Evaluation of T-cell clonality by anti-TRBC1 antibody-based flow cytometry and correlation with T-cell receptor sequencing.

Flow cytometry (FC) incorporating the T-cell receptor ß constant chain-1 (TRBC1) has been recently proposed as a new standard in T-cell clonality assessment. While early studies demonstrated high sensitivity in samples with conspicuous tumour burden, performance in real-world samples, including those with low tumour burden and correlation with molecular methods has been limited. We evaluated TRBC1-FC performance and correlated the results with high-throughput TRB sequencing and a targeted next-generation sequencing gene panel. Our cohort consisted of 90 evaluable samples from 57 patients. TRBC1-FC confirmed T-cell clonality in 37 out of 38 samples (97%) that were involved in a mature T-cell neoplasm (MTCN). T-cell clonality was also identified in nine samples from patients lacking a current or prior diagnosis of MTCN, consistent with the emerging entity T-cell clonality of uncertain significance. TRBC-FC was polyclonal in all samples and negative for disease involvement by standard pathology assessment. However, correlation with TRB sequencing in 17 of these samples identified two cases that harboured the known clonal sequence from index testing, indicating the presence of measurable residual disease not otherwise detected. Our study provides real-world correlative validation of TRBC1-FC, highlighting the strengths and limitations pertinent to its increasing implementation by general diagnostic laboratories.

Persistence of UBTF tandem duplications in remission in acute myeloid leukaemia.

UBTF tandem duplications are recurrent in adult and paediatric acute myeloid leukaemia and have been reported to be associated with a poor prognosis. Co-mutations in WT1 and FLT3 are common while morphological dysplasia is frequent. The role of UBTF-TDs in leukemogenesis is yet to be elucidated; however they have been proposed as early initiating events, making them attractive for assessment of MRD and a potential therapeutic target. We present two cases where the UBTF-TD was observed in remission and discuss the implications of these findings in the clinicobiological understanding of this emerging entity.

GCP16 stabilizes the DHHC9 subfamily of protein acyltransferases through a conserved C-terminal cysteine motif.

Protein S-acylation is a reversible lipid post-translational modification that allows dynamic regulation of processes such as protein stability, membrane association, and localization. Palmitoyltransferase ZDHHC9 (DHHC9) is one of the 23 human DHHC acyltransferases that catalyze protein S-acylation. Dysregulation of DHHC9 is associated with X-linked intellectual disability and increased epilepsy risk. Interestingly, activation of DHHC9 requires an accessory protein-GCP16. However, the exact role of GCP16 and the prevalence of a requirement for accessory proteins among other DHHC proteins remain unclear. Here, we report that one role of GCP16 is to stabilize DHHC9 by preventing its aggregation through formation of a protein complex. Using a combination of size-exclusion chromatography and palmitoyl acyltransferase assays, we demonstrate that only properly folded DHHC9-GCP16 complex is enzymatically active in vitro. Additionally, the ZDHHC9 mutations linked to X-linked intellectual disability result in reduced protein stability and DHHC9-GCP16 complex formation. Notably, we discovered that the C-terminal cysteine motif (CCM) that is conserved among the DHHC9 subfamily (DHHC14, -18, -5, and -8) is required for DHHC9 and GCP16 complex formation and activity in vitro. Co-expression of GCP16 with DHHCs containing the CCM improves DHHC protein stability. Like DHHC9, DHHC14 and DHHC18 require GCP16 for their enzymatic activity. Furthermore, GOLGA7B, an accessory protein with 75% sequence identity to GCP16, improves protein stability of DHHC5 and DHHC8, but not the other members of the DHHC9 subfamily, suggesting selectivity in accessory protein interactions. Our study supports a broader role for GCP16 and GOLGA7B in the function of human DHHCs.

Computational flow cytometry provides accurate assessment of measurable residual disease in chronic lymphocytic leukaemia.

Undetectable measurable residual disease (MRD) is associated with favourable clinical outcomes in chronic lymphocytic leukaemia (CLL). While assessment is commonly performed using multiparameter flow cytometry (MFC), this approach is associated with limitations including user bias and expertise that may not be widely available. Implementation of unsupervised clustering algorithms in the laboratory can address these limitations and have not been previously reported in a systematic quantitative manner. We developed a computational pipeline to assess CLL MRD using FlowSOM. In the training step, a self-organising map was generated with nodes representing the full breadth of normal immature and mature B cells along with disease immunophenotypes. This map was used to detect MRD in multiple validation cohorts containing a total of 456 samples. This included an evaluation of atypical CLL cases and samples collected from two different laboratories. Computational MRD showed high correlation with expert analysis (Pearson's r > 0.99 for typical CLL). Binary classification of typical CLL samples as either MRD positive or negative demonstrated high concordance (>98%). Interestingly, computational MRD detected disease in a small number of atypical CLL cases in which MRD was not detected by expert analysis. These results demonstrate the feasibility and value of automated MFC analysis in a diagnostic laboratory.

Hepatic macrophage mediated immune response in liver steatosis driven carcinogenesis.

