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.

Integrative molecular and functional profiling of ERBB2-amplified breast cancers identifies new genetic dependencies.

Overexpression of the receptor tyrosine kinase ERBB2 (also known as HER2) occurs in around 15% of breast cancers and is driven by amplification of the ERBB2 gene. ERBB2 amplification is a marker of poor prognosis, and although anti-ERBB2-targeted therapies have shown significant clinical benefit, de novo and acquired resistance remains an important problem. Genomic profiling has demonstrated that ERBB2+ve breast cancers are distinguished from ER+ve and 'triple-negative' breast cancers by harbouring not only the ERBB2 amplification on 17q12, but also a number of co-amplified genes on 17q12 and amplification events on other chromosomes. Some of these genes may have important roles in influencing clinical outcome, and could represent genetic dependencies in ERBB2+ve cancers and therefore potential therapeutic targets. Here, we describe an integrated genomic, gene expression and functional analysis to determine whether the genes present within amplicons are critical for the survival of ERBB2+ve breast tumour cells. We show that only a fraction of the ERBB2-amplified breast tumour lines are truly addicted to the ERBB2 oncogene at the mRNA level and display a heterogeneous set of additional genetic dependencies. These include an addiction to the transcription factor gene TFAP2C when it is amplified and overexpressed, suggesting that TFAP2C represents a genetic dependency in some ERBB2+ve breast cancer cells.

Establishment and characterisation of a new breast cancer xenograft obtained from a woman carrying a germline BRCA2 mutation.

BACKGROUND: The BRCA2 gene is responsible for a high number of hereditary breast and ovarian cancers, and studies of the BRCA2 biological functions are limited by the lack of models that resemble the patient's tumour features. The aim of this study was to establish and characterise a new human breast carcinoma xenograft obtained from a woman carrying a germline BRCA2 mutation. METHODS: A transplantable xenograft was obtained by grafting a breast cancer sample into nude mice. The biological and genetic profiles of the xenograft were compared with that of the patient's tumour using histology, immunohistochemistry (IHC), BRCA2 sequencing, comparative genomic hybridisation (CGH), and qRT-PCR. Tumour response to standard chemotherapies was evaluated. RESULTS: Histological profile identified the tumour as a basal-like triple-negative breast cancer. Targeted BRCA2 DNA sequencing of the xenograft showed the presence of the mutation previously identified in the carrier. Comparative genomic hybridisation array profiles of the primary tumour and the xenograft revealed a high number of similar genetic alterations. The therapeutic assessment of the xenograft showed sensitivity to anthracyclin-based chemotherapy and resistance to docetaxel. The xenograft was also highly sensitive to radiotherapy and cisplatin-based treatments. CONCLUSIONS: This study describes a new human breast cancer xenograft obtained from a BRCA2-mutated patient. This xenograft provides a new model for the pre-clinical drug development and for the exploration of the drug response biological basis.

The effect of diclofenac (voltarol) and pethidine on ureteric peristalsis and the isotope renogram.

Diclofenac (a non-steroidal anti-inflammatory drug) and pethidine (a synthetic opiate) are the two analgesics most commonly used to relieve the pain of ureteric colic. Fast frame renography is a non-invasive means of imaging ureteric peristalsis and renal drainage. The aim of this study was to determine the effects of each of these drugs on the drainage pattern of the upper tracts. Twelve normal male volunteers were studied. All underwent a standard fast frame renogram using 75 MBq of technetium-99m-mercaptoacetyltriglycine, and were then administered either 100 mg pethidine or 75 mg diclofenac by intramuscular injection. Fast frame renography was then repeated. Peristalsis was determined from the condensed image of each ureter and the renogram curves were analysed to obtain standard parameters and deconvolution analysis. Diclofenac caused a profound disruption to both ureteric peristalsis and the renogram curve. This effect was not seen after the administration of pethidine. Deconvolution analysis suggests the effects of diclofenac are mediated via a direct effect on drainage rather than by any alteration of blood flow to the kidney. This study suggests that pethidine is the analgesic of choice prior to renography and that inferences about alterations of drainage in the presence of diclofenac should be interpreted with care.

Bladder washout and stone formation in paediatric enterocystoplasty.

OBJECTIVE: To review the incidence of stone formation in our patients with enterocystoplasty to determine the effect of regular bladder washout. METHODS: From 1988 to 1995, a prospective cohort of 30 children underwent enteroplasty with continent diversion. Over the same period, a consecutive group of 30 children had an augmentation alone. All were instructed to wash out their bladder on a weekly basis with sterile water. The frequency of the washouts increased if there were problems with increasing mucus production. Their incidence of stone formation has been compared to a similar group of 30 children performing clean intermittent self catheterisation (CISC) on their native bladders. RESULTS: Five (17%) children with continent diversions formed bladder stones (mean time to formation 35 months, range 13-59 months) were compared with 2 (7%) of children with augmentation. No child performing CISC alone formed stones. CONCLUSIONS: A regime of regular bladder washout in children with enterocystoplasty did not significantly reduce the incidence of stone formation when compared to previously published data.

Resource implications of outpatient referrals to a general urological service.

