Risk factors and clinical impact of thrombosis during induction chemotherapy for pediatric acute lymphoblastic leukemia: A report from CYP-C.

Thromboembolism (TE) is associated with reduced survival in pediatric acute lymphoblastic leukemia (ALL). It has been hypothesized that TE might signal leukemic aggressiveness. The objective was to determine risk factors for TE during ALL induction (TEind ) therapy and whether TEind is associated with treatment refractoriness. This retrospective cohort study using the population-based Cancer in Young People Canada (CYP-C) registry included children <15 years of age diagnosed with ALL (2000-2019) and treated at one of 12 Canadian pediatric centers outside of Ontario. Univariate and multivariable logistic regression models were used to determine risk factors for TEind and whether TEind predicted induction failure and ALL treatment intensification. The impact of TEind on overall and event-free survival was estimated using Cox proportional hazard regression models. The study included 2589 children, of which 45 (1.7%) developed a TEind . Age (<1 year and ≥10 years vs. 1-<10 years), T-cell phenotype, high-risk ALL, and central nervous system involvement were all associated with TEind in univariate analysis. Age and T-cell phenotype remained independent predictors of TEind in multivariable analysis. Induction failure occurred in 53 patients (2.1%). TEind was not associated with induction failure (OR: not estimable) or treatment intensification (adjusted OR [95% CI]: 0.66 [0.26-1.69]). TEind was independently associated with overall survival (adjusted HR [95% CI]: 2.54 [1.20-5.03]) but not event-free survival (adjusted HR [95% CI] 1.86 [0.98-3.51]). In this population-based study of children treated with contemporary chemotherapy protocols, TEind was associated with age and T-cell phenotype and mortality but did not predict induction failure.

Case Report: Propranolol Therapy for Infantile Tremor Syndrome in a Child With Vitamin B12 Deficiency.

Vitamin B12 deficiency in childhood presents with a wide variety of symptoms including anemia, failure to thrive and developmental delays. It is a diagnostic consideration in children who are exclusively breastfed or have minimal solid intake, especially if their mother is vegetarian or has underlying vitamin B12 deficiency. Infantile tremor syndrome (ITS) has been associated with vitamin B12 deficiency. ITS presents with neurological symptoms such as developmental delays and tremors. The tremors seen in ITS can be profound and interfere with daily functioning. Different therapies have been tried for those tremors without much evidence or information regarding their efficacy and dosing regimens. We present the case of a 13-month-old girl with vitamin B12 deficiency who developed ITS with significant tremors after initiation of vitamin B12 therapy. She was treated with propranolol which resulted in significant improvement in her tremors. This case highlights the efficacy and safety of propranolol for the treatment of ITS in the context of vitamin B12 deficiency.

Incidence of childhood cancer in Canada during the COVID-19 pandemic.

BACKGROUND: The COVID-19 pandemic has had a major impact on access to health care resources. Our objective was to estimate the impact of the COVID-19 pandemic on the incidence of childhood cancer in Canada. We also aimed to compare the proportion of patients who enrolled in clinical trials at diagnosis, presented with metastatic disease or had an early death during the first 9 months of the COVID-19 pandemic compared with previous years. METHODS: We conducted an observational study that included children younger than 15 years with a new diagnosis of cancer between March 2016 and November 2020 at 1 of 17 Canadian pediatric oncology centres. Our primary outcome was the monthly age-standardized incidence rates (ASIRs) of cancers. We evaluated level and trend changes using interventional autoregressive integrated moving average models. Secondary outcomes were the proportion of patients who were enrolled in a clinical trial, who had metastatic or advanced disease and who died within 30 days. We compared the baseline and pandemic periods using rate ratios (RRs) and 95% confidence intervals (CIs). RESULTS: Age-standardized incidence rates during COVID-19 quarters were 157.7, 164.6, and 148.0 per million, respectively, whereas quarterly baseline ASIRs ranged between 150.3 and 175.1 per million (incidence RR 0.93 [95% CI 0.78 to 1.12] to incidence RR 1.04 [95% CI 0.87 to 1.24]). We found no statistically significant level or slope changes between the projected and observed ASIRs for all new cancers (parameter estimate [ß], level 4.98, 95% CI -15.1 to 25.04, p = 0.25), or when stratified by cancer type or by geographic area. Clinical trial enrolment rate was stable or increased during the pandemic compared with baseline (RR 1.22 [95% CI 0.70 to 2.13] to RR 1.71 [95% CI 1.01 to 2.89]). There was no difference in the proportion of patients with metastatic disease (RR 0.84 [95% CI 0.55 to 1.29] to RR 1.22 [0.84 to 1.79]), or who died within 30 days (RR 0.16 [95% CI 0.01 to 3.04] to RR 1.73 [95% CI 0.38 to 15.2]). INTERPRETATION: We did not observe a statistically significant change in the incidence of childhood cancer, or in the proportion of children enrolling in a clinical trial, presenting with metastatic disease or who died early during the first 9 months of the COVID-19 pandemic, which suggests that access to health care in pediatric oncology was not reduced substantially in Canada.

