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.

Optical and physical properties of wet-spun films of Na-hyaluronate. Evidence of a phase transition.

The refractive indices, water content, and volume of wet-spun films of Na-hyaluronate have been measured as a function of relative humidity (rh). These data are used with the Lorentz-Lorenz formula to determine the optical polarizabilities of Na-hyaluronate parallel and perpendicular to the helical axis. The analysis reveals a drop in the optical polarizabilities of approximately 20% between 80 and 88% rh, indicating a phase transition.

Haemodynamic response to induction of anaesthesia with ketamine/midazolam.

The haemodynamic responses following induction of anaesthesia with ketamine and midazolam have not been determined previously. Twenty adult patients for elective myocardial revascularization were randomized to two regimens for induction of anaesthesia. Patients in Group I received ketamine, 2 mg.kg-1, and midazolam, 0.2 mg.kg-1 and those in Group II received ketamine, 2 mg.kg-1, and midazolam, 0.4 mg.kg-1. Measurements were recorded at baseline, 1 min post-induction, and at one, three, five and ten minutes after tracheal intubation. Tachycardia and hypertension (greater than 20% increases from awake baseline values) were treated with esmolol, 250 micrograms.kg-1. There were 11 patients in Group I and nine patients in Group II. There were no significant intergroup differences in demographic or haemodynamic variables. Both groups had decreases (P less than 0.05), in stroke volume, pulmonary capillary wedge pressure, and right ventricular end-diastolic volume at multiple study intervals following anaesthetic induction. None of these changes required clinical intervention. Five patients (all in Group II) had hypertensive responses to tracheal intubation. Preoperative hypertension (mean arterial pressure greater than or equal to 100 mmHg) was a predictor (P less than 0.05) of a hypertensive response to intubation, independent of the midazolam dose. Intravenous ketamine and midazolam was associated with a high incidence (25%) of haemodynamic responses to tracheal intubation. The higher dose of midazolam did not provide any haemodynamic advantage.

The treatment of nonstreptococcal pharyngitis with erythromycin: a preliminary study.

This preliminary study evaluated the effectiveness of enteric-coated erythromycin in the treatment of adults with nonstreptococcal pharyngitis. Twenty-six patients, aged 12 or older, with nonstreptococcal pharyngitis were randomly assigned in a double-blind fashion to receive either enteric-coated erythromycin or placebo for 10 days. Each day the patients rated the soreness of their throats and how sick they felt. The use of erythromycin appeared to decrease the median number of days for the feeling of sickness to improve (two days versus four days, P less than .01) as well as to decrease the median number of days for the feeling of sickness to resolve (three-and-a-half days versus five days, P less than .05). This apparent improvement in sickness occurred even though there was no significant decrease in the soreness of the throat in patients treated with erythromycin compared to those patients treated with placebo. Treatment with enteric-coated erythromycin appeared to shorten the time until improvement in this group of adults with nonstreptococcal pharyngitis.

Nephrolithiasis in pregnancy.

Nephrolithiasis is a rare but important condition in pregnant women. The most common presenting complaint is flank pain. Recurrent or persistent urinary tract infections may also be due to nephrolithiasis. Ultrasound is the first-line diagnostic study, but a limited intravenous pyelogram can be obtained when indicated. Spontaneous passage of the stone occurs in approximately two-thirds of patients.

Anti-kidney antibody teratogenesis: evidence for a direct action on the rat conceptus by a mechanism involving the yolk sac.

IgG from anti-rat kidney antiserum (anti-K) was added to 48-h cultures of 9.5-day whole rat conceptuses. At concentrations of 25 micrograms/ml and above, dysmorphogenesis of embryos (optic vesicle defects) was observed. Significant embryonic growth retardation was apparent at 75 micrograms/ml and above. Other abnormalities (neural tube defects, deficits in axial rotation, reduced somite numbers) were seen at 100 and 150 micrograms/ml. In separate experiments, anti-K inhibited pinocytosis of 125I-polyvinylpyrrolidone by isolated yolk sacs. All of these effects of anti-K are similar to those produced by anti-visceral yolk sac antiserum suggesting a similar mechanism of teratogenic action, namely interference with yolk-sac-mediated embryotrophic nutrition.

