MAGENTA: a Multinational patient survey assessing the Awareness, perceptions and unmet needs in GENetic Testing and counselling among patients with breAst cancer.

Objective: Genetic testing and counselling are critical in assessing breast cancer risk and tailoring treatment strategies. However, several barriers hinder patients from opting for genetic testing/counselling, leading to fewer than one-third of patients undergoing testing and even fewer being offered counselling. A granular understanding of these barriers is essential in overcoming them. Methods: A multinational survey developed by patient authors was conducted in 9 countries, to identify the specific local/regional barriers. The survey question pathway was individualized, based on responses to prior questions. Percentage responses to a response option were calculated based on the total number of respondents to that question. Chi-square tests were used to assess the significance of the results, if applicable. Results: The final analysis set (FAS) included 1,176 respondents, with a subset of this responding to all questions. In the FAS, 63% of respondents had undergone testing. Among those who got tested, 70% were offered testing. Among untested respondents, only 40% were offered the test but eventually did not get tested. In the tested population, 44% received counselling, which was significantly higher than 7% (p<0.00001) in the untested group. Among those reporting on awareness, 71% reported awareness level between 'very low' and 'moderate' prior to cancer diagnosis. Most respondents (71%) agreed that all breast cancer patients should undergo testing before treatment initiation. However, Asian patients were less likely to endorse this view compared to respondents from other regions (25% vs ≥50%; p<0.00001). A higher proportion of tested respondents were 'very willing' to get their family members tested (44%) versus untested respondents (11%), with relatively higher willingness among Australian (77%) and Russian respondents (56%), the regional variation being statistically significant (p<0.00001). Conclusions: Critical gaps remain in the access, awareness and perceived value of genetic testing and counselling, with regional variance or difference between the tested and untested groups. Most patients are not offered counselling, which may be associated with the low uptake of testing. Strategic action is needed to drive policy-shaping and improve access to testing and counselling, including raising patient awareness and improving patient experience for better treatment outcomes.

Technical and clinical outcomes in concurrent multivessel occlusions treated with mechanical thrombectomy: insights from the STAR collaboration.

BACKGROUND: Endovascular thrombectomy (EVT) has become the mainstay treatment for large vessel occlusion, with favorable safety and efficacy profile. However, the safety and efficacy of EVT in concurrent multi-territory occlusions (MTVOs) remains unclear. OBJECTIVE: To investigate the prevalence, clinical and technical outcomes of concurrent EVT for MTVOs. METHODS: Data were included from the Stroke Thrombectomy and Aneurysm Registry (STAR) with 32 stroke centers for EVT performed to treat bilateral anterior or concurrent anterior and posterior circulation occlusions between 2017 and 2021. Patients with MTVO were identified, and propensity score matching was used to compare this group with patients with occlusion in a single arterial territory. RESULTS: Of a total of 7723 patients who underwent EVT for acute ischemic stroke, 54 (0.7%) underwent EVT for MTVOs (mean age 69±12.5; female 50%). 28% had bilateral and 72% had anterior and posterior circulations occlusions. The rate of successful recanalization (Thrombolysis in Cerebral Infarction 2b/3), complications, modified Rankin score at 90 days, and mortality was not significantly different between the matched cohorts. Multivariate analysis confirmed that MTVOs were not associated with poor functional outcome, symptomatic intracranial hemorrhage, or longer procedure time. CONCLUSION: Compared with EVT for single vessel occlusions, EVT in appropriately selected patients with MTVOs has a similar efficacy and safety profile.

The Association of Antibiotic Duration With Successful Treatment of Community-Acquired Pneumonia in Children.

