Effectiveness of a Reliable Toxicological Analysis for a Correct Diagnosis of Acute Intoxication in Pediatrics: 2-Year Experience of an Analytical Toxicological Laboratory.

OBJECTIVES: This study aimed to assess the role of the laboratory of toxicology as a support for a correct diagnosis of intoxication through the application of a reliable analytical approach, critically designed to meet pediatric needs. METHODS: Data collected from 360 cases of suspected intoxications in pediatric patients (aged 1 day to 17 years) during the period 2018 to 2019 are presented. Toxicological analyses were performed through different techniques (immunoassay and chromatography) with parameters (limit of detection and cut-off) adjusted according to pediatric needs to produce reliable toxicological data for a wide number of prescription drugs, drugs of abuse, and poisons. RESULTS: We present results about (1) agents involved in suspected poisonings and the methods adopted for a definite analytical diagnosis, (2) the assessment of the concordance of results for analyses proceeded by different techniques, and (3) the percentage of agreement between analytical result and clinical suspicion. CONCLUSIONS: An analytical approach critically designed to minimize misinterpretation of laboratory data and able to provide reliable results for a wide number of substances in a time compatible with the urgency represents a useful support for a correct diagnosis of intoxication in pediatrics.

Eltrombopag second-line therapy in adult patients with primary immune thrombocytopenia in an attempt to achieve sustained remission off-treatment: results of a phase II, multicentre, prospective study.

Up to 30% immune thrombocytopenia (ITP) patients achieve a sustained remission off-treatment (SROT) after discontinuation of thrombopoietin receptor agonists (TPO-RAs). Factors predictive of response are lacking. Patients aged ≥18 years with newly diagnosed or persistent ITP were treated with eltrombopag for 24 weeks. Primary end-point was SROT: the proportion of responders that were able to taper and discontinue eltrombopag maintaining the response during a period of observation (PO) of six months. Secondary end-points included the association between some immunological parameters (TPO serum levels, cytokines and lymphocyte subsets) and response. Fifty-one patients were evaluable. Primary end-point was achieved in 13/51 (25%) treated patients and 13/34 (38%) patients who started the tapering. Baseline TPO levels were not associated with response at week 24 nor with SROT. Higher baseline levels of IL-10, IL-4, TNF-α and osteopontin were negative factors predictive of response (P = 0·001, 0·008, 0·02 and 0·03 respectively). This study confirms that SROT is feasible for a proportion of ITP patients treated with eltrombopag. Some biological parameters were predictive of response.

Chronic lymphocytic leukemia with del13q14 as the sole abnormality: dynamic prognostic estimate by interphase-FISH.

This study analyzed 140 patients with isolated del13q14 on interphase FISH (I-FISH), to identify subsets with a different progression risk and to assess the acquisition of additional chromosomal abnormalities (clonal evolution) in treatment-naïve del13q14 patients. A monoallelic deletion (del13qx1) was detected in 123 cases (88%), a biallelic deletion (del13qx2) in eight and a mosaic of monoallelic and biallelic deletions (del13qx1/del13qx2) in nine. In 33% of cases, deletion encompassed the Rb1 locus The median percentage of abnormal nuclei was 50% (15%-96%), and it was higher in patients with a biallelic/mosaic pattern in comparison with patients with monoallelic deletion. Sixty two patients (44%) have been treated; 5-year treatment free survival rate was 56% and the median treatment free survival was 65 months. The baseline percentage of deleted nuclei, as a continuous variable, was related to progression (HR: 1.02; p = 0.001). According to deletion burden, three groups were identified: 64 cases (46%) had <50% deleted nuclei, 47 (33%) had 50-69% deleted nuclei, and 29 (21%) had ≥70% deleted nuclei. The 5-year untreated rate was 70.5% , 52.6% and 28.7% (p < 0.0001), respectively. In multivariate analysis using IGHV mutational status, presence of a nullisomic clone, CD38 expression and percentage of deleted nuclei as covariates, only IGHV mutational status and the percentage of deleted nuclei were independent risk factors for treatment. In 103 patients serially monitored by I-FISH before starting any treatment, we observed a significant increase in the proportion of del13q14 cells, and this increase affected the risk of subsequent treatment requirement (HR 2.54, p = 0.001). The appearance of a new clone was detected in 16 patients (15.5%) and chromosome 13 was involved in 14 of them. I-FISH monitoring proves worthwhile for a dynamic risk stratification and for planning clinical surveillance.

Does cytogenetic evolution have any prognostic relevance in myelodysplastic syndromes? A study on 153 patients from a single institution.

