footprints of backup repair pathways in homologous recombination deficient cancers [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-06-04.

OBJECTIVES To assess the influence of an infusion of clonidine 1 μg/kg/hr on fentanyl and midazolam requirement in ventilated newborns and infants. DESIGN Prospective, double-blind, randomized controlled multicenter trial. Controlled trials.com/ISRCTN77772144. SETTING Twenty-eight level 3 German PICUs/neonatal ICUs. PATIENTS Ventilated newborns and infants: stratum I (1-28 d), stratum II, (29-120 d), and stratum III (121 d to 2 yr). INTERVENTIONS Patients received clonidine 1 μg/kg/hr or placebo on day 4 after intubation. Fentanyl and midazolam were adjusted to achieve a defined level of analgesia and sedation according to Hartwig score. MEASUREMENTS AND MAIN RESULTS Two hundred nineteen infants were randomized; 212 received study medication, 69.7% were ventilated in the postoperative care and 30.3% for other reasons. Primary endpoint: consumption of fentanyl and midazolam in the 72 hours following the onset of study medication (main observation period) in the overall study population. The confirmatory analysis of the overall population showed no difference in the consumption of fentanyl and midazolam. Explorative age-stratified analysis demonstrated that in stratum I (n = 112) the clonidine group had a significantly lower consumption of fentanyl (clonidine: 2.1 ± 1.8 μg/kg/hr, placebo: 3.2 ± 3.1 μg/kg/hr; p = 0.032) and midazolam (clonidine: 113.0 ± 100.1 μg/kg/hr, placebo: 180.2 ± 204.0 μg/kg/hr; p = 0.030). Strata II (n = 43) and III (n = 46) showed no statistical difference. Sedation and withdrawal-scores were significantly lower in the clonidine group of stratum I (p < 0.001). Frequency of severe adverse events did not differ between groups. CONCLUSIONS Clonidine 1 μg/kg/hr in ventilated newborns reduced fentanyl and midazolam demand with deeper levels of analgesia and sedation without substantial side effects. This was not demonstrated in older infants, possibly due to lower clonidine serum levels.

This work focuses on one component of a larger research effort to develop a simulation tool to model populations of flowing cells. Specifically, in this study a local model of the biochemical interactions between circulating melanoma tumor cells (TC) and substrate adherent polymorphonuclear neutrophils (PMN) is developed. This model provides realistic three-dimensional distributions of bond formation and attendant attraction and repulsion forces that are consistent with the time dependent Computational Fluid Dynamics (CFD) framework of the full system model which accounts local pressure, shear and repulsion forces. The resulting full dynamics model enables exploration of TC adhesion to adherent PMNs, which is a known participating mechanism in melanoma cell metastasis. The model defines the adhesion molecules present on the TC and PMN cell surfaces, and calculates their interactions as the melanoma cell flows past the PMN. Biochemical rates of reactions between individual molecules are determined based on their local properties. The melanoma cell in the model expresses ICAM-1 molecules on its surface, and the PMN expresses the β-2 integrins LFA-1 and Mac-1. In this work the PMN is fixed to the substrate and is assumed fully rigid and of a prescribed shear-rate dependent shape obtained from micro-PIV experiments. The melanoma cell is transported with full six-degrees-of-freedom dynamics. Adhesion models, which represent the ability of molecules to bond and adhere the cells to each other, and repulsion models, which represent the various physical mechanisms of cellular repulsion, are incorporated with the CFD solver. All models are general enough to allow for future extensions, including arbitrary adhesion molecule types, and the ability to redefine the values of parameters to represent various cell types. The model presented in this study will be part of a clinical tool for development of personalized medical treatment programs.