Longitudinal profiling identifies co-occurring BRCA1/2 reversions, TP53BP1, RIF1 and PAXIP1 mutations in PARP inhibitor-resistant advanced breast cancer.

BACKGROUND: Resistance to therapies that target homologous recombination deficiency (HRD) in breast cancer limits their overall effectiveness. Multiple, preclinically validated, mechanisms of resistance have been proposed, but their existence and relative frequency in clinical disease are unclear, as is how to target resistance. PATIENTS AND METHODS: Longitudinal mutation and methylation profiling of circulating tumour (ct)DNA was carried out in 47 patients with metastatic BRCA1-, BRCA2- or PALB2-mutant breast cancer treated with HRD-targeted therapy who developed progressive disease-18 patients had primary resistance and 29 exhibited response followed by resistance. ctDNA isolated at multiple time points in the patient treatment course (before, on-treatment and at progression) was sequenced using a novel >750-gene intron/exon targeted sequencing panel. Where available, matched tumour biopsies were whole exome and RNA sequenced and also used to assess nuclear RAD51. RESULTS: BRCA1/2 reversion mutations were present in 60% of patients and were the most prevalent form of resistance. In 10 cases, reversions were detected in ctDNA before clinical progression. Two new reversion-based mechanisms were identified: (i) intragenic BRCA1/2 deletions with intronic breakpoints; and (ii) intragenic BRCA1/2 secondary mutations that formed novel splice acceptor sites, the latter being confirmed by in vitro minigene reporter assays. When seen before commencing subsequent treatment, reversions were associated with significantly shorter time to progression. Tumours with reversions retained HRD mutational signatures but had functional homologous recombination based on RAD51 status. Although less frequent than reversions, nonreversion mechanisms [loss-of-function (LoF) mutations in TP53BP1, RIF1 or PAXIP1] were evident in patients with acquired resistance and occasionally coexisted with reversions, challenging the notion that singular resistance mechanisms emerge in each patient. CONCLUSIONS: These observations map the prevalence of candidate drivers of resistance across time in a clinical setting, information with implications for clinical management and trial design in HRD breast cancers.

Structured pain management reduces patient discomfort after catheter ablation and rhythm device surgery.

BACKGROUND: The goal was to test the effectiveness of a structured pain management programme after invasive electrophysiological interventions in cardiology including ablation of atrial fibrillation (AF) or ventricular tachycardia (VT) and implantation, or explantation, of pacemakers or implantable cardioverter defibrillators. METHODS: This was a prospective study with a pre-/post-design where a post-intervention group (116 consecutive patients) was compared to a pre-intervention group (102 consecutive patients) after implementation of a structured pain-management programme using the numeric rating scale (NRS 0-10) and classified as moderate-to-severe if NRS > 3. Measurements were recorded every two hours during the first 24 h post-operatively. The location of the pain and the amount of analgesic used were also recorded. RESULTS: The proportion of patients who experienced moderate-to-severe pain after the procedure decreased after initiation of the pain-management program: 47% versus 61%; p = 0.048. This difference was driven primarily by reduced pain late (8-24 h) after the procedure; 16% versus 39%; p < 0.001. The risk to develop late (8-24 h) post-procedural pain was reduced approximately three-fold after implementation of the pain-management programme (OR = 0.32, 95% CI 0.16-0.64, p = 0.001). Multivariate analysis indicated chronic pain, early pain (0-6 h), and type of intervention were associated with late post-interventional pain. In contrast, age, diabetes mellitus, BMI, gender and procedure time were not related. CONCLUSION: The findings illustrate the potential value of a structured pain-management programme. The proportion of patients who experienced moderate-to-severe pain after these electrophysiological procedures decreased significantly. SIGNIFICANCE: This is the first exploratory study that evaluates the impact of a multidisciplinary pain-management programme after cardiac electrophysiological interventions. It demonstrates that significant quality improvement is achievable following simple rules together with patient and staff education. The programme reduces the proportion of patients with moderate-to-severe pain after electrophysiological procedures significantly.

Recombination hotspot in NF1 microdeletion patients.

Neurofibromatosis type 1 (NF1) patients that are heterozygous for an NF1 microdeletion are remarkable for an early age at onset and an excessive burden of dermal neurofibromas. Microdeletions are predominantly maternal in origin and arise by unequal crossover between misaligned NF1REP paralogous sequence blocks which flank the NF1 gene. We mapped and sequenced the breakpoints in several patients and designed primers within each paralog to specifically amplify a 3.4 kb deletion junction fragment. This assay amplified a deletion junction fragment from 25 of the 54 unrelated NF1 microdeletion patients screened. Sequence analysis demonstrated that each of the 25 recombination events occurred in a discrete 2 kb recombination hotspot within each of the flanking NF1REPs. Two recombination events were accompanied by apparent gene conversion. A search for recombination-prone motifs revealed a chi-like sequence; however, it is unknown whether this element stimulates recombination to occur at the hotspot. The deletion-junction assay will facilitate the prospective identification of patients with NF1 microdeletion at this hotspot for genotype-phenotype correlation studies and diagnostic evaluation.

NF1 microdeletion breakpoints are clustered at flanking repetitive sequences.

Neurofibromatosis type 1 patients with a submicroscopic deletion spanning the NF1 tumor suppressor gene are remarkable for an early age at onset of cutaneous neurofibromas, suggesting the deletion of an additional locus that potentiates neurofibromagenesis. Construction of a 3.5 Mb BAC/PAC/YAC contig at chromosome 17q11.2 and analysis of somatic cell hybrids from microdeletion patients showed that 14 of 17 cases had deletions of 1.5 Mb in length. The deletions encompassed the entire 350 kb NF1 gene, three additional genes, one pseudogene and 16 expressed sequence tags (ESTs). In these cases, both proximal and distal breakpoints mapped at chromosomal regions of high identity, termed NF1REPs. These REPs, or clusters of paralogous loci, are 15-100 kb and harbor at least four ESTs and an expressed SH3GL pseudogene. The remaining three patients had at least one breakpoint outside an NF1REP element; one had a smaller deletion thereby narrowing the critical region harboring the putative locus that exacerbates neurofibroma development to 1 Mb. These data show that the likely mechanism of NF1 microdeletion is homologous recombination between NF1REPs on sister chromatids. NF1 microdeletion is the first REP-mediated rearrangement identified that results in loss of a tumor suppressor gene. Therefore, in addition to the germline rearrangements reported here, NF1REP-mediated somatic recombination could be an important mechanism for the loss of heterozygosity at NF1 in tumors of NF1 patients.