Fusion Harboring Mast Cells Can Explain Molecular Positivity in Flow Cytometric MRD Negative Core Binding Factor AML.

Molecular measurable residual disease (MRD) can persist in core binding factor acute myeloid leukemia (AML) in otherwise disease-free patients. Utilizing cell sorting followed by fluorescent in situ hybridization, we show that detection is due to mast cells.

Addressing a Pre-Clinical Pipeline Gap: Development of the Pediatric Acute Myeloid Leukemia Patient-Derived Xenograft Program at Texas Children's Hospital at Baylor College of Medicine.

The survival rate of pediatric acute myeloid leukemia (pAML) is currently around 60%. While survival has slowly increased over the past few decades, the development of novel agents likely to further improve survival for this heterogeneous patient population has been limited by gaps in the pAML pre-clinical pipeline. One of the major hurdles in evaluating new agents for pAML is the lack of pAML patient-derived xenograft (PDX) models. Unlike solid tumors and other types of leukemias, AML is notoriously hard to establish in mouse models, likely due in part to the need for specific human microenvironment elements. Our laboratory at TCH/BCM addressed this gap by establishing a systematic PDX workflow, leveraging advanced immunodeficient hosts and capitalizing on our high volume of pAML patients and close coordination between labs and clinical sections. Patients treated at TCH are offered the chance to participate in specimen banking protocols that allow blood and bone marrow collection as well as the collection of relevant clinical data. All patients who consent and have samples available are trialed for PDX development. In addition, samples from the Children's Oncology Group (COG) are also trialed for PDX generation. Serially transplanting PDX models are validated using short tandem repeat (STR) and characterized using both targeted DNA/RNA next generation sequencing and RNAseq. As of March 2023, this systematic approach has resulted in 26 serially transplanting models. Models have been shared with requesting labs to facilitate external pAML pre-clinical studies. Available PDX models can be located through the BCM PDX Portal. We expect our growing PDX resource to make a significant contribution to expediting the testing of promising novel therapeutics for pAML.

CD74 is expressed in a subset of pediatric acute myeloid leukemia patients and is a promising target for therapy: a report from the Children's Oncology Group.

As curative therapies for pediatric AML remain elusive, identifying potential new treatment targets is vital. We assessed the cell surface expression of CD74, also known as the MHC-II invariant chain, by multidimensional flow cytometry in 973 patients enrolled in the Children's Oncology Group AAML1031 clinical trial. 38% of pediatric AML patients expressed CD74 at any level and a comparison to normal hematopoietic cells revealed a subset with increased expression relative to normal myeloid progenitor cells. Pediatric AML patients expressing high intensity CD74 typically had an immature immunophenotype and an increased frequency of lymphoid antigen expression. Increased CD74 expression was associated with older patients with lower WBC and peripheral blood blast counts, and was enriched for t(8;21), trisomy 8, and CEBPA mutations. Overall, high CD74 expression was associated with low-risk status, however 26% of patients were allocated to high-risk protocol status and 5-year event free survival was 53%, indicating that a significant number of high expressing patients had poor outcomes. In vitro pre-clinical studies indicate that anti-CD74 therapy demonstrates efficacy against AML cells but has little impact on normal CD34+ cells. Together, we demonstrate that CD74 is expressed on a subset of pediatric AMLs at increased levels compared to normal hematopoietic cells and is a promising target for therapy in expressing patients. Given that nearly half of patients expressing CD74 at high levels experience an adverse event within 5 years, and the availability of CD74 targeting drugs, this represents a promising line of therapy worthy of additional investigation.

Urinary calcium indices in primary hyperparathyroidism (PHPT) and familial hypocalciuric hypercalcaemia (FHH): which test performs best?

AIM: Primary hyperparathyroidism (PHPT) is much more common than familial hypocalciuric hypercalcaemia (FHH), but there is considerable overlap in biochemical features. Urine calcium indices help with the differential diagnosis, but their reliability in making this distinction is not clear. The aim of this study was to compare urinary calcium values in patients with PHPT and FHH. METHODS: This was a case-control study of patients with PHPT who had successful surgery and genetically proven FHH between 2011 and 2016. Due to low FHH numbers, patients from neighbouring hospitals and outside study period (2017-2019) were allowed to improve power. Data on demographics and urinary calcium were obtained from electronic records and compared between the two groups. RESULTS: During the study period, 250 patients underwent successful PHPT surgery, while in the FHH arm, 19 genetically proven cases were included. The median (IQR) 24-hour urine calcium excretion (UCE) in the PHPT group was 8.3 (5.6-11.2) mmol/24 hours compared with 3.2 (2.1-6.1) mmol/24 hour in the FHH group (p<0.001). Median (IQR) calcium to creatinine clearance ratio (CCCR) in the PHPT and FHH groups was 0.020 (0.013-0.026) and 0.01 (0.002-0.02), respectively (p=0.001). The sensitivity of urinary tests for PHPT was 96% for UCE (cut-off ≥2.5 mmol/24 hour) and 47% for CCCR (cut-off >0.02). The specificity of the urinary tests for FHH was 29.4% for UCE (cut-off <2.5 mmol/24 hour) and 93% for CCCR (cut-off <0.02). CONCLUSIONS: 24-hour UCE is more sensitive in diagnosing PHPT; however, it is less specific in ruling out FHH as compared with CCCR, when the cut-offs suggested by the International guidelines from the fourth international workshop are used. A significant proportion of patients with PHPT would have also required genetic studies if the guidelines were followed.