BACKGROUND: National guidelines recommend 10 days of antibiotics for children with community-acquired pneumonia (CAP), acknowledging that the outcomes of children hospitalized with CAP who receive shorter durations of therapy have not been evaluated. METHODS: We conducted a comparative effectiveness study of children aged ≥6 months hospitalized at The Johns Hopkins Hospital who received short-course (5-7 days) vs prolonged-course (8-14 days) antibiotic therapy for uncomplicated CAP between 2012 and 2018 using an inverse probability of treatment weighted propensity score analysis. Inclusion was limited to children with clinical and radiographic criteria consistent with CAP, as adjudicated by 2 infectious diseases physicians. Children with tracheostomies; healthcare-associated, hospital-acquired, or ventilator-associated pneumonia; loculated or moderate to large pleural effusion or pulmonary abscess; intensive care unit stay >48 hours; cystic fibrosis/bronchiectasis; severe immunosuppression; or unusual pathogens were excluded. The primary outcome was treatment failure, a composite of unanticipated emergency department visits, outpatient visits, hospital readmissions, or death (all determined to be likely attributable to bacterial pneumonia) within 30 days after completing antibiotic therapy. RESULTS: Four hundred and thirty-nine patients met eligibility criteria; 168 (38%) patients received short-course therapy (median, 6 days) and 271 (62%) received prolonged-course therapy (median, 10 days). Four percent of children experienced treatment failure, with no differences observed between patients who received short-course vs prolonged-course antibiotic therapy (odds ratio, 0.48; 95% confidence interval, .18-1.30). CONCLUSIONS: A short course of antibiotic therapy (approximately 5 days) does not increase the odds of 30-day treatment failure compared with longer courses for hospitalized children with uncomplicated CAP.

Sensitive Detection and Analysis of Neoantigen-Specific T Cell Populations from Tumors and Blood.

Neoantigen-specific T cells are increasingly viewed as important immunotherapy effectors, but physically isolating these rare cell populations is challenging. Here, we describe a sensitive method for the enumeration and isolation of neoantigen-specific CD8+ T cells from small samples of patient tumor or blood. The method relies on magnetic nanoparticles that present neoantigen-loaded major histocompatibility complex (MHC) tetramers at high avidity by barcoded DNA linkers. The magnetic particles provide a convenient handle to isolate the desired cell populations, and the barcoded DNA enables multiplexed analysis. The method exhibits superior recovery of antigen-specific T cell populations relative to literature approaches. We applied the method to profile neoantigen-specific T cell populations in the tumor and blood of patients with metastatic melanoma over the course of anti-PD1 checkpoint inhibitor therapy. We show that the method has value for monitoring clinical responses to cancer immunotherapy and might help guide the development of personalized mutational neoantigen-specific T cell therapies and cancer vaccines.

Crosstalk between PKCα and PI3K/AKT Signaling Is Tumor Suppressive in the Endometrium.

Protein kinase C (PKC) isozymes are commonly recognized as oncoproteins based on their activation by tumor-promoting phorbol esters. However, accumulating evidence indicates that PKCs can be inhibitory in some cancers, with recent findings propelling a shift in focus to understanding tumor suppressive functions of these enzymes. Here, we report that PKCα acts as a tumor suppressor in PI3K/AKT-driven endometrial cancer. Transcriptional suppression of PKCα is observed in human endometrial tumors in association with aggressive disease and poor prognosis. In murine models, loss of PKCα is rate limiting for endometrial tumor initiation. PKCα tumor suppression involves PP2A-family-dependent inactivation of AKT, which can occur even in the context of genetic hyperactivation of PI3K/AKT signaling by coincident mutations in PTEN, PIK3CA, and/or PIK3R1. Together, our data point to PKCα as a crucial tumor suppressor in the endometrium, with deregulation of a PKCα→PP2A/PP2A-like phosphatase signaling axis contributing to robust AKT activation and enhanced endometrial tumorigenesis.

Protein Kinase Cα (PKCα) Is Resistant to Long Term Desensitization/Down-regulation by Prolonged Diacylglycerol Stimulation.