Coupling of cardiac and locomotor rhythms.

The pressure within exercising skeletal muscle rises and falls rhythmically during normal human locomotion, the peak pressure reaching levels that intermittently impede blood flow to the exercising muscle. Speculating that a reciprocal relationship between the timing of peak intramuscular and pulsatile arterial pressures should optimize blood flow through muscle and minimize cardiac load, we tested the hypothesis that heart rate becomes entrained with walking and running cadence at some locomotion speeds, by means of electrocardiography and an accelerometer to provide signals reflecting heart rate and cadence, respectively. In 18 of 25 subjects, 1:1 coupling of heart and step rates was present at one or more speeds on a motorized treadmill, generally at moderate to high exercise intensities. To determine how exercise specific this phenomenon is, and to refute the competing hypothesis that coupling is due to vertical accelerations of the heart during locomotion, we had 12 other subjects cycle on an electronically braked bicycle ergometer. Coupling was found between heart rate and pedaling frequency in 10 of them. Cardiac-locomotor coupling appears to be a normal physiological phenomenon, and its identification provides a fresh perspective from which to study endurance.

Distribution of putative elastic fibers in rabbit temporomandibular joint tissues.

This study describes the general distribution of putative elastic fibers in the connective tissues that comprise the articular disk and some of the adnexal tissues of the rabbit temporomandibular joint. Joints were removed en bloc and processed for light-microscopic study. The fibroelastic tissues of the bilaminar zone of the articular disk and the various attachments of the disk to the mandibular condyle contained numerous elastic fibers. Since these morphologic data indicate the presence of many elastic fibers, we suggest that the rabbit temporomandibular joint may serve as a model system to study the functional consequences of selectively altering the quality or quantity of elastic fibers in these tissues.

Failure of zinc to prevent dysmorphogenesis of cultured rat conceptuses by anti-yolk sac antiserum.

Day 10 rat conceptuses were cultured for 48h in the presence of either cadmium or anti-visceral yolk sac antiserum (AVYS). Cadmium was embryotoxic at concentrations exceeding 0.25 micrograms/ml whilst AVYS caused embryonic dysmorphogenesis, particularly affecting the optic vesicles, at concentrations of 2 microliters/ml and above. The effect of pretreatment with zinc on embryotoxicity caused by cadmium or AVYS was studied. Zinc ameliorated the effects of cadmium but had no effect on AVYS-induced embryonic abnormalities. In a second set of experiments inhibition of 125I-labelled PVP uptake by the yolk sac of cultured whole conceptuses was studied. Cadmium and AVYS both inhibited uptake compared to control cultures. Zinc again ameliorated the effect of cadmium but had no action against AVYS-induced inhibition. These results are in contrast to our previous findings using isolated cultured yolk sacs in which zinc ameliorated the inhibitory effects on 125I-labelled PVP uptake of both cadmium and AVYS. These data show that in experiments using the isolated cultured yolk sac and the intact cultured conceptus, a qualitatively different response in yolk sac behaviour is observed under similar experimental conditions.

The effect of income projection on resident charges for services.

This study examined how family practice residents coded their patient care services, and whether feedback on coding patterns together with income projections would alter coding behavior. Current and future income projections were calculated using assumptions about the practice setting. The collected and calculated data were presented to the residents after the study period, together with a discussion of coding of patient care services and fees. The same data were collected three months and nine months later. There was no significant difference in coding patterns when comparing first- to second- to third-year residents. Six months after the information was presented to the residents, the percentage of brief visits coded decreased significantly, and the percentages of intermediate and comprehensive visits coded increased significantly. Twelve months later, the changes in coding patterns had reverted to the initial findings. This appears to be an effective approach that could be used elsewhere to teach coding of patient care services as part of a practice management curriculum.

The effect of locomotion speed on the anterior tibial intramuscular pressure of normal humans.

