Real-life diagnostic and therapeutic approach to CLL: a 2022 update from an expert panel in Tuscany.

A panel of chronic lymphocytic leukemia (CLL) experts from Tuscany propose a real-life diagnostic and therapeutic approach CLL that considers the role of genomic and somatic prognostic factors in risk stratification and treatment decisions. Safety and efficacy of new agents has been demonstrated now not only in clinical trials but also in many real-world series. The BTK inhibitors, ibrutinib and acalabrutinib, and BH3 mimetic venetoclax are now indicated as first-line therapy and chemoimmunotherapy can be spared to the majority of CLL patients, thus preventing unnecessary hematological and non-hematological toxicity and second primary tumors. For treatment, FISH for 17 p and P53 mutational status are essential. IGHV mutation can be done at diagnosis or before treatment. Echography is the gold standard radiological investigation in CLL, at both diagnosis and response evaluation. Chemotherapy is virtually abandoned. Age, genetic risk, and patient comorbidities have to be carefully evaluated for treatment decision. With the availability of different drugs, there is a need for a uniform and shared approach in daily therapeutic choice. The proposed approach is based on current evidence and guidelines as well as results from clinical trials and daily clinical experience.

Brain Damage in Preterm and Full-Term Neonates: Serum Biomarkers for the Early Diagnosis and Intervention.

The Brain is vulnerable to numerous insults that can act in the pre-, peri-, and post-natal period. There is growing evidence that demonstrate how oxidative stress (OS) could represent the final common pathway of all these insults. Fetuses and newborns are particularly vulnerable to OS due to their inability to active the antioxidant defenses. Specific molecules involved in OS could be measured in biologic fluids as early biomarkers of neonatal brain injury with an essential role in neuroprotection. Although S-100B seems to be the most studied biomarker, its use in clinical practice is limited by the complexity of brain damage etiopathogenesis and the time of blood sampling in relation to the brain injury. Reliable early specific serum markers are currently lacking in clinical practice. It is essential to determine if there are specific biomarkers that can help caregivers to monitor the progression of the disease in order to active an early neuroprotective strategy. We aimed to describe, in an educational review, the actual evidence on serum biomarkers for the early identification of newborns at a high risk of neurological diseases. To move the biomarkers from the bench to the bedside, the assays must be not only be of a high sensitivity but suitable for the very rapid processing and return of the results for the clinical practice to act on. For the best prognosis, more studies should focus on the association of these biomarkers to the type and severity of perinatal brain damage.

Current Concepts of Corticosteroids Use for the Prevention of Bronchopulmonary Dysplasia.

Despite using antenatal steroids, surfactants and protective ventilation, bronchopulmonary dysplasia (BPD) affects 10-89% of preterm infants. Since lung inflammation is central to the BPD pathogenesis, postnatal systemic corticosteroids could reduce the risk of BPD onset in preterm infants, but short and long-term adverse consequences have been underlined in literature after their use (i.e., hyperglycaemia, hypertension, hypertrophic cardiomyopathy, growth failure, gastrointestinal bleeding, cerebral palsy). Alternative therapeutic strategies such as postponing corticosteroid administration, lowering the cumulative dose, giving pulse rather than continuous doses, or individualizing the dose according to the respiratory condition of the infant have been proposed to avoid their adverse effects. Dexamethasone remains the first-line drug for newborns with severe pulmonary disease beyond the second to the third week of life. Hydrocortisone administration in very preterm infants does not appear to be associated with neurotoxic effects, even if its efficacy in preventing and treating BPD has yet been clearly demonstrated. Alternative methods of corticosteroid administration seem promising. A positive effect on BPD prevention occurs when budesonide is nebulized and intratracheally instilled with a surfactant, but more data are required to establish safety and efficacy in preterm newborns. Additional studies are still needed before the chronic lung disease issue, and its related challenges can be solved.

Ibrutinib in patients with relapsed/refractory mantle cell lymphoma: a real-life, retrospective, multicenter trial on behalf of the "RTL" (regional Tuscan lymphoma network).