Obesity confers an independent risk for carcinogenesis. Classically viewed as a genetic disease, owing to the discovery of tumor suppressors and oncogenes, genetic events alone are not sufficient to explain the progression and development of cancers. Tumor development is often associated with metabolic and immunological changes. In particular, obesity is found to significantly increase the mortality rate of liver cancer. As its role is not defined, a fundamental question is whether and how metabolic changes drive the development of cancer. In this review, we will dissect the current literature demonstrating that liver lipid dysfunction is a critical component driving the progression of cancer. We will discuss the involvement of inflammation in lipid dysfunction driven liver cancer development with a focus on the involvement of liver macrophages. We will first discuss the association of steatosis with liver cancer. This will be followed with a literature summary demonstrating the importance of inflammation and particularly macrophages in the progression of liver steatosis and highlighting the evidence that macrophages and macrophage produced inflammatory mediators are critical for liver cancer development. We will then discuss the specific inflammatory mediators and their roles in steatosis driven liver cancer development. Finally, we will summarize the molecular pattern (PAMP and DAMP) as well as lipid particle signals that are involved in the activation, infiltration and reprogramming of liver macrophages. We will also discuss some of the therapies that may interfere with lipid metabolism and also affect liver cancer development.

Upfront tandem autologous non-myeloablative allogeneic stem cell transplant in high-risk multiple myeloma: a long-term single-centre experience.

The role of upfront non-myeloablative allogeneic stem cell transplantation (NMA alloSCT) in high-risk multiple myeloma (HR-MM) is unclear. We evaluated outcomes of NMA alloSCT following autologous stem cell transplant (ASCT) compared with ASCT alone for newly diagnosed HR-MM. Two-year progression-free survival was improved in the ASCT-NMA alloSCT group (44% vs 16%; P = 0.035), with a trend for improved overall survival (P = 0.118). These results suggest that ASCT-NMA alloSCT can be considered as upfront therapy in HR-MM.

Designing Protease-Triggered Protein Cages.

Proteins that self-assemble into enclosed polyhedral cages, both naturally and by design, are garnering attention for their prospective utility in the fields of medicine and biotechnology. Notably, their potential for encapsulation and surface display are attractive for experiments that require protection and targeted delivery of cargo. The ability to control their opening or disassembly would greatly advance the development of protein nanocages into widespread molecular tools. Toward the development of protein cages that disassemble in a systematic manner and in response to biologically relevant stimuli, here we demonstrate a modular protein cage system that is opened by highly sequence-specific proteases, based on sequence insertions at strategically chosen loop positions in the protein cage subunits. We probed the generality of the approach in the context of protein cages built using the two prevailing methods of construction: genetic fusion between oligomeric components and (non-covalent) computational interface design between oligomeric components. Our results suggest that the former type of cage may be more amenable than the latter for endowing proteolytically controlled disassembly. We show that a successfully designed cage system, based on oligomeric fusion, is modular with regard to its triggering protease. One version of the cage is targeted by an asparagine protease implicated in cancer and Alzheimer's disease, whereas the second version is responsive to the blood-clotting protease, thrombin. The approach demonstrated here should guide future efforts to develop therapeutic vectors to treat disease states where protease induction or mis-regulation occurs.

Outcomes of non-myeloablative allogeneic stem cell transplant in older patients with acute myeloid leukaemia in first remission.

The benefits of non-myeloablative stem cell transplant in older patients with acute myeloid leukaemia are unclear. We compare the long-term outcomes of this regimen in those aged 55-65 years in first remission with a chemotherapy only cohort that achieved durable morphologic remission. Five-year overall survival was similar (32% vs 33%, P = 0.90), as was relapse-free survival (23% vs 20%, P = 0.37). There was a trend for decreased relapse that was balanced against increased non-relapse mortality with transplantation.

Rare case of diffuse large B-cell lymphoma presenting with central pontine myelinolysis.

Central pontine myelinolysis (CPM) is commonly associated with osmotic stress and rapid correction of hyponatraemia. It has rarely been reported in conjunction with malignancies. We report a case where CPM was not only associated with a new diagnosis of diffuse large B-cell lymphoma but was also a key presenting feature.

Venetoclax induces rapid elimination of NPM1 mutant measurable residual disease in combination with low-intensity chemotherapy in acute myeloid leukaemia.

Based on promising results in older adults with acute myeloid leukaemia (AML), we treated patients with NPM1mut measurable residual disease (MRD) using off-label venetoclax in combination with low-dose cytarabine or azacitidine. Twelve consecutive patients were retrospectively identified, including five with molecular persistence and seven with molecular relapse/progression. All patients with molecular persistence achieved durable molecular complete remission (CRMRD- ) without transplantation. Six of seven patients with molecular relapse/progression achieved CRMRD- after 1-2 cycles of venetoclax. This paper highlights the promising efficacy of venetoclax-based therapy to reduce the relapse risk in patients with persistent or rising NPM1mut MRD.

An ethylene cross-bridged pentaazamacrocycle and its Cu2+ complex: constrained ligand topology and excellent kinetic stability.