Sustained activation of PKCα is required for long term physiological responses, such as growth arrest and differentiation. However, studies with pharmacological agonists (e.g. phorbol 12-myristate 13-acetate (PMA)) indicate that prolonged stimulation leads to PKCα desensitization via dephosphorylation and/or degradation. The current study analyzed effects of chronic stimulation with the physiological agonist diacylglycerol. Repeated addition of 1,2-dioctanoyl-sn-glycerol (DiC8) resulted in sustained plasma membrane association of PKCα in a pattern comparable with that induced by PMA. However, although PMA potently down-regulated PKCα, prolonged activation by DiC8 failed to engage known desensitization mechanisms, with the enzyme remaining membrane-associated and able to support sustained downstream signaling. DiC8-activated PKCα did not undergo dephosphorylation, ubiquitination, or internalization, early events in PKCα desensitization. Although DiC8 efficiently down-regulated novel PKCs PKCδ and PKCϵ, differences in Ca(2+) sensitivity and diacylglycerol affinity were excluded as mediators of the selective resistance of PKCα. Roles for Hsp/Hsc70 and Hsp90 were also excluded. PMA, but not DiC8, targeted PKCα to detergent-resistant membranes, and disruption of these domains with cholesterol-binding agents demonstrated a role for differential membrane compartmentalization in selective agonist-induced degradation. Chronic DiC8 treatment failed to desensitize PKCα in several cell types and did not affect PKCßI; thus, conventional PKCs appear generally insensitive to desensitization by sustained diacylglycerol stimulation. Consistent with this conclusion, prolonged (several-day) membrane association/activation of PKCα is seen in self-renewing epithelium of the intestine, cervix, and skin. PKCα deficiency affects gene expression, differentiation, and tumorigenesis in these tissues, highlighting the importance of mechanisms that protect PKCα from desensitization in vivo.

Extensively drug-resistant tuberculosis in a young child after travel to India.

Extensively drug-resistant (XDR) tuberculosis is becoming increasingly prevalent worldwide, but little is known about XDR tuberculosis in young children. In this Grand Round we describe a 2-year-old child from the USA who developed pneumonia after a 3 month visit to India. Symptoms resolved with empirical first-line tuberculosis treatment; however, a XDR strain of Mycobacterium tuberculosis grew in culture. In the absence of clinical or microbiological markers, low-radiation exposure pulmonary CT imaging was used to monitor treatment response, and guide an individualised drug regimen. Management was complicated by delays in diagnosis, uncertainties about drug selection, and a scarcity of child-friendly formulations. Treatment has been successful so far, and the child is in remission. This report of XDR tuberculosis in a young child in the USA highlights the risks of acquiring drug-resistant tuberculosis overseas, and the unique challenges in management of tuberculosis in this susceptible population.

Autoinduction of voriconazole metabolism in a child with invasive pulmonary aspergillosis.

Inter- and intra-patient variability in voriconazole pharmacokinetics has been described in children as the result of age-specific differences in hepatic metabolism, saturable nonlinear pharmacokinetics, CYP450 2C19 polymorphisms, decreased bioavailability compared with adults, and drug-drug interactions. We introduce dose-dependent autoinduction of metabolism as another cause for altered voriconazole pharmacokinetics in children and summarize previously published literature on this phenomenon. A 10-year-old girl with severe aplastic anemia developed invasive pulmonary aspergillosis after high-dose cyclophosphamide therapy and required high doses of voriconazole for longer than 2 months. She initially achieved a therapeutic trough of 1.4 µg/ml on voriconazole 11 mg/kg/dose orally every 12 hours but required dose escalations to 9.3 mg/kg/dose orally every 8 hours to maintain a trough above 1 µg/ml. Because there were no changes in concomitant medications, route of administration, adherence, or oral intake, we conclude that the only plausible explanation for the precipitous drop in voriconazole troughs was autoinduction of metabolism, a phenomenon previously reported in adults receiving higher than usual doses or prolonged courses (longer than 2 months). These data highlight the need for continued therapeutic drug monitoring of voriconazole after initial therapeutic troughs are achieved because autoinduction of metabolism can lead to significant declines in subsequent voriconazole troughs, potentially leading to treatment failure.

Antiretroviral treatment strategies in highly treatment experienced perinatally HIV-infected youth.