Enrollment on clinical trials does not improve survival for children with acute myeloid leukemia: A population-based study.

BACKGROUND: It is questionable whether enrollment on clinical trials offers any survival advantage at the population level over standard-of-care treatment. The objectives of this study were to describe the impact of trial enrollment on event-free survival and overall survival in pediatric acute myeloid leukemia (AML) using the Cancer in Young People in Canada (CYP-C) database. METHODS: Children were included if they had had AML newly diagnosed between ages birth and 14 years from 2001 to 2012. CYP-C is a national pediatric cancer population-based database that includes all cases of pediatric cancer diagnosed and treated at 1 of the 17 tertiary pediatric oncology centers in Canada. Univariate and Cox proportional hazards models were used to evaluate the impact of initial trial enrollment on survival. RESULTS: In total, 397 eligible children with AML were included in the analysis, of whom 94 (23.7%) were enrolled on a clinical trial at initial diagnosis. The most common reason for non-enrollment was that no trial was available. The event-free survival rate at 5 years was 57.8% ± 5.2% for those enrolled versus 54.8% ± 2.9% for those not enrolled (P = .75). The overall survival rate at 5 years was 70.1% ± 4.9% for those enrolled versus 66.3% ± 2.8% for those not enrolled (P = .58). Enrollment on a trial was not associated with improved event-free or overall survival in multiple regression analyses. CONCLUSIONS: Enrollment on a clinical trial was not associated with improved survival for children with AML in a population-based cohort. Rationale for trial enrollment should not include the likelihood of benefit compared with non-enrollment.

Thromboembolism Incidence and Risk Factors in Children with Cancer: A Population-Based Cohort Study.

There is conflicting information about the epidemiology of thromboembolism (TE) in paediatric oncology. Objectives were to describe the incidence and risk factors of TE in children with cancer. We included all children with cancer less than 15 years of age diagnosed from 2001 to 2016, treated at one of the 12 Canadian paediatric centres outside of Ontario and entered into the Cancer in Young People-Canada database. Potential risk factors for TE were evaluated using Cox proportional hazards regression stratified by haematological malignancies versus solid tumours. Factors associated with vascular access- and non-vascular access-related TE were compared using chi-square or Fisher's exact tests. Of the 7,471 children included, 283 experienced TE requiring medical intervention; cumulative incidence of TE at 5 years was 3.8 ± 0.2% and 0.36% ± 0.07% for life-threatening or fatal TE. For haematological malignancies, the following factors were associated with TE in multivariable regression: age < 1 year, 5 to 9.99 years and 10 to 14.99 years (relative to age 1-4.99 years), haematopoietic stem cell transplant (hazard ratio [HR] = 1.49, 95% confidence interval [CI], 1.00-2.32), anthracyclines (HR = 2.21, 95% CI, 1.12-4.37) and asparaginase (HR = 1.68, 95% CI, 1.15-2.44). For solid tumours, obesity (HR = 1.92, 95% CI, 1.01-3.68), surgery (HR = 2.70, 95% CI, 1.44-5.08), radiation (HR = 47.51, 95% CI, 24.01-94.01), anthracyclines (HR = 2.74, 95% CI, 1.29-5.82) and platinum agents (HR = 2.26, 95% CI, 1.19-4.28) were associated with TE. Life-threatening and fatal TEs were more common among non-vascular access TEs (14.5% vs. 3.3% p = 0.001). In a population-based cohort, 4% of children with cancer developed a clinically significant TE. Accurate risk stratification tools are needed specific to malignancy type.

