CD371-positive pediatric B-cell acute lymphoblastic leukemia: propensity to lineage switch and slow early response to treatment.

ABSTRACT: In the effort to improve immunophenotyping and minimal residual disease (MRD) assessment in acute lymphoblastic leukemia (ALL), the international Berlin-Frankfurt-Münster (iBFM) Flow Network introduced the myelomonocytic marker CD371 for a large prospective characterization with a long follow-up. In the present study, we aimed to investigate the clinical and biological features of CD371-positive (CD371pos) pediatric B-cell precursor ALL (BCP-ALL). From June 2014 to February 2017, 1812 pediatric patients with newly diagnosed BCP-ALLs enrolled in trial AIEOP-BFM ALL 2009 were evaluated as part of either a screening (n = 843, Italian centers) or validation cohort (n = 969, other iBFM centers). Laboratory assessment at diagnosis consisted of morphological, immunophenotypic, and genetic analysis. Response assessment relied on morphology, multiparametric flow cytometry (MFC), and polymerase chain reaction (PCR)-MRD. At diagnosis, 160 of 1812 (8.8%) BCP-ALLs were CD371pos. This correlated with older age, lower ETV6::RUNX1 frequency, immunophenotypic immaturity (all P < .001), and strong expression of CD34 and of CD45 (P < .05). During induction therapy, CD371pos BCP-ALLs showed a transient myelomonocytic switch (mm-SW: up to 65.4% of samples at day 15) and an inferior response to chemotherapy (slow early response, P < .001). However, the 5-year event-free survival was 88.3%. Among 420 patients from the validation cohort, 27 of 28 (96.4%) cases positive for DUX4-fusions were CD371pos. In conclusion, in the largest pediatric cohort, CD371 is the most sensitive marker of transient mm-SW, whose recognition is essential for proper MFC MRD assessment. CD371pos is associated to poor early treatment response, although a good outcome can be reached after MRD-based ALL-related therapies.

Asparagine transport through SLC1A5/ASCT2 and SLC38A5/SNAT5 is essential for BCP-ALL cell survival and a potential therapeutic target.

B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) blasts strictly depend on the transport of extra-cellular asparagine (Asn), yielding a rationale for L-asparaginase (ASNase) therapy. However, the carriers used by ALL blasts for Asn transport have not been identified yet. Exploiting RS4;11 cells as BCP-ALL model, we have found that cell Asn is lowered by either silencing or inhibition of the transporters ASCT2 or SNAT5. The inhibitors V-9302 (for ASCT2) and GluγHA (for SNAT5) markedly lower cell proliferation and, when used together, suppress mTOR activity, induce autophagy and cause a severe nutritional stress, leading to a proliferative arrest and a massive cell death in both the ASNase-sensitive RS4;11 cells and the relatively ASNase-insensitive NALM-6 cells. The cytotoxic effect is not prevented by coculturing leukaemic cells with primary mesenchymal stromal cells. Leukaemic blasts of paediatric ALL patients express ASCT2 and SNAT5 at diagnosis and undergo marked cytotoxicity when exposed to the inhibitors. ASCT2 expression is positively correlated with the minimal residual disease at the end of the induction therapy. In conclusion, ASCT2 and SNAT5 are the carriers exploited by ALL cells to transport Asn, and ASCT2 expression is associated with a lower therapeutic response. ASCT2 may thus represent a novel therapeutic target in BCP-ALL.

Phenotypic profiling of CD34+ cells by advanced flow cytometry improves diagnosis of juvenile myelomonocytic leukemia.

Diagnostic criteria for juvenile myelomonocytic leukemia (JMML) are currently well defined, however in some patients diagnosis still remains a challenge. Flow cytometry is a well established tool for diagnosis and follow-up of hematological malignancies, nevertheless it is not routinely used for JMML diagnosis. Herewith, we characterized the CD34+ hematopoietic precursor cells collected from 31 children with JMML using a combination of standardized EuroFlow antibody panels to assess the ability to discriminate JMML cells from normal/reactive bone marrow cell as controls (n=29) or from cells of children with other hematological diseases mimicking JMML (n=9). CD34+ precursors in JMML showed markedly reduced B-cell and erythroid-committed precursors compared to controls, whereas monocytic and CD7+ lymphoid precursors were significantly expanded. Moreover, aberrant immunophenotypes were consistently present in CD34+ precursors in JMML, while they were virtually absent in controls. Multivariate logistic regression analysis showed that combined assessment of the number of CD34+CD7+ lymphoid precursors and CD34+ aberrant precursors or erythroid precursors had a great potential in discriminating JMMLs versus controls. Importantly our scoring model allowed highly efficient discrimination of truly JMML versus patients with JMML-like diseases. In conclusion, we show for the first time that CD34+ precursors from JMML patients display a unique immunophenotypic profile which might contribute to a fast and accurate diagnosis of JMML worldwide by applying an easy to standardize single eight-color antibody combination.