BACKGROUND: Relapsed or refractory (R/R) mantle-cell lymphoma (MCL) patients have a poor prognosis and their management is challenging, in absence of a golden standard as salvage treatment. Bruton's tyrosine kinase inhibitor ibrutinib represents an effective treatment for R/R MCL patients. We investigated ibrutinib efficacy and safety in daily clinical practice, together with factors that could predict disease outcome. PATIENTS AND METHODS: We retrospectively analyzed 69 consecutive R/R MCL patients managed in 10 Tuscan onco-hematological centers. The treatment regimen consisted of oral, continuous, single-agent ibrutinib, maximum dosage of 560 mg once per day, until disease progression. RESULTS: Overall response rate was 62.3%, with a CR rate of 39.1%. After a median follow-up of 15.6 months, 40/69 patients (58%) were alive, the main cause of death was progressive disease (PD, 22/69 cases, 31.9%). Median progression-free survival (PFS) and overall survival (OS) were 17 and 34.8 months. Inferior PFS was associated with >1 prior line of therapy and B symptoms. Ibrutinib refractoriness was associated with inferior OS, median OS after ibrutinib failure was only 5 months. DISCUSSION AND CONCLUSION: In this real-life setting ibrutinib treatment prolonged survival in R/R MCL patients, without unexpected adverse events. Patients receiving ibrutinib as 2nd line regimen had the most favorable outcome.

Management of Infants with Brief Resolved Unexplained Events (BRUE) and Apparent Life-Threatening Events (ALTE): A RAND/UCLA Appropriateness Approach.

Unexpected events of breath, tone, and skin color change in infants are a cause of considerable distress to the caregiver and there is still debate on their appropriate management. The aim of this study is to survey the trend in prevention, decision-making, and management of brief resolved unexplained events (BRUE)/apparent life-threatening events (ALTE) and to develop a shared protocol among hospitals and primary care pediatricians regarding hospital admission criteria, work-up and post-discharge monitoring of patients with BRUE/ALTE. For the study purpose, a panel of 54 experts was selected to achieve consensus using the RAND/UCLA appropriateness method. Twelve scenarios were developed: one addressed to primary prevention of ALTE and BRUE, and 11 focused on hospital management of BRUE and ALTE. For each scenario, participants were asked to rank each option from '1' (extremely inappropriate) to '9' (extremely appropriate). Results derived from panel meeting and discussion showed several points of agreement but also disagreement with different opinion emerged and the need of focused education on some areas. However, by combining previous recommendations with expert opinion, the application of the RAND/UCLA appropriateness permitted us to drive pediatricians to reasoned and informed decisions in term of evaluation, treatment and follow-up of infants with BRUE/ALTE, reducing inappropriate exams and hospitalisation and highlighting priorities for educational interventions.

Sudden Infant Death Syndrome: Beyond Risk Factors.

Sudden infant death syndrome (SIDS) is defined as "the sudden death of an infant under 1 year of age which remains unexplained after thorough investigation including a complete autopsy, death scene investigation, and detailed clinical and pathological review". A significant decrease of SIDS deaths occurred in the last decades in most countries after the beginning of national campaigns, mainly as a consequence of the implementation of risk reduction action mostly concentrating on the improvement of sleep conditions. Nevertheless, infant mortality from SIDS still remains unacceptably high. There is an urgent need to get insight into previously unexplored aspects of the brain system with a special focus on high-risk groups. SIDS pathogenesis is associated with a multifactorial condition that comprehends genetic, environmental and sociocultural factors. Effective prevention of SIDS requires multiple interventions from different fields. Developing brain susceptibility, intrinsic vulnerability and early identification of infants with high risk of SIDS represents a challenge. Progress in SIDS research appears to be fundamental to the ultimate aim of eradicating SIDS deaths. A complex model that combines different risk factor data from biomarkers and omic analysis may represent a tool to identify a SIDS risk profile in newborn settings. If high risk is detected, the infant may be referred for further investigations and follow ups. This review aims to illustrate the most recent discoveries from different fields, analyzing the neuroanatomical, genetic, metabolic, proteomic, environmental and sociocultural aspects related to SIDS.

