Detecting measurable residual disease beyond 10-4 by an IGHV leader-based NGS approach improves prognostic stratification in CLL.

The sensitivity of conventional techniques for reliable quantification of minimal/measurable residual disease (MRD) in chronic lymphocytic leukemia (CLL) is limited to MRD 10-4. Measuring MRD <10-4 could help to further distinguish between patients with CLL with durable remission and those at risk of early relapse. We herein present an academically developed immunoglobulin heavy-chain variable (IGHV) leader-based next-generation sequencing (NGS) assay for the quantification of MRD in CLL. We demonstrate, based on measurements in contrived MRD samples, that the linear range of detection and quantification of our assay reaches beyond MRD 10-5. If provided with sufficient DNA input, MRD can be detected down to MRD 10-6. There was high interassay concordance between measurements of the IGHV leader-based NGS assay and allele-specific oligonucleotide quantitative polymerase chain reaction (PCR) (r = 0.92 [95% confidence interval {CI}, 0.86-0.96]) and droplet digital PCR (r = 0.93 [95% CI, 0.88-0.96]) on contrived MRD samples. In a cohort of 67 patients from the CLL11 trial, using MRD 10-5 as a cutoff, undetectable MRD was associated with superior progression-free survival (PFS) and time to next treatment. More important, deeper MRD measurement allowed for additional stratification of patients with MRD <10-4 but ≥10-5. PFS of patients in this MRD range was significantly shorter, compared with patients with MRD <10-5 (hazard ratio [HR], 4.0 [95% CI, 1.6-10.3]; P = .004), but significantly longer, compared with patients with MRD ≥10-4 (HR, 0.44 [95% CI, 0.23-0.87]; P = .018). These results support the clinical utility of the IGHV leader-based NGS assay.

New rabies viral resources for multi-scale neural circuit mapping.

Comparisons and linkage between multiple imaging scales are essential for neural circuit connectomics. Here, we report 20 new recombinant rabies virus (RV) vectors that we have developed for multi-scale and multi-modal neural circuit mapping tools. Our new RV tools for mesoscale imaging express a range of improved fluorescent proteins. Further refinements target specific neuronal subcellular locations of interest. We demonstrate the discovery power of these new tools including the detection of detailed microstructural changes of rabies-labeled neurons in aging and Alzheimer's disease mouse models, live imaging of neuronal activities using calcium indicators, and automated measurement of infected neurons. RVs that encode GFP and ferritin as electron microscopy (EM) and fluorescence microscopy reporters are used for dual EM and mesoscale imaging. These new viral variants significantly expand the scale and power of rabies virus-mediated neural labeling and circuit mapping across multiple imaging scales in health and disease.

Spontaneous Extrusion of a Conjunctivolith Containing Herpes Virus Confirmed by Electron Microscopy and Energy Dispersive Spectroscopy.

: This report describes the spontaneous extrusion from between the eyelids of a presumed conjunctivolith in a patient with resolving severe herpes zoster ophthalmicus. A 57-year-old man presented for ophthalmologic assessment and management due to severe left herpes zoster ophthalmicus. At one subsequent ophthalmologic assessment, a conjunctivolith spontaneously egressed the lateral commissure of the OS when the lateral fornix was inspected. The conjunctivolith was retrieved from the floor of the consulting room. Electron microscopic analysis and energy dispersive spectroscopy was undertaken to determine its composition. Scanning electron microscopy showed that the conjunctivolith was composed of carbon, calcium, and oxygen. Transmission electron microscopy diagnosed Herpes virus within the conjunctivolith. Conjunctivoliths, or possible lacrimal gland stones, are a very rare phenomenon, and their etiology is currently unclear. In this case, there was likely to have been an association between herpes zoster ophthalmicus and the conjunctivolith.

Analysis of an Australian death database of people with intellectual disability living out of the family home: Place of death and associated variables.

This study reports on a five-year data set about the deaths of 599 individuals in New South Wales Australia, who at the time of their death were living in out-of-home care. Analysis aimed to: i) gain a clearer understanding of place of death for people with intellectual disability; and ii) identify and analyse associated variables to investigate how well they predict place of death for this population. Hospital admissions, polypharmacy and living situation were the strongest standalone predictors of place of death. A hospital death was more likely if the target population were subject to polypharmacy, lived in a group home, had a moderate intellectual disability or had GORD. Death, and place of death, is an issue requiring individual consideration. This study has identified some of the variables that need attention when supporting people with intellectual disability to have a good death.