Improving predictive accuracy in primary biliary cholangitis: A new genetic risk score.

BACKGROUND AND AIMS: Genetic variants influence primary biliary cholangitis (PBC) risk. We established and tested an accurate polygenic risk score (PRS) using these variants. METHODS: Data from two Italian cohorts (OldIT 444 cases, 901 controls; NewIT 255 cases, 579 controls) were analysed. The latest international genome-wide meta-analysis provided effect size estimates. The PRS, together with human leukocyte antigen (HLA) status and sex, was included in an integrated risk model. RESULTS: Starting from 46 non-HLA genes, 22 variants were selected. PBC patients in the OldIT cohort showed a higher risk score than controls: -.014 (interquartile range, IQR, -.023, .005) versus -.022 (IQR -.030, -.013) (p < 2.2 × 10-16). For genetic-based prediction, the area under the curve (AUC) was .72; adding sex increased the AUC to .82. Validation in the NewIT cohort confirmed the model's accuracy (.71 without sex, .81 with sex). Individuals in the top group, representing the highest 25%, had a PBC risk approximately 14 times higher than that of the reference group (lowest 25%; p < 10-6). CONCLUSION: The combination of sex and a novel PRS accurately discriminated between PBC cases and controls. The model identified a subset of individuals at increased risk of PBC who might benefit from tailored monitoring.

Adverse events in single-arm clinical trials with non-fatal time-to-event efficacy endpoint: from clinical questions to methods for statistical analysis.

BACKGROUND: In any single-arm trial on novel treatments, assessment of toxicity plays an important role as occurrence of adverse events (AEs) is relevant for application in clinical practice. In the presence of a non-fatal time-to-event(s) efficacy endpoint, the analysis should be broadened to consider AEs occurrence in time. The AEs analysis could be tackled with two approaches, depending on the clinical question of interest. Approach 1 focuses on the occurrence of AE as first event. Treatment ability to protect from the efficacy endpoint event(s) has an impact on the chance of observing AEs due to competing risks action. Approach 2 considers how treatment affects the occurrence of AEs in the potential framework where the efficacy endpoint event(s) could not occur. METHODS: In the first part of the work we review the strategy of analysis for these two approaches. We identify theoretical quantities and estimators consistent with the following features: (a) estimators should address for the presence of right censoring; (b) theoretical quantities and estimators should be functions of time. In the second part of the work we propose the use of alternative methods (regression models, stratified Kaplan-Meier curves, inverse probability of censoring weighting) to relax the assumption of independence between the potential times to AE and to event(s) in the efficacy endpoint for addressing Approach 2. RESULTS: We show through simulations that the proposed methods overcome the bias due to the dependence between the two potential times and related to the use of standard estimators. CONCLUSIONS: We demonstrated through simulations that one can handle patients selection in the risk sets due to the competing event, and thus obtain conditional independence between the two potential times, adjusting for all the observed covariates that induce dependence.

How should childhood acute lymphoblastic leukemia relapses in low-income and middle-income countries be managed: The AHOPCA-ALL study group experience.