The effects of new outpatient referrals on the dynamics of global provision in a surgical service has not previously been defined. Because of managerial pressure to reduce the time interval between general practitioner referral and first specialist assessment, many services are now faced with additional outpatient loads without any clear idea of the effect that this additional burden will have on overall practice. In an attempt to define the logistic implications of a new outpatient load, 293 patients, referred from primary care to a general urological service, were followed for a further two interactions with the secondary care team. 'One-stop' visits with in-clinic investigation and an active discharge policy were employed to assist with efficient patient management. Of the original patients, 28% required investigations not available in the clinic, with cost and logistic implications for support services. In all, 32% of the patients needed further follow-up appointments, despite the active discharge policy. This necessitated 95 people being seen in additional clinic time. Of the patients referred, 37% needed inpatient treatment; 46% being day case procedures, the remainder constituting a variable case mix. This work necessitated 7.1 operating sessions and an additional 75 inpatient bed days for every 100 new patients referred. A model for determining the resource requirements for a surgical outpatient load is proposed.

Identification of patients requiring out-patient follow-up after transurethral prostatectomy: is there a role for nurse-led screening of post-operative outcomes by telephone?

OBJECTIVE: To determine the most efficient method to follow patients after transurethral prostatectomy (TURP) such that only those patients suffering significant post-operative problems are reviewed. PATIENTS AND METHODS: The study comprised two parts: (1) a retrospective review of the case notes of 100 consecutive patients who underwent TURP under one consultant to determine whether any factors could be identified pre- or post-operatively by which those patients most likely to require clinic review could be selected and; (2) a prospective review of the succeeding 100 patients undergoing TURP, using a telephone 'screening' call made by the urological research nurse 3 months after the operation. Patients who requested follow-up and those patients with malignancy or admitted in high-pressure chronic retention were reviewed in the out-patient department. RESULTS: In the first part, 17 patients (17%) required an out-patient review for malignancy. Only nine patients (11%) with benign histology required further treatment after TURP; this subgroup could not be identified on the basis of their pre- or post-operative symptoms. In the second part, 23 patients were not reviewed by telephone; 14 had carcinoma of the prostate, eight had no telephone and one could not be contacted after seven attempts. Of the remaining 77 contacted by phone, 61 (79%) declined further clinic review and 16 (21%) requested follow-up for persistent problems. A mean of two calls was made per patient and the mean duration of each call was 6.3 min. CONCLUSIONS: Based on pre- or post-operative symptoms at the time of discharge, there is no reliable method of identifying those patients who have a poor result after TURP. Telephone screening of patients at 3 months identified successfully those patients who required an out-patient review and enabled resources to be targeted towards this difficult group of patients.

EMLA cream anaesthesia for frenuloplasty.

OBJECTIVE: To evaluate the use of a eutectic mixture of local anaesthetics (EMLA) cream as the sole anaesthetic for frenuloplasty. PATIENTS AND METHODS: Fifteen patients (mean age 25.6 years, range 19-41) were given topical EMLA cream 30 min before frenuloplasty; a Tegaderm dressing was applied to keep the anaesthetic in place. The level of anaesthesia was assessed on arrival in theatre using pin-prick testing and the patients were asked to grade their level of pain during the procedure using a visual analogue scale. If the level of anaesthesia was inadequate for the operation to be performed, then supplementary infiltrative anaesthesia was administered. RESULTS: Fourteen patients had pain scores of zero and were fully anaesthetized during the procedure. One patient's Tegaderm dressing was displaced soon after application and he was inadequately anaesthetized on testing, had a pain score of 4 and required infiltrative lignocaine before proceeding with frenuloplasty. CONCLUSIONS: EMLA cream is a well-tolerated and reliable anaesthetic for frenuloplasty. Using a condom to keep the cream in place would reduce the small failure rate associated with the displacement of the Tegaderm dressing.

Quantitative analysis of the acute response to haemorrhage of the renin-angiotensin-vasoconstrictor feedback loop in areflexic dogs.

The importance of the renin-angiotensin-vasoconstrictor system during haemorrhagic hypotension was quantitated in 44 areflexic dogs by determining the ability of the arterial pressure to recover following haemorrhage to 8.8 kPa (66 mm Hg). In 30 animals with intact kidneys, the arterial pressure following haemorrhage rose to a new steady-state level averaging 11.7 kPa (88 mm Hg), which represented 65.3 +/- 1.8 (SE) % compensation. In 18 nephrectomized animals only 24.4 +/- 1.5% compensation occurred. The system exhibited a relatively rapid time course for pressure compensation with the new steady state occurring 19.2 +/- 2.0 min after haemorrhage. Following reinfusion of blood the pressure returned to the pre-haemorrhage in 19.2 +/- 3.0 min. Arterial renin activity was significantly elevated following haemorrhage in the intact kidney group and unchanged in the anephric group. The arterial pressure compensation of two animals with intact kidneys was significantly reduced when the angiotensin-converting enzyme inhibitor (SQ 20881) was infused before the haemorrhage. The results are consistent with a renin-angiotensin-vasoconstrictor mechanism of arterial pressure compensation and indicate that this mechanism possesses sufficient gain and time response characteristics to play a homeostatically significatn role during haemorrhagic hypotension.