Manual Therapy Facilitates Homeostatic Adaptation to Bone Microstructural Declines Induced by a Rat Model of Repetitive Forceful Task.

The effectiveness of manual therapy in reducing the catabolic effects of performing repetitive intensive force tasks on bones has not been reported. We examined if manual therapy could reduce radial bone microstructural declines in adult female Sprague-Dawley rats performing a 12-week high-repetition and high-force task, with or without simultaneous manual therapy to forelimbs. Additional rats were provided 6 weeks of rest after task cessation, with or without manual therapy. The control rats were untreated or received manual therapy for 12 weeks. The untreated TASK rats showed increased catabolic indices in the radius (decreased trabecular bone volume and numbers, increased osteoclasts in these trabeculae, and mid-diaphyseal cortical bone thinning) and increased serum CTX-1, TNF-α, and muscle macrophages. In contrast, the TASK rats receiving manual therapy showed increased radial bone anabolism (increased trabecular bone volume and osteoblast numbers, decreased osteoclast numbers, and increased mid-diaphyseal total area and periosteal perimeter) and increased serum TNF-α and muscle macrophages. Rest, with or without manual therapy, improved the trabecular thickness and mid-diaphyseal cortical bone attributes but not the mineral density. Thus, preventive manual therapy reduced the net radial bone catabolism by increasing osteogenesis, while rest, with or without manual therapy, was less effective.

Blocking CTGF/CCN2 reduces established skeletal muscle fibrosis in a rat model of overuse injury.

Tissue fibrosis is a hallmark of overuse musculoskeletal injuries and contributes to functional declines. We tested whether inhibition of CCN2 (cellular communication network factor 2, previously known as connective tissue growth factor, CTGF) using a specific antibody (termed FG-3019 or pamrevlumab) reduces established overuse-induced muscle fibrosis in a clinically relevant rodent model of upper extremity overuse injury. Young adult rats performed a high repetition high force (HRHF) reaching and lever-pulling task for 18 weeks, after first being shaped for 6 weeks to learn this operant task. Rats were then euthanized (HRHF-Untreated), or rested and treated for 6 weeks with FG-3019 (HRHF-Rest/FG-3019) or a human IgG as a vehicle control (HRHF-Rest/IgG). HRHF-Untreated and HRHF-Rest/IgG rats had higher muscle levels of several fibrosis-related proteins (TGFß1, CCN2, collagen types I and III, and FGF2), and higher muscle numbers of alpha SMA and pERK immunopositive cells, compared to control rats. Each of these fibrogenic changes was restored to control levels by the blocking of CCN2 signaling in HRHF-Rest/FG-3019 rats, as were HRHF task-induced increases in serum CCN2 and pro-collagen I intact N-terminal protein. Levels of cleaved CCN3, an antifibrotic protein, were lowered in HRHF-Untreated and HRHF-Rest/IgG rats, compared to control rats, yet elevated back to control levels in HRHF-Rest/FG-3019 rats. Significant grip strength declines observed in HRHF-Untreated and HRHF-Rest/IgG rats, were restored to control levels in HRHF-Rest/FG-3019 rats. These results are highly encouraging for use of FG-3019 for therapeutic treatment of persistent skeletal muscle fibrosis, such as those induced with chronic overuse.

Unplanned implant removal in locally advanced breast cancer.

Study conducted to determine frequency and timing of unplanned breast implant removal after mastectomy, reconstruction, and postmastectomy radiation (PMRT). From 2010-2017, 52 patients underwent mastectomy, reconstruction, and PMRT. With median follow-up of 3.1 years, 23 patients (44%) experienced implant removal. Implant removal occurred in 9 (17%) patients before starting PMRT and 14 (27%) patients after starting PMRT. Implant removal rates were similar for hypofractionated PMRT compared with standard fractionation and for proton compared with photon PMRT. Implant removal is common for women undergoing mastectomy and reconstruction followed by PMRT. The risk is clinically significant even before starting radiation.