Characterization of renal cell carcinoma-associated constitutional chromosome abnormalities by genome sequencing.

Constitutional translocations, typically involving chromosome 3, have been recognized as a rare cause of inherited predisposition to renal cell carcinoma (RCC) for four decades. However, knowledge of the molecular basis of this association is limited. We have characterized the breakpoints by genome sequencing (GS) of constitutional chromosome abnormalities in five individuals who presented with RCC. In one individual with constitutional t(10;17)(q11.21;p11.2), the translocation breakpoint disrupted two genes: the known renal tumor suppressor gene (TSG) FLCN (and clinical features of Birt-Hogg-Dubé syndrome were detected) and RASGEF1A. In four cases, the rearrangement breakpoints did not disrupt known inherited RCC genes. In the second case without chromosome 3 involvement, the translocation breakpoint in an individual with a constitutional t(2;17)(q21.1;q11.2) mapped 12 Kb upstream of NLK. Interestingly, NLK has been reported to interact indirectly with FBXW7 and a previously reported RCC-associated translocation breakpoint disrupted FBXW7. In two cases of constitutional chromosome 3 translocations, no candidate TSGs were identified in the vicinity of the breakpoints. However, in an individual with a constitutional chromosome 3 inversion, the 3p breakpoint disrupted the FHIT TSG (which has been reported previously to be disrupted in two apparently unrelated families with an RCC-associated t(3;8)(p14.2;q24.1). These findings (a) expand the range of constitutional chromosome rearrangements that may be associated with predisposition to RCC, (b) confirm that chromosome rearrangements not involving chromosome 3 can predispose to RCC, (c) suggest that a variety of molecular mechanisms are involved the pathogenesis of translocation-associated RCC, and (d) demonstrate the utility of GS for investigating such cases.

A reassessment of the phylogeny and circumscription of Zaluzianskya (Scrophulariaceae).

The genus Zaluzianskya (Scrophulariaceae s.s.) encompasses a diversity of floral and ecological traits. However, this diversity, as described by the current taxonomic circumscription of Zaluzianskya, is an underestimate. We present molecular data suggesting that this genus requires expansion via incorporation of species from other genera and recognition of unnamed cryptic species. This study advances prior molecular phylogenies of the southern African genus through the addition of DNA regions and 51 populations that had not previously been sampled in a published phylogeny. A total of 82 species of Zaluzianskya and related genera are included, adding 48 to those previously sampled. Results are presented from analyses of five DNA regions, including nuclear ITS and four rapidly evolving chloroplast regions (trnL-trnF, rpl16, rps16, and trnS-trnfM). Our primary finding is that the genus Phyllopodium is polyphyletic as currently circumscribed, with some species placed within Zaluzianskya and others grouping with Polycarena, indicating the need for further phylogenetic work on these genera. Preliminary support for the incorporation of Reyemia into Zaluzianskya is reinforced here by the first molecular analysis to include both species of Reyemia and a strong sampling of species across Zaluzianskya and major clades of tribe Limoselleae. The two disjunct, tropical African species of Zaluzianskya are also confirmed as members of this genus. Finally, a broad sampling of 21 populations of Z. microsiphon establishes their phylogenetic division into two to five separate lineages. Hybridization, coevolution, and cryptic speciation may each play a role in the evolution of Z. microsiphon. Further resolution within a clade comprising sections Nycterinia and Macrocalyx is needed to better understand their relationships.

Evaluation of safety climate and employee injury rates in healthcare.

OBJECTIVES: Safety climates that support safety-related behaviour are associated with fewer work-related injuries, and prior research in industry suggests that safety knowledge and motivation are strongly related to safety performance behaviours; this relationship is not well studied in healthcare settings. METHODS: We performed analyses of survey results from a Veterans Health Administration (VHA) Safety Barometer employee perception survey, conducted among VHA employees in 2012. The employee perception survey assessed 6 safety programme categories, including management participation, supervisor participation, employee participation, safety support activities, safety support climate and organisational climate. We examined the relationship between safety climate from the survey results on VHA employee injury and illness rates. RESULTS: Among VHA facilities in the VA New England Healthcare System, work-related injury rate was significantly and inversely related to overall employee perception of safety climate, and all 6 safety programme categories, including employee perception of employee participation, management participation, organisational climate, supervisor participation, safety support activities and safety support climate. CONCLUSIONS: Positive employee perceptions of safety climate in VHA facilities are associated with lower work-related injury and illness rates. Employee perception of employee participation, management participation, organisational climate, supervisor participation, safety support activities and safety support climate were all associated with lower work-related injury rates. Future implications include fostering a robust safety climate for patients and healthcare workers to reduce healthcare worker injuries.