BACKGROUND: There is limited information on antiretroviral (ARV) regimens and outcomes in perinatally HIV (PHIV)-infected youth. Substantial drug resistance after long-term ARV use and nonadherence hinder efforts to design suppressive regimens for PHIV-infected youth. This study compares clinical outcomes by expected activity of the prescribed ARV regimens. METHODS: A retrospective cohort study of 13- to 24-year-old PHIV-infected youth on stable ARV regimens for ≥6 months was conducted at a pediatric HIV clinic. ARV regimens were retrospectively categorized as optimal or suboptimal based on accumulated genotypic resistance before study regimen initiation. RESULTS: Fifty-two patients with similar baseline characteristics met inclusion criteria (21 optimal and 31 suboptimal regimens). Patients receiving optimal regimens had significantly higher increases in CD4 than those given suboptimal regimens by week 48 of treatment (+62 versus +8 cells/mm, respectively; P = 0.04) and by the end of study period (+93 versus -1 cells/mm, respectively; P = 0.03). There were no significant differences between the groups in decline of viral load, frequency of opportunistic infections or hospitalizations or accumulation of resistance mutations. Overall, 60% of the optimal and 45% of the suboptimal groups had nonadherence during the study regimen (P = 0.3). CONCLUSIONS: PHIV-infected youth receiving optimal regimens had greater CD4 improvements but no difference in virologic outcomes compared with those receiving suboptimal regimens. In a patient population with significant nonadherence, providers must weigh the immunologic benefits of initiating an optimal regimen versus the potential risks of further resistance accumulation limiting future treatment options.

Antiretroviral therapy adherence and drug-drug interactions in the aging HIV population.

It is estimated that by 2015 more than half of all HIV-infected individuals in the United States will be 50 years of age or older. As this population ages, the frequency of non-AIDS related comorbidities increases, which includes cardiovascular, metabolic, gastrointestinal, genitourinary and psychiatric disorders. As a result, medical management of the aging HIV population can be complicated by polypharmacy and higher pill burden, leading to poorer antiretroviral therapy (ART) adherence. Adherence to ART is generally better in older populations when compared to younger populations; however, cognitive impairment in elderly patients can impair adherence, leading to worse treatment outcomes. Practical monitoring tools can improve adherence and increase rates of viral load suppression. Several antiretroviral drugs exhibit inhibitory and/or inducing effects on cytochrome P450 isoenzymes, which are responsible for the metabolism of many medications used for the treatment of comorbidities in the aging HIV population. The combination of ART with polypharmacy significantly increases the chance of potentially serious drug-drug interactions (DDIs), which can lead to drug toxicity, poorer ART adherence, loss of efficacy of the coadministered medication, or virologic breakthrough. Increasing clinicians awareness of common DDIs and the use of DDI programs can prevent coadministration of potentially harmful combinations in elderly HIV-infected individuals. Well designed ART adherence interventions and DDI studies are needed in the elderly HIV population.

Severe prolonged sedation associated with coadministration of protease inhibitors and intravenous midazolam during bronchoscopy.

STUDY OBJECTIVE: To determine whether human immunodeficiency virus (HIV)-positive patients who received intravenous midazolam during an inpatient bronchoscopy procedure were more likely to experience severe prolonged sedation if they were taking antiretroviral therapy that included a protease inhibitor versus those who were not taking any antiretroviral therapy. DESIGN: Retrospective cohort study. SETTING: Tertiary care academic medical center. PATIENTS: Two hundred forty-one HIV-positive adults who received intravenous midazolam while undergoing bronchoscopy between January 1, 2003, and December 31, 2006, were analyzed; 51 patients were taking an antiretroviral regimen that included a protease inhibitor (exposed group), whereas 190 patients were not taking any antiretroviral agents (nonexposed group). MEASUREMENTS AND MAIN RESULTS: Patient demographics, medication administration records, and bronchoscopy data were collected from electronic databases and patient medical records. The exposed and nonexposed groups had similar demographic characteristics except that patients in the exposed group had lower HIV viral loads and were less likely to have altered mental status or respiratory distress before bronchoscopy. In addition, the exposed group had a higher proportion of males and patients with hepatitis B or C virus coinfection. The incidence of severe prolonged sedation was 9.80% in the exposed group versus 1.58% in the nonexposed group (relative risk [RR] 6.21, 95% confidence interval [CI] 1.53-25.12). Specific protease inhibitors associated with severe prolonged sedation were atazanavir-ritonavir and lopinavir-ritonavir. Length of hospital stay was approximately 3 days longer in the exposed group compared with the nonexposed group. CONCLUSION: Although the interaction between intravenous midazolam and protease inhibitors is well known, this study was the first systematic evaluation, to our knowledge, of the risk of severe prolonged sedation in a cohort of hospitalized HIV-positive patients. Coadministration of protease inhibitors with intravenous midazolam was associated with severe prolonged sedation as well as increased length of hospital stay. Therefore, concomitant use of these drugs should be closely monitored, or alternative sedatives for procedural sedation should be considered.