BACKGROUND: Children with relapsed acute lymphoblastic leukemia (ALL) in low-income and middle-income countries rarely survive. The Pediatric Hematology-Oncology Association of Central America (AHOPCA) developed the AHOPCA-ALL REC 2014 protocol to improve outcomes in resource-constrained settings without access to stem cell transplantation. METHODS: The AHOPCA-ALL REC 2014 protocol was based on a modified frontline induction phase 1A, a consolidation therapy with six modified R-blocks derived from the ALL-Berlin-Frankfurt-Munster REZ 2002 protocol and intermittent maintenance therapy. Children with B-lineage ALL were eligible after a late medullary relapse, an early or late combined relapse, or any extramedullary relapses. Those with T-lineage ALL were eligible after early and late extramedullary relapses, as were those with both B-lineage and T-lineage relapses occurring at least 3 months after therapy abandonment. RESULTS: The study population included 190 patients with T-lineage (n = 3) and B-lineage (n = 187) ALL. Of those with B-lineage ALL, 25 patients had a very early extramedullary relapse, 40 had an early relapse (32 extramedullary and 8 combined), and 125 had a late relapse (34 extramedullary, 19 combined, and 72 medullary). The main cause of treatment failure was second relapse (52.1%). The 3-year event-free survival rate (± standard error) was 25.9% ± 3.5%, and the 3-year overall survival rate was 36.7% ± 3.8%. The 3-year event-free survival rate was 47.2% ± 4.7% for late relapses. The most frequently reported toxicity was grade 3 or 4 infection. Mortality during treatment occurred in 17 patients (8.9%), in most cases because of infectious complications. CONCLUSIONS: Selected children with relapsed ALL in Central America can be cured with second-line regimens even without access to consolidation with stem cell transplantation. Children in low-income and middle-income countries who have lower risk relapses of ALL should be treated with curative intent.

Improving outcomes of in situ split liver transplantation in Italy over the last 25 years.

BACKGROUND & AIMS: Split liver transplant(ation) (SLT) is still considered a challenging procedure that is by no means widely accepted. We aimed to present data on 25-year trends in SLT in Italy, and to investigate if, and to what extent, outcomes have improved nationwide during this time. METHODS: The study included all consecutive SLTs performed from May 1993 to December 2019, divided into three consecutive periods: 1993-2005, 2006-2014, and 2015-2019, which match changes in national allocation policies. Primary outcomes were patient and graft survival, and the relative impact of each study period. RESULTS: SLT accounted for 8.9% of all liver transplants performed in Italy. A total of 1,715 in situ split liver grafts were included in the analysis: 868 left lateral segments (LLSs) and 847 extended right grafts (ERGs). A significant improvement in patient and graft survival (p <0.001) was observed with ERGs over the three periods. Predictors of graft survival were cold ischaemia time (CIT) <6 h (p = 0.009), UNOS status 2b (p <0.001), UNOS status 3 (p = 0.009), and transplant centre volumes: 25-50 cases vs. <25 cases (p = 0.003). Patient survival was significantly higher with LLS grafts in period 2 vs. period 1 (p = 0.008). No significant improvement in graft survival was seen over the three periods, where predictors of graft survival were CIT <6 h (p = 0.007), CIT <6 h vs. ≥10 h (p = 0.019), UNOS status 2b (p = 0.038), and UNOS status 3 (p = 0.009). Retransplantation was a risk factor in split liver graft recipients, with significantly worse graft and patient survival for both types of graft (p <0.001). CONCLUSIONS: Our analysis showed Italian SLT outcomes to have improved over the last 25 years. These results could help to dispel reservations regarding the use of this procedure. IMPACT AND IMPLICATIONS: Split liver transplant(ation) (SLT) is still considered a challenging procedure and is by no means widely accepted. This study included all consecutive in situ SLTs performed in Italy from May 1993 to December 2019. With more than 1,700 cases, it is one of the largest series, examining long-term national trends in in situ SLT since its introduction. The data presented indicate that the outcomes of SLT improved during this 25-year period. Improvements are probably due to better recipient selection, refinements in surgical technique, conservative graft-to-recipient matching, and the continuous, yet carefully managed, expansion of donor selection criteria under a strict mandatory split liver allocation policy. These results could help to dispel reservations regarding the use of this procedure.

Effect of two additional doses of intrathecal methotrexate during induction therapy on serious infectious toxicity in pediatric patients with acute lymphoblastic leukemia.