Neuroprem: the Neuro-developmental outcome of very low birth weight infants in an Italian region.

INTRODUCTION: The survival of preterm babies has increased worldwide, but the risk of neuro-developmental disabilities remains high, which is of concern to both the public and professionals. The early identification of children at risk of neuro-developmental disabilities may increase access to intervention, potentially influencing the outcome. AIMS: Neuroprem is an area-based prospective cohort study on the neuro-developmental outcome of very low birth weight (VLBW) infants that aims to define severe functional disability at 2 years of age. METHODS: Surviving VLBW infants from an Italian network of 7 neonatal intensive care units (NICUs) were assessed for 24 months through the Griffiths Mental Developmental Scales (GMDS-R) or the Bayley Scales of Infant and Toddler Development (BSDI III) and neuro-functional evaluation according to the International Classification of Disability and Health (ICF-CY). The primary outcome measure was severe functional disability at 2 years of age, defined as cerebral palsy, a BSDI III cognitive composite score < 2 standard deviation (SD) or a GMDS-R global quotients score < 2 SD, bilateral blindness or deafness. RESULTS: Among 211 surviving VLBW infants, 153 completed follow-up at 24 months (72.5%). Thirteen patients (8.5%) developed a severe functional disability, of whom 7 presented with cerebral palsy (overall rate of 4.5%). Patients with cerebral palsy were all classified with ICF-CY scores of 3 or 4. BSDI III composite scores and GMDS-R subscales were significantly correlated with ICF-CY scores (p < 0.01). CONCLUSION: Neuroprem represents an Italian network of NICUs aiming to work together to ensure preterm neuro-developmental assessment. This study updates information on VLBW outcomes in an Italian region, showing a rate of cerebral palsy and major developmental disabilities in line with or even lower than those of similar international studies. Therefore, Neuroprem provides encouraging data on VLBW neurological outcomes and supports the implementation of a preterm follow-up programme from a national network perspective.

Flow Cytometry Assessment of CD26+ Leukemic Stem Cells in Peripheral Blood: A Simple and Rapid New Diagnostic Tool for Chronic Myeloid Leukemia.

BACKGROUND: Recent investigations in chronic myeloid leukemia (CML) have focused on the identification and characterization of leukemic stem cells (LSCs). These cells reside within the CD34+ /CD38─ /Lin─ fraction and score positive for CD26 (dipeptidylpeptidase IV) a marker, expressed in both bone marrow (BM) and peripheral blood (PB) samples, that discriminates CML cells from normal hematopoietic stem cells (HSCs) or from LSCs of other myeloid neoplasms. CD26 evaluation could be a useful tool to improve the identification of CML LCSs by using flow-cytometry assay. METHODS: CD26+ LSCs have been isolated from EDTA PB and BM samples of patients with leucocytosis suspected for CML. Analysis of LSCs CML has been performed by using custom-made lyophilized pre-titrated antibody mixture test and control tube and a CD45+ /CD34+ /CD38- /CD26+ panel as a strict flow cytometric gating strategy. RESULTS: The expression of CD26 on CD34+ /CD38- population was detectable in 211/211 PB and 84/84 BM samples of subsequently confirmed BCR-ABL+ CP-CML patients. None of the 32 samples suspicious for CML but scoring negative for circulating CD26+ LSCs were diagnosed as CML after conventional cytogenetic and molecular testing. To validate our results, we checked for PB CD26+ LSCs in patients affected by other hematological disorders and they all scored negative for CD26 expression. CONCLUSIONS: We propose flow cytometry evaluation of CD26 expression on PB CD34+ /CD38- population as a new rapid, reproducible, and powerful diagnostic tool for the diagnosis of CML. © 2019 The Authors. Cytometry Part B: Clinical Cytometry published by Wiley Periodicals, Inc. on behalf of International Clinical Cytometry Society.

Infections with multiple high-risk HPV types are associated with high-grade and persistent low-grade intraepithelial lesions of the cervix.