Structural and mutational studies of a hyperthermophilic intein from DNA polymerase II of Pyrococcus abyssi.

Protein splicing is a precise self-catalyzed process in which an intein excises itself from a precursor with the concomitant ligation of the flanking polypeptides (exteins). Protein splicing proceeds through a four-step reaction but the catalytic mechanism is not fully understood at the atomic level. We report the solution NMR structures of the hyperthermophilic Pyrococcus abyssi PolII intein, which has a noncanonical C-terminal glutamine instead of an asparagine. The NMR structures were determined to a backbone root mean square deviation of 0.46 Å and a heavy atom root mean square deviation of 0.93 Å. The Pab PolII intein has a common HINT (hedgehog intein) fold but contains an extra ß-hairpin that is unique in the structures of thermophilic inteins. The NMR structures also show that the Pab PolII intein has a long and disordered loop in place of an endonuclease domain. The N-terminal Cys-1 amide is hydrogen bonded to the Thr-90 hydroxyl in the conserved block-B TXXH motif and the Cys-1 thiol forms a hydrogen bond with the block F Ser-166. Mutating Thr-90 to Ala dramatically slows N-terminal cleavage, supporting its pivotal role in promoting the N-S acyl shift. Mutagenesis also showed that Thr-90 and His-93 are synergistic in catalyzing the N-S acyl shift. The block F Ser-166 plays an important role in coordinating the steps of protein splicing. NMR spin relaxation indicates that the Pab PolII intein is significantly more rigid than mesophilic inteins, which may contribute to the higher optimal temperature for protein splicing.

Inhibition of angiogenesis and suppression of colorectal cancer metastatic to the liver using the Sleeping Beauty Transposon System.

BACKGROUND: Metastatic colon cancer is one of the leading causes of cancer-related death worldwide, with disease progression and metastatic spread being closely associated with angiogenesis. We investigated whether an antiangiogenic gene transfer approach using the Sleeping Beauty (SB) transposon system could be used to inhibit growth of colorectal tumors metastatic to the liver. RESULTS: Liver CT26 tumor-bearing mice were hydrodynamically injected with different doses of a plasmid containing a transposon encoding an angiostatin-endostatin fusion gene (Statin AE) along with varying amounts of SB transposase-encoding plasmid. Animals that were injected with a low dose (10 µg) of Statin AE transposon plasmid showed a significant decrease in tumor formation only when co-injected with SB transposase-encoding plasmid, while for animals injected with a higher dose (25 µg) of Statin AE transposon, co-injection of SB transposase-encoding plasmid did not significantly affect tumor load. For animals injected with 10 µg Statin AE transposon plasmid, the number of tumor nodules was inversely proportional to the amount of co-injected SB plasmid. Suppression of metastases was further evident in histological analyses, in which untreated animals showed higher levels of tumor cell proliferation and tumor vascularization than animals treated with low dose transposon plasmid. CONCLUSION: These results demonstrate that hepatic colorectal metastases can be reduced using antiangiogenic transposons, and provide evidence for the importance of the transposition process in mediating suppression of these tumors.

Exogenous and endogenous TLR ligands activate anti-chromatin and polyreactive B cells.

Autoreactive B cells may become activated in a T-independent manner via synergistic engagement of the BCR and TLRs. Using the VH3H9 Ig H chain transgene to track anti-chromatin B cells, we demonstrate that VH3H9/Vlambda1 anti-chromatin B cells proliferate in response to stimulatory oligodeoxynucleotides containing CpG motifs, suggesting that these autoreactive B cells are responsive to TLR9 signaling. Strikingly, some VH3H9 B cells, but not the well-characterized VH3H9/Vlambda1 B cells, proliferate spontaneously in culture medium. This proliferation is blocked by inhibitory CpG oligodeoxynucleotides, implicating the TLR9 (or possibly TLR7) pathway. Most hybridomas generated from the proliferating cells are polyreactive, and one exhibits binding to nuclear Ags but not to the other Ags tested. Thus, B cells carrying autoreactive and/or polyreactive specificities may be susceptible to T cell-independent activation via dual engagement of the BCR and TLRs.