Although initial central nervous system (CNS) involvement is rarely detected in childhood acute lymphoblastic leukemia (ALL), risk-adapted CNS-directed therapy is essential for all patients. Treatment intensity depends on the initial CNS status. In the AIEOP-BFM ALL 2009 trial, patients with cytomorphologic detection of leukemic blasts in initial cerebrospinal fluid were classified as CNS2 or CNS3 and received five intrathecal doses of methotrexate (MTX) in induction therapy compared to patients with CNS1 status (no blasts detected) who received three doses. The impact of additional intrathecal (IT) MTX on systemic toxicity in induction therapy is unknown. Between June 1st 2010 and February 28th 2017, a total of 6,136 ALL patients aged 1-17 years were enrolled onto the AIEOP-BFM ALL 2009 trial. The effect of three versus five doses of IT MTX during induction therapy on the incidence of severe infectious complications was analyzed. Among 4,706 patients treated with three IT MTX doses, 77 (1.6%) had a life-threatening infection during induction as compared to 59 of 1,350 (4.4%) patients treated with five doses (P<0.001; Odds Ratio 2.86 [95% Confidence Interval 1.99-4.13]). In a multivariate regression model, treatment with additional IT MTX proved to be the strongest risk factor for life-threatening infections (Odds Ratio 2.85 [1.96-4.14]). Fatal infections occurred in 16 (0.3%) and 38 (1.6%) patients treated with three or five IT MTX doses, respectively (P<0.001). As the relevance of additional intrathecal MTX in induction for relapse prevention in CNS2 patients is unclear, doses of intrathecal therapy have been reduced for these patients. (Clinicaltrials.gov identifiers: NCT01117441 and NCT00613457).

Rhabdomyosarcoma in low- and middle-income countries: A report from the Asociacion de Hemato-oncología Pediatrica de Centro América (AHOPCA).

This report describes the results of an observational study dedicated to rhabdomyosarcoma developed by the Asociación de Hemato-oncología Pediatrica de Centro América (AHOPCA) between 2001 and 2018. Overall, 337 previously untreated patients < 18 years old were included in the analysis; 58% had unresected disease, and 19% were metastatic at diagnosis. With a median follow-up of 6.6 years, five-year event-free and overall survival rates were 30% and 33%, respectively. Local progression/relapse was the main cause of treatment failure.

Peri-intubation Cardiovascular Collapse in Patients Who Are Critically Ill: Insights from the INTUBE Study.

Rationale: Cardiovascular instability/collapse is a common peri-intubation event in patients who are critically ill. Objectives: To identify potentially modifiable variables associated with peri-intubation cardiovascular instability/collapse (i.e., systolic arterial pressure <65 mm Hg [once] or <90 mm Hg for >30 minutes; new/increased vasopressor requirement; fluid bolus >15 ml/kg, or cardiac arrest). Methods: INTUBE (International Observational Study to Understand the Impact and Best Practices of Airway Management In Critically Ill Patients) was a multicenter prospective cohort study of patients who were critically ill and undergoing tracheal intubation in a convenience sample of 197 sites from 29 countries across five continents from October 1, 2018, to July 31, 2019. Measurements and Main Results: A total of 2,760 patients were included in this analysis. Peri-intubation cardiovascular instability/collapse occurred in 1,199 out of 2,760 patients (43.4%). Variables associated with this event were older age (odds ratio [OR], 1.02; 95% confidence interval [CI], 1.02-1.03), higher heart rate (OR, 1.008; 95% CI, 1.004-1.012), lower systolic blood pressure (OR, 0.98; 95% CI, 0.98-0.99), lower oxygen saturation as measured by pulse oximetry/FiO2 before induction (OR, 0.998; 95% CI, 0.997-0.999), and the use of propofol as an induction agent (OR, 1.28; 95% CI, 1.05-1.57). Patients with peri-intubation cardiovascular instability/collapse were at a higher risk of ICU mortality with an adjusted OR of 2.47 (95% CI, 1.72-3.55), P < 0.001. The inverse probability of treatment weighting method identified the use of propofol as the only factor independently associated with cardiovascular instability/collapse (OR, 1.23; 95% CI, 1.02-1.49). When administered before induction, vasopressors (OR, 1.33; 95% CI, 0.84-2.11) or fluid boluses (OR, 1.17; 95% CI, 0.96-1.44) did not reduce the incidence of cardiovascular instability/collapse. Conclusions: Peri-intubation cardiovascular instability/collapse was associated with an increased risk of both ICU and 28-day mortality. The use of propofol for induction was identified as a modifiable intervention significantly associated with cardiovascular instability/collapse.Clinical trial registered with clinicaltrials.gov (NCT03616054).