Key indicators of repetitive overuse-induced neuromuscular inflammation and fibrosis are prevented by manual therapy in a rat model.

BACKGROUND: We examined the effectiveness of a manual therapy consisting of forearm skin rolling, muscle mobilization, and upper extremity traction as a preventive treatment for rats performing an intensive lever-pulling task. We hypothesized that this treatment would reduce task-induced neuromuscular and tendon inflammation, fibrosis, and sensorimotor declines. METHODS: Sprague-Dawley rats performed a reaching and lever pulling task for a food reward, 2 h/day, 3 days/week, for 12 weeks, while simultaneously receiving the manual therapy treatment 3 times per week for 12 weeks to either the task-involved upper extremities (TASK-Tx), or the lower extremities as an active control group (TASK-Ac). Results were compared to similarly treated control rats (C-Tx and C-Ac). RESULTS: Median nerves and forearm flexor muscles and tendons of TASK-Ac rats showed higher numbers of inflammatory CD68+ and fibrogenic CD206+ macrophages, particularly in epineurium, endomysium and epitendons than TASK-Tx rats. CD68+ and CD206+ macrophages numbers in TASK-Tx rats were comparable to the non-task control groups. TASK-Ac rats had more extraneural fibrosis in median nerves, pro-collagen type I levels and immunoexpression in flexor digitorum muscles, and fibrogenic changes in flexor digitorum epitendons, than TASK-Tx rats (which showed comparable responses as control groups). TASK-Ac rats showed cold temperature, lower reflexive grip strength, and task avoidance, responses not seen in TASK-Tx rats (which showed comparable responses as the control groups). CONCLUSIONS: Manual therapy of forelimbs involved in performing the reaching and grasping task prevented the development of inflammatory and fibrogenic changes in forearm nerves, muscle, and tendons, and sensorimotor declines.

Does talking about end of life with adults with intellectual disability cause emotional discomfort or psychological harm?

BACKGROUND: Caregivers often avoid involving people with intellectual disability in end-of-life discussions and activities. One reason is fear that the person may become upset or psychologically harmed. METHODS: Pre and post a 6-month intervention about end of life, we assessed depression, anxiety, and fear of death among intervention (n = 24) and comparison (n = 20) participants with intellectual disability. End-of-life 'encounters' (conversations/activities about end of life) were monitored, including comfort ratings. RESULTS: Overall, 79% of encounters were rated very comfortable/somewhat comfortable. Participants initiated 69% of encounters. There was no significant pre-post change in depression or fear of death. Anxiety improved significantly. CONCLUSIONS: This is the first controlled, longitudinal study providing robust evidence about whether discussing end of life leads to emotional discomfort or psychological harm. Data showed adults with intellectual disability can safely engage in conversations/activities about end of life. The high percentage of participant-initiated encounters showed participants wanted to talk about end of life.

Impact of adjuvant trastuzumab on locoregional failure rates in a randomized clinical trial: North Central Cancer Treatment Group N9831 (alliance) study.