We examined the hypothesis that anterior tibial intramuscular pressure increases with the speed of locomotion. A solidstate pressure transducer was placed near the tip of an intramuscular fluid-filled catheter and each of ten normal subjects walked, jogged and ran barefoot, at 1.5, 2.5 and 3.5 m s-1 respectively, on a treadmill to which they had been previously familiarized. The pressure signals during ten gait cycles from each subject were sampled at 600 Hz and were averaged at each speed. The peak pressures increased in magnitude as a function of increasing locomotion speed, while the mean pressure remained constant.

Reliability of walking endurance with an incremental treadmill test.

Treadmill walking endurance is the major outcome measure for intervention studies on patients with intermittent claudication. The measurement properties of this test have not, however, been well documented. We assessed the test-retest reliability of treadmill walking endurance in 10 normal subjects with a mean (+/- 1SD) age of 27.5 (+/- 3.3) years. On four occasions, at intervals of at least one week, each subject walked on a motorized treadmill to the limits of his or her capabilities. With the standard protocol, subjects began at a very low exercise intensity (0.25 m/sec, 0% grade), which increased in either speed or grade at ninety-second intervals through a maximum of fifteen stages to 2.75 m/sec and 20% grade. On the final occasion, 5 of the subjects were tested twice with a fifteen-minute interval, using a truncated protocol starting at stage five (1.25 m/sec, 0% grade). Within-subject standard deviations were low, averaging twenty-three seconds (2.1%). Reliability was very high for the first three tests with the standard protocol (r greater than or equal to 0.98, p less than 0.0001) and for the 5 subjects tested and retested with the short interval and truncated protocol (r = 0.99, p less than 0.0002), while the correlation between the standard protocol and the truncated protocol was somewhat lower (r greater than 0.81, p less than 0.01). For young normal subjects the measurement of walking endurance with an incremental treadmill test is highly reliable, but the limits of reliability need to be considered when conclusions are drawn from such measures.

Differential effect of zinc on teratogen-induced inhibition of pinocytosis by cultured rat yolk sac.

Pinocytosis as measured by the uptake of 125I labelled PVP by the isolated cultured day 12 rat yolk sac was observed to be linear over a 4 h incubation period and to proceed at a rate of approximately 2.5 microliters/mg protein/h. Cadmium, anti-visceral yolk sac antibody (AVYS) and trypan blue all inhibited pinocytosis in a concentration-dependent fashion when added to the culture medium, although at low concentrations trypan blue was slightly stimulatory. The effect of zinc on the inhibition of pinocytosis by these three teratogens was studied. It was observed that zinc ameliorated the inhibitory effects of cadmium and AVYS, but had no effect on inhibition by trypan blue. These results indicate that the previously demonstrated protective action of zinc against cadmium-induced yolk sac dysfunction is not specific to that agent but extends to inhibition of pinocytosis by AVYS, and further suggest that, because of its refractoriness to zinc, trypan blue-induced inhibition of pinocytosis by yolk sac occurs by a mechanism different from that effected by cadmium and AVYS.

In-vitro teratogenicity of retinoids.

Mid-gestation rat conceptuses were cultured for 48 h in serum containing the retinoids all-trans-retinoic acid (TRA), 13-cis-retinoic acid (13-CRA), etretinate (ETR), etretin or one of six retinamides at concentrations ranging from 0.5 to 400 micrograms/ml. TRA was toxic at a concentration of 0.5 microgram/ml. 13-CRA and etretin caused abnormal development at 1.0 microgram/ml. However, the six retinamides were less toxic and adverse developmental effects were only evident at concentrations of 50 or 100 micrograms/ml. ETR was without effect at 100 micrograms/ml, the highest dose level of this compound tested. In vivo, TRA, 13-CRA and ETR are highly teratogenic. In this culture system, TRA and 13-CRA caused abnormal development at very low concentrations but in contrast, ETR was non-toxic at 100 micrograms/ml. Therefore these findings indicate that in vivo, maternal pharmacokinetics, and bioactivation in particular, play a major role in inducing abnormal development. Cis/trans isomerization was not a major determinant of toxicity. However, there appeared to be a relationship between abnormal development and the actual or estimated pKa values of the 10 retinoids tested.