Rigid and topologically constrained ethylene cross-bridged tetraazamacrocycles have been increasingly utilised for thirty years as they form remarkably stable transition metal complexes for catalysis, biomedical imaging, and inorganic drug molecule applications. Extending these benefits to pentaazamacrocycles has been achieved and a first transition metal complex prepared and structurally characterized.

The Role of Patient Advocacy and the Declining Rate of Clitoroplasty in 46,XX Patients With Congenital Adrenal Hyperplasia.

Prior to the 1990s, most 46,XX infants with clitoromegaly secondary to congenital adrenal hyperplasia were treated with feminizing genitoplasty to make their cosmetic appearance congruent with their genotypic sex. A 2006 consensus statement for the management of intersex disorders accepted input from patient advocates and did not support purely cosmetic surgery for clitoromegaly. This study examined the extent to which the desired change was implemented in practice. Retrospective chart review was performed at a single Midwestern tertiary care medical center for patients born between 1979 and 2013. Of 45 virilized patients, 40 had clitoromegaly and 39 had urogenital sinus or posterior labial fusion. Twenty-seven (67.5%) patients underwent clitoroplasty and 33 (84.6%) underwent perineoplasty, including vaginoplasty, urethroplasty, imperforate vagina repair, and/or posterior labial fusion repair. There was a linear decline in the rate of clitoroplasty over time for the patient cohort. This study demonstrates the power of patient advocacy to improve medical practice.

Calcipotriol Targets LRP6 to Inhibit Wnt Signaling in Pancreatic Cancer.

UNLABELLED: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy in need of more effective treatment approaches. One potential therapeutic target is Wnt/ß-catenin signaling, which plays important roles in PDAC tumor initiation and progression. Among Wnt inhibitors with suitable in vivo biologic activity is vitamin D, which is known to antagonize Wnt/ß-catenin signaling in colorectal cancer and have antitumor activity in PDAC. For this study, the relationship between vitamin D signaling, Wnt/ß-catenin activity, and tumor cell growth in PDAC was investigated through the use of calcipotriol, a potent non-hypercalcemic vitamin D analogue. PDAC tumor cell growth inhibition by calcipotriol was positively correlated with vitamin D receptor expression and Wnt/ß-catenin activity. Furthermore, vitamin D and Wnt signaling activity were found to be reciprocally linked through feedback regulation. Calcipotriol inhibited autocrine Wnt/ß-catenin signaling in PDAC cell lines in parallel with decreased protein levels of the low-density lipoprotein receptor-related protein 6 (LRP6), a requisite coreceptor for ligand-dependent canonical Wnt signaling. Decrease in LRP6 protein seen with calcipotriol was mediated through a novel mechanism involving transcriptional upregulation of low-density lipoprotein receptor adaptor protein 1 (LDLRAP1). Finally, changes in LRP6 or LDLRAP1 expression directly altered Wnt reporter activity, supporting their roles as regulators of ligand-dependent Wnt/ß-catenin signaling. IMPLICATIONS: This study provides a novel biochemical target through which vitamin D signaling exerts inhibitory effects on Wnt/ß-catenin signaling, as well as potential biomarkers for predicting and following tumor response to vitamin D-based therapy.

Leptin-receptor-expressing neurons in the dorsomedial hypothalamus and median preoptic area regulate sympathetic brown adipose tissue circuits.

Brown adipose tissue (BAT) thermogenesis is critical to maintain homoeothermia and is centrally controlled via sympathetic outputs. Body temperature and BAT activity also impact energy expenditure, and obesity is commonly associated with decreased BAT capacity and sympathetic tone. Severely obese mice that lack leptin or its receptor (LepRb) show decreased BAT capacity, sympathetic tone, and body temperature and thus are unable to adapt to acute cold exposure (Trayhurn et al., 1976). LepRb-expressing neurons are found in several hypothalamic sites, including the dorsomedial hypothalamus (DMH) and median preoptic area (mPOA), both critical sites to regulate sympathetic, thermoregulatory BAT circuits. Specifically, a subpopulation in the DMH/dorsal hypothalamic area (DHA) is stimulated by fever-inducing endotoxins or cold exposure (Dimicco and Zaretsky, 2007; Morrison et al., 2008). Using the retrograde, transsynaptic tracer pseudorabies virus (PRV) injected into the BAT of mice, we identified PRV-labeled LepRb neurons in the DMH/DHA and mPOA (and other sites), thus indicating their involvement in the regulation of sympathetic BAT circuits. Indeed, acute cold exposure induced c-Fos (as a surrogate for neuronal activity) in DMH/DHA LepRb neurons, and a large number of mPOA LepRb neurons project to the DMH/DHA. Furthermore, DMH/DHA LepRb neurons (and a subpopulation of LepRb mPOA neurons) project and synaptically couple to rostral raphe pallidus neurons, consistent with the current understanding of BAT thermoregulatory circuits from the DMH/DHA and mPOA (Dimicco and Zaretsky, 2007; Morrison et al., 2008). Thus, these data present strong evidence that LepRb neurons in the DMH/DHA and mPOA mediate thermoregulatory leptin action.