BACKGROUND: The goal of this study was to assess the impact of trastuzumab on locoregional failure. METHODS: The analysis included 2763 patients with HER2-positive (HER2+) breast cancer who were randomly assigned to adjuvant doxorubicin (A), cyclophosphamide (C), paclitaxel (T) and trastuzumab (H) (arm A, AC→T [n = 922]; arm B, AC→T→H [n = 988]; arm C, AC→T+H→H [n = 853]). Radiotherapy was given after AC→T concurrently with H. Radiotherapy was given after lumpectomy (L) or after mastectomy (M) with ≥4 positive lymph nodes but was optional for 1 to 3 positive lymph nodes. Locoregional failures at 10 years (LFR10) as first events were compared using competing risk analysis. RESULTS: The median follow-up was 13.0 years. The first site of failure was local-only in 96 cases, locoregional in 16 cases, regional in 32 cases, and not specified in 2 cases; LFR10 was 4.8% (95% CI 4.1%-5.7%). LFR10 was 5.5% (95% CI 4.3%-7.2%), 4.9% (95% CI 3.7%-6.4%), and 2.8% (95% CI 1.9%-4.1%) in arms A, B, and C (B vs A: hazard ratio [HR] 0.91, P = .62; C vs A: HR 0.72, P = .12). For estrogen receptor-positive patients, LFR10 was 3.7% (95% CI 2.8%-4.8%) and for estrogen receptor-negative patients, it was 6.1% (95% CI 5.0%-7.4%; HR 0.61, P = .004). Local treatment included L+RT (n = 1044 [38%]), M+RT (n = 1025 [37%]), and M (n = 694 [25%]). LFR10 was 6.% (95% CI 5.0%-7.8%), 3.0% (95% CI 2.1%-4.3%), and 5.5% (95% CI 4.0%-7.4%) for L+RT, M+RT, and M, respectively (M+RT vs L+RT: HR 0.43, P < .001; M vs L+RT: HR 0.88, P = .57). For 1 to 3 positive lymph nodes, LFR10 was 6.5% (95% CI 4.8%-8.9%), 4.1% (95% CI 2.4%-7.0%), and 4.3% (95% CI 2.9%-6.5%) in L+RT, M+RT, and M, respectively (M vs L+RT: HR 0.68, P = .14; M vs M+RT: HR 1.2, P = .6). CONCLUSION: Low 10-year LFRs were seen regardless of trastuzumab use. Differences in local therapy in patients with 1 to 3 positive lymph nodes did not appear to improve local control. Local therapy studies for HER2+ and other tumor characteristics are important as the role of local therapies continues to evolve.

Early Plasma Matrix Metalloproteinase Profiles. A Novel Pathway in Pediatric Acute Respiratory Distress Syndrome.

RATIONALE: MMPs (Matrix metalloproteinases) and their endogenous tissue inhibitors may contribute to lung injury through extracellular matrix degradation and modulation of inflammation and fibrosis. OBJECTIVES: To test for an association between MMP pathway proteins and inflammation, endothelial dysfunction, and clinical outcomes. METHODS: We measured MMPs in plasma collected on acute respiratory distress syndrome (ARDS) Day 1 from 235 children at five hospitals between 2008 and 2017. We used latent class analysis to identify patients with distinct MMP profiles and then associated those profiles with markers of inflammation (IL-1RA, -6, -8, -10, and -18; macrophage inflammatory protein-1α and -1ß; tumor necrosis factor-α and -R2), endothelial injury (angiopoietin-2, von Willebrand factor, soluble thrombomodulin), impaired oxygenation (PaO2/FiO2 [P/F] ratio, oxygenation index), morbidity, and mortality. MEASUREMENTS AND MAIN RESULTS: In geographically distinct derivation and validation cohorts, approximately one-third of patients demonstrated an MMP profile characterized by elevated MMP-1, -2, -3, -7, and -8 and tissue inhibitor of metalloproteinase-1 and -2; and depressed active and total MMP-9. This MMP profile was associated with multiple markers of inflammation, endothelial injury, and impaired oxygenation on Day 1 of ARDS, and conferred fourfold increased odds of mortality or severe morbidity independent of the P/F ratio and other confounders (95% confidence interval, 2.1-7.6; P < 0.001). Logistic regression using both the P/F ratio and MMP profiles was superior to the P/F ratio alone in prognosticating mortality or severe morbidity (area under the receiver operating characteristic curve, 0.75; 95% confidence interval, 0.68-0.82 vs. area under the receiver operating characteristic curve, 0.66; 95% confidence interval, 0.58-0.73; P = 0.009). CONCLUSIONS: Pediatric patients with ARDS have specific plasma MMP profiles associated with inflammation, endothelial injury, morbidity, and mortality. MMPs may play a role in the pathobiology of children with ARDS.

The experience of mothers supporting self-determination of adult sons and daughters with intellectual disability.

BACKGROUND: The right of people with disability to be self-determining, to live a life of their choosing, is increasingly recognized and promoted. For adults with intellectual disability, support to enable self-determination may be required. This is often provided by family, yet little is understood about the experience of providing such support. METHODS: An interpretative phenomenological analysis (IPA) of eight individual, semi-structured interviews with mothers was conducted, to understand the meaning given to their experience of supporting self-determination of their adult son or daughter with intellectual disability. RESULTS: Three superordinate themes were identified: (a) support context; (b) continuum of support roles; and (c) mother's personal concerns. CONCLUSION: Mothers of adults with intellectual disability experience an ongoing sense of responsibility to balance competing rights and concerns as they support self-determination. This complex, interdependent relationship results in roles that may facilitate, guide, influence and at times restrict choice and control.