Osteonecrosis in paediatric acute lymphoblastic leukaemia: Incidence, risk factors, radiological patterns and evolution in a single-centre cohort.

Osteonecrosis (ON) is a well-known sequela of paediatric acute lymphoblastic leukaemia (ALL) treatment. Incidence differs substantially among studies and the clinical significance of radiological findings is not fully established. We analysed 256 consecutive patients with ALL treated in our Institution between October 2010 and December 2016. Within the cohort, 41 developed ON, with a mean 5-year cumulative incidence of 18.5 (standard error, SE, 5.7)% overall. The mean (SE) 5-year cumulative incidence of ON was 12.7 (2.1)% after censoring upon stem cell transplantation (SCT) and/or relapse. Patients aged ≥10 years and patients allocated to the high-risk stratum had a 10-fold and fivefold higher risk of ON respectively. The risk of ON was more than double in relapsed patients, whereas no significant impact of gender, immunophenotype and SCT was demonstrated. Multiple lesions (median four joints involved per patient) were detected by magnetic resonance imaging in all but one patient, with the knee being the most affected joint. Lesions affecting convex joint surfaces experienced the worst evolution, whereas most lesions on diaphyses and concave surfaces remained radiologically stable or disappeared during follow-up. ON has a high prevalence in paediatric ALL, presenting with multiple lesions. Lesions involving convex surfaces were at higher risk of radiological deterioration.

One-year pulmonary impairment after severe COVID-19: a prospective, multicenter follow-up study.

BACKGROUND: Long-term pulmonary sequelae following hospitalization for SARS-CoV-2 pneumonia is largely unclear. The aim of this study was to identify and characterise pulmonary sequelae caused by SARS-CoV-2 pneumonia at 12-month from discharge. METHODS: In this multicentre, prospective, observational study, patients hospitalised for SARS-CoV-2 pneumonia and without prior diagnosis of structural lung diseases were stratified by maximum ventilatory support ("oxygen only", "continuous positive airway pressure (CPAP)" and "invasive mechanical ventilation (IMV)") and followed up at 12 months from discharge. Pulmonary function tests and diffusion capacity for carbon monoxide (DLCO), 6 min walking test, high resolution CT (HRCT) scan, and modified Medical Research Council (mMRC) dyspnea scale were collected. RESULTS: Out of 287 patients hospitalized with SARS-CoV-2 pneumonia and followed up at 1 year, DLCO impairment, mainly of mild entity and improved with respect to the 6-month follow-up, was observed more frequently in the "oxygen only" and "IMV" group (53% and 49% of patients, respectively), compared to 29% in the "CPAP" group. Abnormalities at chest HRCT were found in 46%, 65% and 80% of cases in the "oxygen only", "CPAP" and "IMV" group, respectively. Non-fibrotic interstitial lung abnormalities, in particular reticulations and ground-glass attenuation, were the main finding, while honeycombing was found only in 1% of cases. Older patients and those requiring IMV were at higher risk of developing radiological pulmonary sequelae. Dyspnea evaluated through mMRC scale was reported by 35% of patients with no differences between groups, compared to 29% at 6-month follow-up. CONCLUSION: DLCO alteration and non-fibrotic interstitial lung abnormalities are common after 1 year from hospitalization due to SARS-CoV-2 pneumonia, particularly in older patients requiring higher ventilatory support. Studies with longer follow-ups are needed.

Machine learning in primary biliary cholangitis: A novel approach for risk stratification.

BACKGROUND & AIMS: Machine learning (ML) provides new approaches for prognostication through the identification of novel subgroups of patients. We explored whether ML could support disease sub-phenotyping and risk stratification in primary biliary cholangitis (PBC). METHODS: ML was applied to an international dataset of PBC patients. The dataset was split into a derivation cohort (training set) and a validation cohort (validation set), and key clinical features were analysed. The outcome was a composite of liver-related death or liver transplantation. ML and standard survival analysis were performed. RESULTS: The training set was composed of 11,819 subjects, while the validation set was composed of 1,069 subjects. ML identified four clusters of patients characterized by different phenotypes and long-term prognosis. Cluster 1 (n = 3566) included patients with excellent prognosis, whereas Cluster 2 (n = 3966) consisted of individuals at worse prognosis differing from Cluster 1 only for albumin levels around the limit of normal. Cluster 3 (n = 2379) included young patients with florid cholestasis and Cluster 4 (n = 1908) comprised advanced cases. Further sub-analyses on the dynamics of albumin within the normal range revealed that ursodeoxycholic acid-induced increase of albumin >1.2 x lower limit of normal (LLN) is associated with improved transplant-free survival. CONCLUSIONS: Unsupervised ML identified four novel groups of PBC patients with different phenotypes and prognosis and highlighted subtle variations of albumin within the normal range. Therapy-induced increase of albumin >1.2 x LLN should be considered a treatment goal.