BACKGROUND: Infections with multiple human papillomavirus (HPV) types (mHPV) in Papanicolaou tests have been reported but the histologic correlation and clinical meaning remains debatable. METHODS: The authors prospectively tested 37 HPV types using the Linear Array HPV Genotyping Test and correlated the results to cytology and histology findings in 260 women evaluated from June 2009 to October 2011 and followed for up to 60 months. RESULTS: HPV was detected in 148 of 235 samples (63%) and high-risk HPV was detected in 132 samples (56%). mHPV infection was found to be twice as common as single HPV (sHPV) infection and was detected more frequently in low-grade squamous intraepithelial lesion (LSIL) (48 of 83 samples [58%]) and high-grade squamous intraepithelial lesion or invasive carcinoma (HSIL + (26 of 47 samples [55%]) compared with other categories (P<.001). Of 34 LSIL/cervical intraepithelial neoplasia 1 (CIN1) index cases, 13 of 21 patients with mHPV (61.9%) persisted on CIN1, whereas no histologic abnormality was detected during follow-up in all 12 patients with sHPV infection (high risk or low risk) (P<.001). Eighteen of 20 patients with HSIL/cervical intraepithelial neoplasia 2 (CIN2) (90%) and high-risk mHPV persisted on HSIL+/CIN2 + whereas 6 of 11 patients with sHPV infection did not demonstrate HSIL+/CIN2 + on follow-up (54.5%) (P = .066). Approximately 40% of women with HSIL were infected by high-risk HPV types other than types 16 or 18. CONCLUSIONS: High-risk mHPV infection identified patients with persistent LSIL/CIN1 and may to help identify patients at higher risk of disease progression to HSIL+/CIN2+. Longer follow-up will clarify the role of mHPV testing in patient care. Cancer Cytopathol 2017;125:138-143. © 2016 American Cancer Society.

Identifying High-Risk Chronic Lymphocytic Leukemia: A Pathogenesis-Oriented Appraisal of Prognostic and Predictive Factors in Patients Treated with Chemotherapy with or without Immunotherapy.

Chronic lymphocytic leukemia (CLL) displays an extremely variable clinical behaviour. Accurate prognostication and prediction of response to treatment are important in an era of effective first-line regimens and novel molecules for high risk patients. Because a plethora of prognostic biomarkers were identified, but few of them were validated by multivariable analysis in comprehensive prospective studies, we applied in this survey stringent criteria to select papers from the literature in order to identify the most reproducible prognostic/predictive markers. Each biomarker was analysed in terms of reproducibility across the different studies with respect to its impact on time to first treatment (TTFT), progression free survival (PFS), overall survival (OS) and response to treatment. We were able to identify the following biomarkers as the most reliable in guiding risk stratification in the daily clinical practice: 17p-/TP53 mutations, IGHV unmutated configuration, short telomeres and 11q-. However, the method for measuring telomere length was not validated yet and 11q- was predictive of inferior OS only in those patients who did not receive FCR-like combinations. Stage and lymphocytosis were predictive of shorter TTFT and age, high serum thymidine kinase levels and poor performance status were predictive of shorter OS. Using our criteria no parameter was found to independently predict for inferior response to treatment.

Sleep breathing disorders in 40 Italian patients with Myotonic dystrophy type 1.

The aim of this study was to estimate the prevalence and nature of sleep breathing disorders in Myotonic dystrophy type 1 (DM1). We wanted to determine whether there is a relationship between sleep breathing disorders and clinical parameters such as pulmonary function, degree of neuromuscular impairment, daytime sleepiness, and fatigue. This will help assess the prevalence of DM1 patients requiring nocturnal ventilatory treatments. We studied a random sample of 40 unrelated patients and found that 22/40 patients had obstructive sleep apnoea. Of these 22 patients, five showed also periodic breathing and four showed sleep hypoventilation. Nine patients were put on nocturnal ventilation following clinical and instrumental evaluations. Our study reveals that obstructive sleep apnoea is very common in these patients, but cannot be predicted on the basis of clinical-neurological features and diurnal functional respiratory tests. Our data emphasize that a periodical evaluation by polysomnography should be mandatory to ascertain, and treat if necessary, the presence of obstructive sleep apnoea, periodic breathing or nocturnal hypoventilation.