Enrolling children with acute lymphoblastic leukaemia on a clinical trial improves event-free survival: a population-based study.

BACKGROUND: The objectives of this study were to describe the impact of trial enrollment at diagnosis on event-free and overall survival in paediatric acute lymphoblastic leukaemic (ALL) using a population-based approach. METHODS: We conducted a retrospective cohort study that included children newly diagnosed with ALL between 1 and 14 years of age. The data source was the Cancer in Young People in Canada (CYP-C) national paediatric cancer population-based database. We conducted univariate and multiple Cox proportional hazards models. RESULTS: There were 2569 children with ALL; 1408 (54.8%) were enrolled on a clinical trial at initial diagnosis. Event-free survival at 5 years was 89.8%±0.9 vs 84.1%±1.2. (P<0.0001) for those enrolled and not enrolled on a clinical trial, respectively. Overall survival at 5 years was higher for those enrolled (94.1%±0.7) vs not enrolled (90.5%±1.0; P=0.001). In a model that adjusted for demographic, leukaemic and socioeconomic factors, enrollment on trials was significantly associated with better event-free survival (hazard ratio (HR) 0.67, 95% confidence interval (CI) 0.47-0.95; P=0.023), but not overall survival (HR 0.69, 95% CI 0.44-1.08; P=0.102). CONCLUSIONS: Event-free survival was significantly better in children with ALL enrolled on a clinical trial. Future research should identify barriers to clinical trial enrollment for children with ALL.

Most children with cancer are not enrolled on a clinical trial in Canada: a population-based study.

BACKGROUND: Primary objective was to describe the proportion of children newly diagnosed with cancer enrolled on a therapeutic clinical trial. Secondary objectives were to describe reasons for non-enrollment and factors associated with enrollment on trials. METHODS: In this retrospective cohort study, we included children newly diagnosed with cancer between 0 and 14 years of age and diagnosed from 2001 to 2012. We used data from the Cancer in Young People in Canada (CYP-C) national pediatric cancer population-based database. CYP-C captures all cases of pediatric cancer (0-14 years) diagnosed and treated at one of the 17 tertiary pediatric oncology centers in Canada. Non-enrollment was evaluated using univariate and multiple logistic regression analysis. RESULTS: There were 9204 children with cancer included, of whom 2533 (27.5%) were enrolled on a clinical trial. The most common reasons cited for non-enrollment were lack of an available trial (52.2%) and physician choice (11.2%). In multiple regression, Asian and Arab/west Asian race were associated with lower enrollment (P = 0.006 and P = 0.032 respectively). All cancer diagnoses were more likely to be enrolled compared to astrocytoma and children with acute lymphoblastic leukemia had an almost 18-fold increased odds of enrollment compared to astrocytoma (P < 0.0001). Greater distance from the tertiary care center was independently associated with non-enrollment (P < 0.0001). CONCLUSIONS: In Canada, 27.5% of children with cancer are enrolled onto therapeutic clinical trials and lack of an available trial is the most common reason contributing to non-enrollment. Future research should better understand reasons for lack of trial availability and physician preferences to not offer trials.

Evaluation of the need for chest X-rays in the management of asymptomatic, intraluminal vascular access device occlusion in childhood cancer.