Posterior fossa syndrome in a population of children and young adults with medulloblastoma: a retrospective, multicenter Italian study on incidence and pathophysiology in a histologically homogeneous and consecutive series of 136 patients.

INTRODUCTION: Posterior fossa syndrome (PFS) is a set of debilitating complications that can occur after surgery for posterior fossa tumors. This study aimed to assess the preoperative radiological and surgical risk factors for the onset of PFS in a histologically homogeneous population of children with medulloblastoma and compare it to a similar population of young adults. METHODS: Included patients underwent posterior fossa surgery for medulloblastoma at 11 Italian neurosurgical wards (2003-2019) and were referred to Fondazione IRCCS Istituto Nazionale dei Tumori in Milan (INT) for postoperative treatments. We collected patients' pre- and post-operative clinical, surgical and radiological data from the INT charts. To compare the distribution of variables, we used the Mann-Whitney and Fisher tests for continuous and categorical variables, respectively. RESULTS: 136 patients (109 children and 27 young adults) were included in the study. Among children, 29 (27%) developed PFS, and all of them had tumors at midline site with invasion of the fourth ventricle. Radiological evidence of involvement of the right superior (39% versus 12%; p = 0.011) or middle cerebellar peduncles (52% versus 18%; p = 0.002) seemed more common in children who developed PFS. Young adults showed an expected lower incidence of PFS (4 out of 27; 15%), that may be due to anatomical, physiological and oncological elements. CONCLUSIONS: This study confirmed some factors known to be associated with PFS onset and shed light on other debated issues. Our findings enhance an already hypothesized role of cerebellar language lateralization. The analysis of a population of young adults may shed more light on the often-neglected existence of PFS in non-pediatric patients.

Development and validation of a predictive model to identify the active phase of labor.

BACKGROUND: The diagnosis of the active phase of labor is a crucial clinical decision, thus requiring an accurate assessment. This study aimed to build and to validate a predictive model, based on maternal signs and symptoms to identify a cervical dilatation ≥4 cm. METHODS: A prospective study was conducted from May to September 2018 in a II Level Maternity Unit (development data), and from May to September 2019 in a I Level Maternity Unit (validation data). Women with singleton, term pregnancy, cephalic presentation and presence of contractions were consecutively enrolled during the initial assessment to diagnose the stage of labor. Women < 18 years old, with language barrier or induction of labor were excluded. A nomogram for the calculation of the predictions of cervical dilatation ≥4 cm on the ground of 11 maternal signs and symptoms was obtained from a multivariate logistic model. The predictive performance of the model was investigated by internal and external validation. RESULTS: A total of 288 assessments were analyzed. All maternal signs and symptoms showed a significant impact on increasing the probability of cervical dilatation ≥4 cm. In the final logistic model, "Rhythm" (OR 6.26), "Duration" (OR 8.15) of contractions and "Show" (OR 4.29) confirmed their significance while, unexpectedly, "Frequency" of contractions had no impact. The area under the ROC curve in the model of the uterine activity was 0.865 (development data) and 0.927 (validation data), with an increment to 0.905 and 0.956, respectively, when adding maternal signs. The Brier Score error in the model of the uterine activity was 0.140 (development data) and 0.097 (validation data), with a decrement to 0.121 and 0.092, respectively, when adding maternal signs. CONCLUSION: Our predictive model showed a good performance. The introduction of a non-invasive tool might assist midwives in the decision-making process, avoiding interventions and thus offering an evidenced-base care.

Monocyte-macrophage polarization and recruitment pathways in the tumour microenvironment of B-cell acute lymphoblastic leukaemia.