Comparing effects of rest with or without a NK1RA on fibrosis and sensorimotor declines induced by a voluntary moderate demand task.

OBJECTIVES: Fibrosis is one contributing factor in motor dysfunction and discomfort in patients with overuse musculoskeletal disorders. We pharmacologically targeted the primary receptor for Substance P, neurokinin-1, using a specific antagonist (NK1RA) in a rat model of overuse with the goal of improving tissue fibrosis and discomfort. METHODS: Female rats performed a low repetition, high force (LRHF) grasping task for 12 weeks, or performed the task for 12 weeks before being placed on a four week rest break, with or without simultaneous NK1RA treatment. Results were compared to control rats (untreated, or treated 4 weeks with NK1RA or vehicle). RESULTS: Rest improved LRHF-induced declines in grip strength, although rest plus NK1RA treatment (Rest/NK1RA) rescued it. Both treatments improved LRHF-induced increases in muscle TGFß1 and collagen type 1 levels, forepaw mechanical hypersensitivity (Rest/NK1RA more effectively), macrophage influx into median nerves, and enhanced collagen deposition in forepaw dermis. Only Rest/NK1RA reduced muscle hypercellularity. However, LRHF+4wk Rest /NK1RA rats showed hyposensitivity to noxious hot temperatures. CONCLUSIONS: While the NK1RA induced hot temperature hyposensitivity should be taken into consideration if this or related drug were used long-term, the NK1RA more effectively reduced muscle hypercellularity and improved grip strength and forepaw mechanical hypersensitivity.

Phosphorylation of STAT2 on serine-734 negatively regulates the IFN-α-induced antiviral response.

Serine phosphorylation of STAT proteins is an important post-translational modification event that, in addition to tyrosine phosphorylation, is required for strong transcriptional activity. However, we recently showed that phosphorylation of STAT2 on S287 induced by type I interferons (IFN-α and IFN-ß), evoked the opposite effect. S287-STAT2 phosphorylation inhibited the biological effects of IFN-α. We now report the identification and characterization of S734 on the C-terminal transactivation domain of STAT2 as a new phosphorylation site that can be induced by type I IFNs. IFN-α-induced S734-STAT2 phosphorylation displayed different kinetics to that of tyrosine phosphorylation. S734-STAT2 phosphorylation was dependent on STAT2 tyrosine phosphorylation and JAK1 kinase activity. Mutation of S734-STAT2 to alanine (S734A) enhanced IFN-α-driven antiviral responses compared to those driven by wild-type STAT2. Furthermore, DNA microarray analysis demonstrated that a small subset of type I IFN stimulated genes (ISGs) was induced more by IFNα in cells expressing S734A-STAT2 when compared to wild-type STAT2. Taken together, these studies identify phosphorylation of S734-STAT2 as a new regulatory mechanism that negatively controls the type I IFN-antiviral response by limiting the expression of a select subset of antiviral ISGs.

Electronic patient-reported outcomes and toxicities during radiotherapy for head-and-neck cancer.

PURPOSE: To characterize quality of life (QOL) using real-time, electronic patient-reported outcomes (ePROs) and to evaluate adverse events (AEs) and supportive care during head-and-neck radiotherapy (RT) and concurrent chemoradiotherapy (CCRT). METHODS: Sixty-five patients undergoing head-and-neck RT completed electronic, real-time, 12-item linear analog self-assessments (LASA) at baseline, before biweekly appointments, and at the last week of RT. Changes in QOL domains between time points were calculated. Clinical data were collected from the institutional medical record. AEs were recorded at the same time points as the LASA and graded. RESULTS: During head-and-neck RT, most patients had clinically meaningful decreases in all QOL domains except level of support, financial concerns, and legal concerns. QOL domains with the most prevalent, clinically meaningful decreases were fatigue (75.4% of patients; 95% CI, 62.9-84.9%), social activity (70.8%; 95% CI, 58.0-81.1%), and overall QOL (70.8%; 95% CI, 58.0-81.1%). All patients had grade 2 AEs; 35.4% had grade 3 (50.0%, CCRT; 12.0%, RT; P = .002). Weight loss averaged 5.5 kg (6.9 kg, CCRT; 2.8 kg, RT; P < .001). Intravenous hydration was needed in 52.3% (77.5%, CCRT; 12.0%, RT; P < .001); feeding tube placement 40.0% (57.5%, CCRT; 12.0%, RT; P = .001); emergency department visits without hospitalization, 10.8%; and emergent hospitalization, 27.7% (37.5%, CCRT; 12.0%, RT; P = .04). CONCLUSIONS: Head-and-neck RT, particularly CCRT, negatively impacts patients' overall QOL, social activity, and fatigue, with frequent grade 3 AEs, weight loss, intravenous hydration, feeding tube placement, ED visits, and hospitalization. Real-time ePROs allow providers to monitor QOL at multiple time points during RT, potentially allowing early intervention to improve QOL and mitigate AEs.