The present study was designed to establish the incidence of cytogenetic evolution (CE), defined as the acquisition of chromosomal defects during the course of MDS, in order to correlate it with the WHO classification and IPSS score, and to assess its impact on overall survival (OS) and risk of MDS/AML evolution (progression-free interval, PFI) by means of Cox models for time-dependent covariates. Adjustments for known risk factors were achieved by performing a bivariable analysis. The study was carried out in 153 MDS patients who were followed for a median period of 45.2 months. Disease progression occurred in 42.4% of patients after a 65.2-month median PFI, while CE occurred in 30.7% of patients. Our study shows that (1) CE was more common in advanced than in early MDS, and advanced MDS presented secondary chromosomal defects distinct from those of early MDS; (2) CE significantly affected OS and PFI independently of other prognostic variables; (3) del(7)(q31q34) was the only secondary chromosomal defect which significantly affected PFI; trisomy 8 had only a moderate influence.

Efficacy of intraventricular amikacin treatment in pan-resistant Pseudomonas aeruginosa postsurgical meningitis.

BACKGROUND: We describe a case of pan-resistant Pseudomonas aeruginosa postsurgical meningitis associated with the presence of an external ventricular device. We changed therapy twice; finally, by using amikacin and a continuous infusion of cefepime, we obtained clinical improvement. CASE PRESENTATION: A female patient, who underwent surgery for a cavernous angioma, presented with meningitis. Cerebrospinal fluid culture revealed a multidrug-resistant Pseudomonas aeruginosa, initially sensitive only to colistin. We successfully used intrathecal amikacin and intravenous cefepime continuous infusion plus intravenous amikacin after two previous ineffective therapeutic approaches. CONCLUSION: The evaluation of the antibiotic concentration and the bactericidal activity in cerebrospinal fluid may contribute to the choice of the drug in cases of multidrug-resistant meningitis.

Alternative splicing of hTERT: a further mechanism for the control of active hTERT in acute myeloid leukemia.

hTERT component is the key regulator of telomerase. Alternatively spliced variants of hTERT generate different telomerase activity. The goal of the study was to determine the role of different hTERT isoforms in the regulation of telomerase expression in AML patients. Among the 97 studied patients, 45 had a complex karyotype and 52 a normal karyotype. hTERT isoforms expression was determined in bone marrow samples by q-RT-PCR, using SYBR Green I. hTERT expression was lower in AML patients than controls (median 2.5 vs. 10.1, p = .003), though no difference was observed between the complex and normal karyotype (median 3.2 vs. 2.3, p = .37). High trans-dominant negative isoform expression increased the response rate by two. High expression of inactive product (-α - ß) was shown to increase the risk of relapse by about three times. In conclusion, our data suggest an intriguing link between the control of hTERT isoforms expression and AML outcome.

MDS/AML del(11)(q14) Share Common Morphological Features Despite Different Chromosomal Breakpoints.

In myelodysplatic syndromes and acute myeloid leukemia (MDS/AML) deletion of the 11q14 region is a rare chromosomal defect (incidence: 0.6-1.0%), included within the intermediate risk criteria by the International Prognostic Scoring System. No fluorescence in situ hybridization (FISH) study has yet been performed to identify a common breakpoint region (CBR). In our study through FISH with bacterial artificial chromosomes and commercial probes, we analyzed seven patients with MDS/AML harboring 11q14 deletion on conventional cytogenetic analysis. FISH revealed deletions in five patients and amplifications in two. Three patients with deletion carried a CBR, two had a deletion involving a more centromeric breakpoint. These five patients exhibited multilineage dysplasia, blast cells with large round nuclei, loose chromatin, small and abundant nucleoli, and vacuolated cytoplasm with very thin Auer bodies. In conclusion, the morphological features which occur independently of the extent of the deletion are of multilineage dysplasia in MDS and leukemic blasts strongly reactive to peroxidase in AML; despite the variable size of the deleted area, some patients harbor a CBR.

Clinical relevance of cytogenetics in myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are a group of heterogeneous stem cell disorders with different clinical behaviors and outcomes. Conventional cytogenetics (CC) studies have demonstrated that the majority of MDS patients harbor clonal chromosome defects. The probability of discovering a chromosomal abnormality has been increased by fluorescence in situ hybridization (FISH), which has revealed that about 15% of patients with a normal chromosome pattern on CC may instead present cryptic defects. Cytogenetic abnormalities, except for the interstitial long-arm deletion of chromosome 5 (5q-), are not specific for any French-American-British (FAB)/World Health Organization (WHO) MDS subtypes, demonstrate the clonality of the disease, and identify peculiar morphological entities, thus confirming clinical diagnosis. In addition, chromosome abnormalities are independent prognostic factors predicting overall survival and the likelihood of progression in acute myeloid leukemia.