Adaptor protein-2 sigma subunit mutations causing familial hypocalciuric hypercalcaemia type 3 (FHH3) demonstrate genotype-phenotype correlations, codon bias and dominant-negative effects.

The adaptor protein-2 sigma subunit (AP2σ2) is pivotal for clathrin-mediated endocytosis of plasma membrane constituents such as the calcium-sensing receptor (CaSR). Mutations of the AP2σ2 Arg15 residue result in familial hypocalciuric hypercalcaemia type 3 (FHH3), a disorder of extracellular calcium (Ca(2+) o) homeostasis. To elucidate the role of AP2σ2 in Ca(2+) o regulation, we investigated 65 FHH probands, without other FHH-associated mutations, for AP2σ2 mutations, characterized their functional consequences and investigated the genetic mechanisms leading to FHH3. AP2σ2 mutations were identified in 17 probands, comprising 5 Arg15Cys, 4 Arg15His and 8 Arg15Leu mutations. A genotype-phenotype correlation was observed with the Arg15Leu mutation leading to marked hypercalcaemia. FHH3 probands harboured additional phenotypes such as cognitive dysfunction. All three FHH3-causing AP2σ2 mutations impaired CaSR signal transduction in a dominant-negative manner. Mutational bias was observed at the AP2σ2 Arg15 residue as other predicted missense substitutions (Arg15Gly, Arg15Pro and Arg15Ser), which also caused CaSR loss-of-function, were not detected in FHH probands, and these mutations were found to reduce the numbers of CaSR-expressing cells. FHH3 probands had significantly greater serum calcium (sCa) and magnesium (sMg) concentrations with reduced urinary calcium to creatinine clearance ratios (CCCR) in comparison with FHH1 probands with CaSR mutations, and a calculated index of sCa × sMg/100 × CCCR, which was ≥ 5.0, had a diagnostic sensitivity and specificity of 83 and 86%, respectively, for FHH3. Thus, our studies demonstrate AP2σ2 mutations to result in a more severe FHH phenotype with genotype-phenotype correlations, and a dominant-negative mechanism of action with mutational bias at the Arg15 residue.

Genetic heterogeneity in Cornelia de Lange syndrome (CdLS) and CdLS-like phenotypes with observed and predicted levels of mosaicism.

BACKGROUND: Cornelia de Lange syndrome (CdLS) is a multisystem disorder with distinctive facial appearance, intellectual disability and growth failure as prominent features. Most individuals with typical CdLS have de novo heterozygous loss-of-function mutations in NIPBL with mosaic individuals representing a significant proportion. Mutations in other cohesin components, SMC1A, SMC3, HDAC8 and RAD21 cause less typical CdLS. METHODS: We screened 163 affected individuals for coding region mutations in the known genes, 90 for genomic rearrangements, 19 for deep intronic variants in NIPBL and 5 had whole-exome sequencing. RESULTS: Pathogenic mutations [including mosaic changes] were identified in: NIPBL 46 [3] (28.2%); SMC1A 5 [1] (3.1%); SMC3 5 [1] (3.1%); HDAC8 6 [0] (3.6%) and RAD21 1 [0] (0.6%). One individual had a de novo 1.3 Mb deletion of 1p36.3. Another had a 520 kb duplication of 12q13.13 encompassing ESPL1, encoding separase, an enzyme that cleaves the cohesin ring. Three de novo mutations were identified in ANKRD11 demonstrating a phenotypic overlap with KBG syndrome. To estimate the number of undetected mosaic cases we used recursive partitioning to identify discriminating features in the NIPBL-positive subgroup. Filtering of the mutation-negative group on these features classified at least 18% as 'NIPBL-like'. A computer composition of the average face of this NIPBL-like subgroup was also more typical in appearance than that of all others in the mutation-negative group supporting the existence of undetected mosaic cases. CONCLUSIONS: Future diagnostic testing in 'mutation-negative' CdLS thus merits deeper sequencing of multiple DNA samples derived from different tissues.

Improving communication among attending physicians, long-term care facilities, residents, and residents' families.

Effective bidirectional communication between attending physicians and long-term care facilities is of critical importance to ensure timely, appropriate, and high-quality care that is responsive to residents' needs, values, and preferences. Ongoing communication with residents and residents' families is essential to the establishment of mutual trust and respect. This earned trust and respect in turn promotes frank discussions among long-term care practitioners and the facility staff who can then better guide residents and families through difficult care decisions.

Information exchange.

The Royal College of Nursing has established a group to look at standards, nutrition and the older adult. The aim of the group is to devise national guidelines by a process of analysis and consensus in which standards set locally are compared with those written by the expert group.