B-cell acute lymphoblastic leukaemia (B-ALL) reprograms the surrounding bone marrow (BM) stroma to create a leukaemia-supportive niche. To elucidate the contribution of immune cells to the leukaemic microenvironment, we investigated the involvement of monocyte/macrophage compartments, as well as several recruitment pathways in B-ALL development. Immunohistochemistry analyses showed that CD68-expressing macrophages were increased in leukaemic BM biopsies, compared to controls and predominantly expressed the M2-like markers CD163 and CD206. Furthermore, the "non-classical" CD14+ CD16++ monocyte subset, expressing high CX3CR1 levels, was significantly increased in B-ALL patients' peripheral blood. CX3CL1 was shown to be significantly upregulated in leukaemic BM plasma, thus providing an altered migratory pathway possibly guiding NC monocyte recruitment into the BM. Additionally, the monocyte/macrophage chemoattractant chemokine ligand 2 (CCL2) strongly increased in leukaemic BM plasma, possibly because of the interaction of leukaemic cells with mesenchymal stromal cells and vascular cells and due to a stimulatory effect of leukaemia-related inflammatory mediators. C5a, a macrophage chemoattractant and M2-polarizing factor, further appeared to be upregulated in the leukaemic BM, possibly as an effect of PTX3 decrease, that could unleash complement cascade activation. Overall, deregulated monocyte/macrophage compartments are part of the extensive BM microenvironment remodelling at B-ALL diagnosis and could represent valuable targets for novel treatments to be coupled with classical chemotherapy.

Measurement of Gamma Glutamyl Transferase to Determine Risk of Liver Transplantation or Death in Patients With Primary Biliary Cholangitis.

BACKGROUND & AIMS: Gamma-glutamyltransferase (GGT) is a serum marker of cholestasis. We investigated whether serum level of GGT is a prognostic marker for patients with primary biliary cholangitis (PBC). METHODS: We analyzed data from patients with PBC from the Global PBC Study Group, comprising 14 centers in Europe and North America. We obtained measurements of serum GGT at baseline and time points after treatment. We used Cox model hazard ratios to evaluate the association between GGT and clinical outcomes, including liver transplantation and liver-related death. RESULTS: Of the 2129 patients included in our analysis, 281 (13%) had a liver-related clinical endpoint. Mean age at diagnosis was 53 years and 91% of patients were female patients. We found a correlation between serum levels of GGT and alkaline phosphatase (ALP) (r = 0.71). Based on data collected at baseline and yearly for up to 5 years, higher serum levels of GGT were associated with lower hazard for transplant-free survival. Serum level of GGT at 12 months after treatment higher than 3.2-fold the upper limit of normal (ULN) identified patients who required liver transplantation or with liver-related death at 10 years with an area under the receiver operating characteristic curve of 0.70. The risk of liver transplantation or liver-related death in patients with serum level of GGT above 3.2-fold the ULN, despite level of ALP lower than 1.5-fold the ULN, was higher compared to patients with level of GGT lower than 3.2-fold the ULN and level of ALP lower than 1.5-fold the ULN (P < .05). Including information on level of GGT increased the prognostic value of the Globe score. CONCLUSIONS: Serum level of GGT can be used to identify patients with PBC at risk for liver transplantation or death, and increase the prognostic value of ALP measurement. Our findings support the use of GGT as primary clinical endpoint in clinical trials. In patients with low serum level of ALP, a high level of GGT identifies those who might require treatment of metabolic disorders or PBC treatment escalation.

Ordinal Outcomes Are Superior to Binary Outcomes for Designing and Evaluating Clinical Trials in Compensated Cirrhosis.