ABL1 amplification in T-cell acute lymphoblastic leukemia.

ABL1 amplification, due to a cryptic episomal translocation NUP214/ABL1, is a novel finding in T-cell acute lymphoblastic leukemia (ALL). Here we report on the incidence and clinical features of this genetic defect in a series of 30 consecutive adult T-cell ALL patients. Multiple copies of the ABL1 gene were detected in two patients (6.6%), one with the karyotype 46,XY,t(1;3)(p36;p21),del(6)(q23)/46,XY and the other without analyzable metaphases. Metaphase/interphase fluorescence in situ hybridization (FISH) detected multiple uncountable signals corresponding to ABL1 in mitotic cells and nuclei from both patients. In one patient, no signals corresponded with the 9p21 chromosomal region, which contains the p16INK4a gene, and in the other one signal was observed. Quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) demonstrated that in these patients ABL1 gene expression was 14- and 18-fold greater than in normal controls, and returned to normal levels only when complete remission was achieved. We reached the following conclusions: (1) FISH is the only technique that promptly identifies T-cell ALL patients with ABL1 amplification, (2) quick identification with FISH is fundamental in the clinic because this T-cell ALL subset is imatinib sensitive but may become resistant due to development of additional mutations, and (3) ABL1 quantitative RT-PCR may be easily applied to monitor minimal residual disease.

Molecular genetics of acute myeloid leukemia.

Recurring chromosomal abnormalities are detected in most patients with acute myeloid leukemia (AML). They may be associated with a distinct AML FAB subtype or may identify distinct clinicobiological entities within the same FAB subtype. Therefore, cytogenetic investigation has a pivotal role in AML diagnosis. In addition, it is one of the most valuable prognostic determinants of the disease, as recently demonstrated. The development of new molecular techniques, such as reverse transcriptase polymerase chain reaction and fluorescence in situ hybridization, has allowed perfect definition of the chromosome regions containing genes with a crucial role in normal hemopoiesis and leukemia. Understanding the action of such genes provides new insights into AML pathogenesis and has led us to envisage new therapeutic options.

Molecularly targeted therapy in acute myeloid leukemia.

Meaningful progress has been made toward clarifying the molecular steps in the pathogenesis of acute myeloid leukemia (AML). Chromosome studies have established that translocations/inversions are the most common cytogenetic defects in AML. Cloning of chromosome breakpoints has shown that genes involved in the chromosome abnormalities are transcription factors, functional loss of which alters chromatin configuration and results in the disruption of myeloid differentiation. However, transgenic animal models have demonstrated that AML-specific translocations/inversions alone are insufficient to cause overt leukemia, which occurs only when point mutations affecting receptor tyrosine kinases (RTKs) develop. Therefore, development of AML is now considered a two-step process in which RTK mutations provide a proliferative and a survival advantage to a clonal cell population already marked by impaired differentiation. In addition, more accurate definition of such genetic lesions has led to a more precise insight as to how such lesions interact with cellular signaling pathways that are aberrantly regulated in AML. All these new data have profound clinical and therapeutic implications and will surely translate into the development of molecules that target specific mutations or signal transduction pathways.

Detection of TET2 abnormalities by fluorescence in situ hybridization in 41 patients with myelodysplastic syndrome.

TET2 haplo-insufficiency occurs through different molecular mechanisms and is promptly revealed by array comparative genomic hybridization, single nucleotide polymorphism (SNP) array, and next-generation sequencing (NGS). Fluorescence in situ hybridization (FISH) can effectively demonstrate TET2 deletions and is often used to validate molecular results. In the present study 41 MDS patients with and without 4q abnormalities were analyzed with a series of bacterial artificial chromosome (BAC) probes spanning the 4q22.3-q25 region. On conventional cytogenetic (CC) studies, a structural defect of the long arm of chromosome 4 (4q) was observed in seven patients. In three, one each with a t(1;4)(p21;q24), an ins(5;4)(q23;q24qter), and a t(4;17)(q31;p13) as the sole chromosomal abnormality, FISH with the RP11-356L5 and RP11-16G16 probes, which cover the TET2 locus, produced one signal only. Unexpectedly, this same result was achieved in 3 of the remaining 34 patients. Thus, a TET2 deletion was observed in a total of six patients (14.6%). TET2 deletion was not correlated with any particular clinical findings or outcome. These findings demonstrate that 1) FISH is an effective and economical method to reveal cryptic abnormalities of band 4q22-q24 resulting in TET2 deletions; 2) in these patients, TET2 deletion is the unifying genetic event; and 3) the different breakpoints within the 4q22-q25 region suggest that deletions are not mediated by repetitive sequences.