BACKGROUND: Venous access device (VAD) occlusion from intraluminal thrombus is a common complication during childhood cancer treatment. Current practice at many institutions is to assess VAD position with a chest X-ray (CXR) prior to intraluminal administration of tissue plasminogen activator (tPA). We aimed to determine the utility of this practice. PROCEDURE: A retrospective chart review of children with newly diagnosed cancer with a VAD, treated at The Hospital for Sick Children between 2010 and 2011, was performed. Episodes of line occlusion were identified both by reviewing patient CXRs for indication and identifying tPA doses dispensed. These episodes were reviewed to determine whether CXR findings resulted in management other than tPA. Cases in which the X-ray resulted in a change in management were further reviewed to determine whether administration of tPA could have resulted in potential patient harm. RESULTS: A total of 330 patients with newly diagnosed cancer with VADs were identified. Eighty-five (25.8%) patients experienced 123 episodes of VAD occlusion. VAD occlusions occurred more frequently in patients with tunneled external central venous lines (16/39, 41.5%) and peripherally inserted central catheters (PICC) (27/73, 37.0%) versus PORT (42/216, 19.4%; P = 0.001). There were nine (8.1%) episodes of VAD occlusion evaluated with a CXR in which the findings led to a change in management other than administering tPA. In each case, multiple specialists independently concluded that administration of tPA would have been unlikely to cause patient harm. CONCLUSION: Routine CXRs prior to the administration of tPA for asymptomatic VAD occlusion can safely be omitted.

Cancer incidence, morbidity, and survival in Canadian first nation children: a Manitoba population-based study from the cancer in young people in Canada (CYP-C) registry.

BACKGROUND: Health disparities between Canadian First Nation (FN) people and the rest of the national population exist. No studies have specifically documented cancer-related health outcomes in Canadian FN children. The purpose of this study was to describe the incidence of pediatric malignancies in Manitoba FN children, and to compare morbidity patterns and survival between FN and non-FN children with cancer in the Canadian province of Manitoba. PROCEDURE: A retrospective, population-based review of all children (0-14.99 years) diagnosed with malignancy (2001-2008) in Manitoba, Canada was undertaken using the Cancer in Young People in Canada registry. FN children were compared to the non-FN population for markers of morbidity and survival. RESULTS: The average annual age-standardized incidence rate for all childhood cancers in FN children was 132 per 1,000,000 per year. 240 children were included in the morbidity and survival analyses (38 FN; 202 non-FN). No differences were found between FN and non-FN children in time from first presentation of symptoms to consultation with an oncology specialist or diagnosis, or number of hospital admissions / total days of admission for treatment complications. Overall survival was inferior for FN children in univariable analysis (P = 0.048) but not when risk group was included in a multivariable analysis (P = 0.15). No difference in event free survival or cumulative incidence of relapse was identified. CONCLUSION: The estimated incidence of childhood cancers in the Manitoba FN population is similar to provincial incidence rates. No differences in morbidity patterns or survival were found between Manitoba FN and non-FN children with cancer.

Structural and functional characterization of Salmonella enterica serovar Typhimurium YcbL: an unusual Type II glyoxalase.

YcbL has been annotated as either a metallo-ß-lactamase or glyoxalase II (GLX2), both members of the zinc metallohydrolase superfamily, that contains many enzymes with a diverse range of activities. Here, we report crystallographic and biochemical data for Salmonella enterica serovar Typhimurium YcbL that establishes it as GLX2, which differs in certain structural and functional properties compared with previously known examples. These features include the insertion of an α-helix after residue 87 in YcbL and truncation of the C-terminal domain, which leads to the loss of some recognition determinants for the glutathione substrate. Despite these changes, YcbL has robust GLX2 activity. A further difference is that the YcbL structure contains only a single bound metal ion rather than the dual site normally observed for GLX2s. Activity assays in the presence of various metal ions indicate an increase in activity above basal levels in the presence of manganous and ferrous ions. Thus, YcbL represents a novel member of the GLX2 family.

Novel structural features in two ZHX homeodomains derived from a systematic study of single and multiple domains.