Exploratory study of the association of volumetric modulated arc therapy (VMAT) plan robustness with local failure in head and neck cancer.

This work is to show which is more relevant to cause local failures (LFs) due to patient setup uncertainty between the planning target volume (PTV) underdosage and the potential target underdosage subject to patient setup uncertainties in head and neck (H&N) cancer treated with volumetric-modulated arc therapy (VMAT). Thirteen LFs in 10 H&N patients treated by VMAT were analyzed. Measures have been taken to minimize the chances of insufficient target delineation for these patients and the patients were clinically determined to have LF based on the PET/CT scan results by an experienced radiologist and then reviewed by a second experienced radiation oncologist. Two methods were used to identify the possible locations of LF due to underdosage: (a) examining the standard VMAT plan, in which the underdosed volume in the nominal dose distribution (UVN) was generated by subtracting the volumes receiving the prescription doses from PTVs, and (b) plan robustness analysis, in which in addition to the nominal dose distribution, six perturbed dose distributions were created by translating the CT iso-center in three cardinal directions by the PTV margin. The coldest dose distribution was represented by the minimum of the seven doses in each voxel. The underdosed volume in the coldest dose distribution (UVC) was generated by subtracting the volumes receiving the prescription doses in the coldest dose distribution from the volumes receiving the prescription doses in the nominal dose distribution. UVN and UVC were subsequently examined for spatial association with the locations of LF. The association was tested using the binominal distribution and the Fisher's exact test of independence. We found that of 13 LFs, 11 were associated with UVCs (P = 0.011), while three were associated with UVNs (P = 0.99). We concluded that the possible target underdosage due to patient setup uncertainties appeared to be a more relevant factor associated with LF in VMAT for H&N cancer than the compromised PTV coverage at least for the patients included in this study.

[Loss of BRCA associated protein 1 expression in malignant mesothelioma and its diagnostic application].

Objective: To investigate the expression of BRCA-associated protein 1 (BAP1) in malignant mesothelioma, non-small cell lung cancer and carcinosarcoma, and its application in the differential diagnosis. Methods: Twenty-two cases of malignant mesothelioma including 17 epithelioid type, 2 sarcomatoid type and 3 biphasic type were collected.As the study control, 80 non-small cell lung cancers infringement pleural membrane(including 40 lung adenocarcinomas and 40 lung squamous cell carcinomas) and 15 carcinosarcomas were included. BAP1 expression was detected using immunohistochemical method. A differential diagnosis antibody panel, including calretinin, WT1, CK5/6, D2-40, CAM5.2, CEA, TTF1, Napsin A, p63 and p40 was tested in all cases. Results: All 80 cases of non-small cell lung cancer and 15 cases of carcinosarcoma were BAP1 positive. In contrast, 64% (14/22) of malignant mesotheliomas lost BAP1 expression (P<0.01). Addition of BAP1 to the mesothelioma marker panel, the diagnostic accuracy of malignant mesothelioma was enhanced to 93%. Focal expression of BAP1 in tumors suggested multiclonal evolution of mesothelioma. Conclusions: Loss of BAP1 expression helps to confirm the diagnosis of malignant mesothelioma whereas all non-small cell lung cancer expresses BAP1. It is therefore recommended that BAP1 can be used in conjunction with other immunohistochemical markers to improve the diagnostic accuracy of malignant mesothelioma.