IGHV unmutated status influences outcome more than IGHV1-69 gene usage per se in patients with chronic lymphocytic leukemia.

In this study, IGHV1-69 gene usage was detected in 46 out of 379 cases (12%) of chronic lymphocytic leukemia (CLL). In comparison with patients using alternative immunoglobulin heavy-chain variable (IGHV) genes, patients with IgHV1-69 CLLs more often presented at advanced stage, lacked somatic hypermutation (unmutated cases, 87% vs. 35%; P = .00001), and expressed unfavorable biologic characteristics. In 12 patients (26%), common amino acid motifs within the heavy-chain third complementarity-determining region were identified, allowing assignment to previously reported stereotyped subsets. In our study, treatment-free survival of patients with unmutated IGVH1-69 did not differ significantly from that of patients expressing unmutated alternative IGHV genes. As such, IGHV1-69 gene usage per se did not seem to be predictive of progressive disease, progression being primarily related to the unmutated IGHV profile.

World Health Organization classification in combination with cytogenetic markers improves the prognostic stratification of patients with de novo primary myelodysplastic syndromes.

This study correlated chromosomal defects with French-American-British (FAB)/World Health Organization (WHO) classification subtypes, proposed a revised International Prognostic Scoring System (IPSS) cytogenetic grouping; and established which classification, when used with the IPSS cytogenetic categories, best predicted clinical outcome in the myelodysplastic syndromes (MDS). A higher prevalence of chromosomal defects and distinct defects were observed in patients with multi-lineage dysplasia and a blast cell percentage >10%. Abnormalities of the long arm of chromosome 3, del(7)(q31q35), trisomy 8, del(11)(q14q23), del(12p) and 20q- could be segregated from their respective IPSS cytogenetic categories and used to develop new cytogenetic subgroups. Clinical parameters, FAB/WHO classification, IPSS score and standard or revised cytogenetic categories were statistically relevant for overall survival (OS) and progression-free intervals (PFI) and were included within five distinct multivariate models compared by the Akaike Information Criterion. To predict OS, the best models included age, WHO classification and standard or revised IPSS cytogenetic categories; to predict PFI, the best model included the same variables and revised cytogenetic categories. In conclusion, (i) the WHO classification was associated with a more homogeneous cytogenetic pattern than the FAB classification, (ii) WHO classification and standard/revised IPSS cytogenetic categories were much more effective than IPSS for predicting MDS clinical outcome, (iii) revised cytogenetic subgroups predicted PFI more effectively than standard categories.

Long-term follow up with conventional cytogenetics and band 13q14 interphase/metaphase in situ hybridization monitoring in monoclonal gammopathies of undetermined significance.

One-third of patients with monoclonal gammopathy of undetermined significance (MGUS) may progress to multiple myeloma (MM) and may develop a long arm deletion of chromosome 13 (13q-). As the incidence of 13q-, time of development and prognostic impact in MGUS patients is still under debate, we decided to perform serial sequential conventional cytogenetics (CC) and metaphase/interphase fluorescence in situ hybridization (FISH) analyses on bone marrow mononuclear cells obtained from 18 asymptomatic, untreated MGUS patients. Median follow up was 30 months (range 6-72). Interphase FISH identified a 13q14 deletion in five out of 18 patients (on clinical diagnosis in one patient and during the follow up in the remaining four patients). Subsequently, metaphase FISH and CC also identified the deletion in four out of five patients. All five of the patients progressed to MM 6-12 months after 13q- identification, without developing any FISH determined JH rearrangements. MM progression also occurred in two other karyotypically normal patients. We conclude that: (i) the extent of the 13q deletion does not vary during the clinical outcome; (ii)13q- plays a crucial role in MGUS/MM pathogenesis and confers a proliferative advantage to clonal plasma cells being initially demonstrated by interphase FISH and only afterwards by metaphase FISH and CC; and (iii) association of 13q- with t(4;14)(p16.3;q32) remains to be demonstrated. However, a transition from MGUS to MM may also occur in patients with normal karyotypes or other abnormalities, suggesting the possibility of distinct pathogenetic pathways.