BACKGROUND: Zhx1 to 3 (zinc-fingers and homeoboxes) form a set of paralogous genes encoding multi-domain proteins. ZHX proteins consist of two zinc fingers followed by five homeodomains. ZHXs have biological roles in cell cycle control by acting as co-repressors of the transcriptional regulator Nuclear Factor Y. As part of a structural genomics project we have expressed single and multi-domain fragments of the different human ZHX genes for use in structure determination. RESULTS: A total of 30 single and multiple domain ZHX1-3 constructs selected from bioinformatics protocols were screened for soluble expression in E. coli using high throughput methodologies. Two homeodomains were crystallized leading to structures for ZHX1 HD4 and ZHX2 HD2. ZHX1 HD4, although closest matched to homeodomains from 'homez' and 'engrailed', showed structural differences, notably an additional C-terminal helix (helix V) which wrapped over helix I thereby making extensive contacts. Although ZHX2 HD2-3 was successfully expressed and purified, proteolysis occurred during crystallization yielding crystals of just HD2. The structure of ZHX2 HD2 showed an unusual open conformation with helix I undergoing 'domain-swapping' to form a homodimer. CONCLUSIONS: Although multiple-domain constructs of ZHX1 selected by bioinformatics studies could be expressed solubly, only single homeodomains yielded crystals. The crystal structure of ZHX1 HD4 showed additional hydrophobic interactions relative to many known homeodomains via extensive contacts formed by the novel C-terminal helix V with, in particular, helix I. Additionally, the replacement of some charged covariant residues (which are commonly observed to form salt bridges in non-homeotherms such as the Drosophila 'engrailed' homeodomain), by apolar residues further increases hydrophobic contacts within ZHX1 HD4, and potentially stability, relative to engrailed homeodomain. ZHX1 HD4 helix V points away from the normally observed DNA major groove binding site on homeodomains and thus would not obstruct the putative binding of nucleic acid. In contrast, for ZHX2 HD2 the observed altered conformation involving rearrangement of helix I, relative to the canonical homeodomain fold, disrupts the normal DNA binding site, although protein-protein binding is possible as observed in homodimer formation.

The tandem zinc-finger region of human ZHX adopts a novel C2H2 zinc finger structure with a C-terminal extension.

Binding of the nuclear factor-Y complex (NF-Y) to the inverted CCAAT-box interferes with transcription activation through nucleosome reorganization. The three homologous proteins forming the zinc-fingers and homeoboxes (ZHX) family interact with the activation domain of NF-Ya to repress transcription. Each ZHX-protein contains two generic C2H2 zinc-fingers (ZNF1 and ZNF2) followed by five homeodomains. Although the proteins have been related to the occurrence of certain cancers, the function and structure of the individual ZHX domains are still unknown. Here, we determined the structure of the tandem zinc-finger region of human ZHX1. Folding and secondary structure predictions combined with expression screening revealed that the C-terminal extension (E) to ZNF2 could form a single domain with the two hZHX1 zinc-fingers. We therefore decided to determine the solution structure of the zinc-fingers followed by this extension. We show that both zinc-fingers adopt canonical betabetaalpha-folds in which a zinc ion is coordinated by two cysteine and two histidine residues. The C-terminal extension to ZNF2 forms two beta-strands to make a beta-sheet with the beta-strands of this zinc-finger. The ZNF1 and ZNF2-E domains do not show evident contacts and their mutual orientation seems variable. The high degree of sequence conservation among ZHX family members permitted us to prepare homology models for ZHX2 and ZHX3, revealing distinct surface characteristics for each family member. Implications of these structural features for ZHX-functioning in transcription regulation are discussed.

The structure of NMB1585, a MarR-family regulator from Neisseria meningitidis.

The structure of the MarR-family transcription factor NMB1585 from Neisseria meningitidis has been solved using data extending to a resolution of 2.1 A. Overall, the dimeric structure resembles those of other MarR proteins, with each subunit comprising a winged helix-turn-helix (wHtH) domain connected to an alpha-helical dimerization domain. The spacing of the recognition helices of the wHtH domain indicates that NMB1585 is pre-configured for DNA binding, with a putative inducer pocket that is largely occluded by the side chains of two aromatic residues (Tyr29 and Trp53). NMB1585 was shown to bind to its own promoter region in a gel-shift assay, indicating that the protein acts as an auto-repressor.

Dinucleotide-sensing proteins: linking signaling networks and regulating transcription.

Differential binding of dinucleotides to key regulatory proteins can modulate their interactions with other proteins and, in some cases, can signal fluctuations in the cellular redox state, to produce changes in transcription and physiological state. The dinucleotide-binding proteins human HSCARG and yeast transcription repressor Gal80p are examples that offer exciting glimpses into the potential for dinucleotide-sensing proteins to couple fluctuations in dinucleotide ratios to changes in transcription and to act as networking agents linking different classes of signaling molecules.