A decreased positivity for CD90 on human mesenchymal stromal cells (MSCs) is associated with a loss of immunosuppressive activity by MSCs.

Biologic and clinical interest in human mesenchymal stromal cells (hMSC) has risen over the last years, mainly due to their immunosuppressive properties. In this study, we investigated the basis of immunomodulant possible variability using hMSC from different sources (amniotic membrane, chorion, and bone marrow from either healthy subjects or patients with hematological malignancies, HM) and having discordant positivity for several immunological markers. The CD90+ hMSC reduced lymphoproliferative response in phytohemagglutinin (PHA) activated peripheral blood mononuclear cells (PBMC) via sHLA-G and IL-10 up-modulation. On the contrary, hMSC showing a significantly lower expression for CD90 antigen, elicited a lymphoproliferative allogeneic response in PHA/PBMCs without any increase in soluble HLA-G and IL-10 levels. These data seems to suggest that CD90 molecule may be considered a novel predictive marker for hMSC inhibitory ability, and might cooperate with HLA-G molecule in regulating suppressive versus stimulatory properties of hMSC. These results may have clinical implication in either transplantation or in regenerative medicine fields.

CXCR4 pos circulating progenitor cells coexpressing monocytic and endothelial markers correlating with fibrotic clinical features are present in the peripheral blood of patients affected by systemic sclerosis.

There is still controversy regarding the role of circulating endothelial and progenitor cells (CECs/CEPs) in the pathogenesis of systemic sclerosis (SSc). Using a sequential Boolean gating strategy based on a 4-color flow cytometric protocol, an increased number of CD31(pos)/CD184(pos)(CXCR4)/CD34(pos)/CD45(pos) and CD31(pos)/CD117(pos) (c-kit-R) /CD34(pos)/ CD45(pos) hematopoietic circulating progenitor cells (HCPCs) was detected in SSc patients compared with healthy subjects. In SSc, no circulating mature and progenitor endothelial cells were observed, while an enhanced generation of erythroid progenitor cells was found to be correlated with the presence of CD117+ HCPCs. The presence of freshly detected CXCR4posHCPC was correlated either to the in vitro cultured spindle-shaped endothelial like cells (SELC) with an endo/myelomonocytic profile or to SDF-1 and VEGF serum level. These data are related to more fibrotic clinical features of the disease, thus supporting a possible role of these cells in fibrosis.

Transient periventricular echodensities in preterms and neurodevelopmental outcome.

Little is known about the clinical evolution and neurologic sequelae of transient periventricular echodensities in the neonatal period. The aim of our study was to assess the neurodevelopmental outcome in preterm infants with transient periventricular echodensities. Cerebral ultrasonography was performed within the first 72 hours of life on all preterms with a < or = 37 weeks' gestational age who were admitted consecutively to the Neonatal Intensive Care Unit of the University of Parma from January 2001 to December 2002. Cerebral ultrasonography was performed at least twice within the 14th postnatal day and was repeated weekly until 40 weeks' postconceptional age. Transient aspecific echodensities were defined as areas in the periventricular region brighter than the choroid plexus persisting less than 14 days. One hundred sixty-four preterm infants were selected and divided into three groups: (1) 78 preterm infants without ultrasound abnormalities, (2) 50 preterm infants with transient periventricular echodensities, and (3) 36 preterm infants with persistent echodensities. Developmental outcome was assessed at 44 weeks' postconceptional age, after 1 month from the discharge and at the corrected ages of 3, 6, 9, and 12 months using the Griffiths Mental Developmental Scale. Group 1 and 2 infants showed normal neurodevelopment in 88.5% and 94% of cases, respectively, whereas the preterm infants belonging to group 3 had a favorable outcome in 22.2% (P < .001) of cases only. In conclusion, our study demonstrates how infants with transient echodensities show a neurodevelopmental outcome that is entirely identical to infants with a steadily negative ultrasound finding.

Immunophenotypic heterogeneity of bone marrow-derived mesenchymal stromal cells from patients with hematologic disorders: correlation with bone marrow microenvironment.