BACKGROUND AND AIMS: Prevention of decompensation is a primary therapeutic target in patients with compensated cirrhosis (CC). However, a major problem is the large sample size and long follow-up required to demonstrate a significant treatment effect because of the relatively low baseline risk. For this reason, it has been recently suggested that ordinal outcomes may be used in this area to gain power and reduce sample size. The aim of this study was to assess the applicability of ordinal outcomes in cirrhosis. APPROACH AND RESULTS: An inception cohort of 202 patients with CC (no ascites, gastrointestinal bleeding, encephalopathy, or jaundice) without esophageal varices was included, and 5-year outcome is reported. Etiology was mostly viral and alcoholic, and there were no dropouts. Ordinal outcome was set according to six grades with a previously established prognostic ordinality: grade 1 = no disease progression; grade 2 = development of varices; grade 3 = bleeding alone; grade 4 = nonbleeding single decompensation; grade 5 = more than one decompensating event; and grade 6 = death. At the 60-month time point, patients were distributed in grades 1 through 6 as follows: 129, 43, 2, 7, 5, and 16, respectively. Emulation of a clinical trial performed by dividing patients based on baseline platelet count into two groups (cutoff, 150 × 109 /L) demonstrated a statistically significant outcome difference between groups when using ordinal outcomes not detectable by binary logistic or chi-square or time-to-event analyses. Additionally, using ordinal outcomes in a hypothetical study to prevent decompensation resulted in sample-size estimates 3-to 4-fold lower than using a binary composite endpoint. CONCLUSIONS: Compared to traditional binary outcomes, the use of ordinal outcomes in trials of cirrhosis decompensation may provide more power and thus may require a smaller sample size.

Sampling strategies to evaluate the prognostic value of a new biomarker on a time-to-event end-point.

BACKGROUND: The availability of large epidemiological or clinical data storing biological samples allow to study the prognostic value of novel biomarkers, but efficient designs are needed to select a subsample on which to measure them, for parsimony and economical reasons. Two-phase stratified sampling is a flexible approach to perform such sub-sampling, but literature on stratification variables to be used in the sampling and power evaluation is lacking especially for survival data. METHODS: We compared the performance of different sampling designs to assess the prognostic value of a new biomarker on a time-to-event endpoint, applying a Cox model weighted by the inverse of the empirical inclusion probability. RESULTS: Our simulation results suggest that case-control stratified (or post stratified) by a surrogate variable of the marker can yield higher performances than simple random, probability proportional to size, and case-control sampling. In the presence of high censoring rate, results showed an advantage of nested case-control and counter-matching designs in term of design effect, although the use of a fixed ratio between cases and controls might be disadvantageous. On real data on childhood acute lymphoblastic leukemia, we found that optimal sampling using pilot data is greatly efficient. CONCLUSIONS: Our study suggests that, in our sample, case-control stratified by surrogate and nested case-control yield estimates and power comparable to estimates obtained in the full cohort while strongly decreasing the number of patients required. We recommend to plan the sample size and using sampling designs for exploration of novel biomarker in clinical cohort data.

Helmet CPAP to treat hypoxic pneumonia outside the ICU: an observational study during the COVID-19 outbreak.

BACKGROUND: Respiratory failure due to COVID-19 pneumonia is associated with high mortality and may overwhelm health care systems, due to the surge of patients requiring advanced respiratory support. Shortage of intensive care unit (ICU) beds required many patients to be treated outside the ICU despite severe gas exchange impairment. Helmet is an effective interface to provide continuous positive airway pressure (CPAP) noninvasively. We report data about the usefulness of helmet CPAP during pandemic, either as treatment, a bridge to intubation or a rescue therapy for patients with care limitations (DNI). METHODS: In this observational study we collected data regarding patients failing standard oxygen therapy (i.e., non-rebreathing mask) due to COVID-19 pneumonia treated with a free flow helmet CPAP system. Patients' data were recorded before, at initiation of CPAP treatment and once a day, thereafter. CPAP failure was defined as a composite outcome of intubation or death. RESULTS: A total of 306 patients were included; 42% were deemed as DNI. Helmet CPAP treatment was successful in 69% of the full treatment and 28% of the DNI patients (P < 0.001). With helmet CPAP, PaO2/FiO2 ratio doubled from about 100 to 200 mmHg (P < 0.001); respiratory rate decreased from 28 [22-32] to 24 [20-29] breaths per minute, P < 0.001). C-reactive protein, time to oxygen mask failure, age, PaO2/FiO2 during CPAP, number of comorbidities were independently associated with CPAP failure. Helmet CPAP was maintained for 6 [3-9] days, almost continuously during the first two days. None of the full treatment patients died before intubation in the wards. CONCLUSIONS: Helmet CPAP treatment is feasible for several days outside the ICU, despite persistent impairment in gas exchange. It was used, without escalating to intubation, in the majority of full treatment patients after standard oxygen therapy failed. DNI patients could benefit from helmet CPAP as rescue therapy to improve survival. TRIAL REGISTRATION: NCT04424992.