Structural analysis of the recognition of the negative regulator NmrA and DNA by the zinc finger from the GATA-type transcription factor AreA.

Amongst the most common protein motifs in eukaryotes are zinc fingers (ZFs), which, although largely known as DNA binding modules, also can have additional important regulatory roles in forming protein:protein interactions. AreA is a transcriptional activator central to nitrogen metabolism in Aspergillus nidulans. AreA contains a GATA-type ZF that has a competing dual recognition function, binding either DNA or the negative regulator NmrA. We report the crystal structures of three AreA ZF-NmrA complexes including two with bound NAD(+) or NADP(+). The molecular recognition of AreA ZF-NmrA involves binding of the ZF to NmrA via hydrophobic and hydrogen bonding interactions through helices alpha1, alpha6 and alpha11. Comparison with an earlier NMR solution structure of AreA ZF-DNA complex by overlap of the AreA ZFs shows that parts of helices alpha6 and alpha11 of NmrA are positioned close to the GATA motif of the DNA, mimicking the major groove of DNA. The extensive overlap of DNA with NmrA explains their mutually exclusive binding to the AreA ZF. The presence of bound NAD(+)/NADP(+) in the NmrA-AreaA ZF complex, however, causes minimal structural changes. Thus, any regulatory effects on AreA function mediated by the binding of oxidised nicotinamide dinucleotides to NmrA in the NmrA-AreA ZF complex appear not to be modulated via protein conformational rearrangements.

Structural basis for the improved drug resistance profile of new generation benzophenone non-nucleoside HIV-1 reverse transcriptase inhibitors.

Owing to the emergence of resistant virus, next generation non-nucleoside HIV reverse transcriptase inhibitors (NNRTIs) with improved drug resistance profiles have been developed to treat HIV infection. Crystal structures of HIV-1 RT complexed with benzophenones optimized for inhibition of HIV mutants that were resistant to the prototype benzophenone GF128590 indicate factors contributing to the resilience of later compounds in the series (GW4511, GW678248). Meta-substituents on the benzophenone A-ring had the designed effect of inducing better contacts with the conserved W229 while reducing aromatic stacking interactions with the highly mutable Y181 side chain, which unexpectedly adopted a "down" position. Up to four main-chain hydrogen bonds to the inhibitor also appear significant in contributing to resilience. Structures of mutant RTs (K103N, V106A/Y181C) with benzophenones showed only small rearrangements of the NNRTIs relative to wild-type. Hence, adaptation to a mutated NNRTI pocket by inhibitor rearrangement appears less significant for benzophenones than other next-generation NNRTIs.

Structural basis for drug resistance mechanisms for non-nucleoside inhibitors of HIV reverse transcriptase.

The selection of drug resistant virus is a significant obstacle to the continued successful treatment of HIV infection. Reverse transcriptase is the target for numerous approved anti-HIV drugs including both nucleoside inhibitor (NRTI) and non-nucleosides (NNRTI). The many available crystal structures of RT reveal that, generally, in relation to their binding sites NRTI resistance mutations are generally more distally positioned, whilst for NNRTIs mutations are clustered. Such clustering implies a direct stereochemical basis for NNRTI resistance mechanisms, which is indeed observed in many cases such as the loss of key ring stacking interactions with inhibitors via mutations at Tyr181 and Tyr188. However, there are also indirect resistance mechanisms observed, e.g. V108I (via perturbation of Tyr188 and Tyr181) and K103N (apo-enzyme stabilisation). The resistance mechanism can be NNRTI-dependent as is the case for K101E where either indirect (nevirapine) or direct effects (efavirenz) apply. Structural studies have contributed to the design of newer generation NNRTIs and identified a number of features which may contribute to their much improved resistance profiles. Such factors include reduced interactions with Tyr181, the presence of inhibitor/main-chain H-bonds and ability to undergo conformational flexing and rearrangement within the mutated drug site.