The immunophenotypic analysis of ex vivo-expanded mesenchymal stromal cells (MSC) has so far been confined to single or dual staining analysis in normal subjects. In this study, using a four-color cytofluorimetric protocol, we demonstrated that cultured MSC derived from the bone marrow of patients with hematologic malignancies showed alterations in the expression of CD105, CD90, CD184, and HLA-DR molecules. The decrease in the percentage of CD105+ and CD90+ MSC correlated with an increased bone marrow angiogenesis. This paper provides evidence that multiparametric flow cytometry is essential for the establishment of a standardized protocol to identify various MSCs subsets and aberrant phenotypes.

Alteration of the amniotic fluid and neonatal outcome.

Quantitative and qualitative alterations of the amniotic fluid complicate 7% of the pregnancies. Polyhydramnios complicates 1-3% while oligohydramnios involves 3-5% of the pregnancies. The most common causes of polyhydramnios are fetal abnormalities, maternal diabetes and twin pregnancies, but are idiopathic in the 60%. Perinatal mortality has been reported to range between 10-30% while the risk of preterm birth reaches up to 22% in pregnancies complicated by polyhydramnios. The neonatal outcome, in cases where polyhydramnios is due to fetal-neonatal abnormalities, depends on the underlying pathology. Polyhydramnios due to defects in intestinal canalisation in particular, has been correlated to good neonatal prognosis. In our experience no early postoperative deaths occurred in a group of 16 newborns consequtively admitted to our unit in the last two years, with abnormalities of the gastrointestinal tract with need of surgery within the second week of life. Most cases of oligohydramnios are due to premature rupture of membranes, other causes are fetal abnormalities, such as urinary tract malformations, or chromosomopaties and drugs e.g. NSAID's. Oligohydramnios of mild entities is often associated to preterm birth, fetal growth restriction. In some cases of oligohydramnios, neonatal survival is highly conditioned by pulmonary hypoplasia which develops with rates that range between 13 and 21%. Neonatal prognosis is often disastrous in cases with severe oligohydramnios, which however could be improved by amnioinfusion, which restores an amniotic fluid volume sufficient in reducing the adverse environmental effects and in prolonging, where possible, pregnancy. Beside the quantity also the quality of the amniotic fluid may be related to the neonatal outcome. Finding of some inflammatory factors (interleukines) in the amniotic fluid seems to be significantly correlated to periventricular leucomalacia (PVL), cerebral paralysis and long-term neurological abnormalities, both in the preterm and term neonate. Therefore, increase of the cytokines in the amniotic fluid could give information not only of the infection but also regarding the risk of developing neurological sequelae in neonatal period. Diagnosis and therapy for pathologies that alter the amniotic fluid have progressed, however efforts have still to be made in the identification and search for those quantitative-qualitative alterations of the amniotic fluid, for their potential implications on neonatal outcome.

"In vitro" evaluation of bone marrow angiogenesis in myelodysplastic syndromes: a morphological and functional approach.

In myelodysplastic syndromes (MDS), ineffective hematopoiesis and cytopenia may arise either from increased susceptibility to apoptosis of hematopoietic cells, or possibly from alterations of bone marrow stroma that contributes to defective development of marrow lineages. In order to test the significance of the endothelial growth in MDS patients, two in vitro assays are presented in this study: a long-term culture method for the detection of human bone marrow endothelial colonies (CFU-En) (77 patients) and a human primary model for the evaluation of the influence of a "bone marrow conditioned medium" on the formation of new vessels in a culture matrix ("angio-kit assay") (in 24 out 77 patients). The in vitro growth pattern of bone marrow CFU-En as well as the generation of microvessels in the angio-kit system was increased in RA, RARS and RAEB in comparison with controls. No CFU-En were detected in RAEB-T. The occurrence of large islands, formed by clusters of endothelial cells, unable to generate microcapillaries was also reported. Chromosomal abnormalities did not correlate with the angiogenetic pattern. These in vitro assays may constitute an useful approach to the analysis of angiogenesis in MDS.