The phenotypic landscape of primary biliary cholangitis and autoimmune hepatitis variants.

Autoimmune Hepatitis (AIH) and Primary Biliary Cholangitis (PBC) stand as distinct diseases, yet occasionally intertwine with overlapping features, posing diagnostic and management challenges. This recognition traces back to the 1970s, with initial case reports highlighing this complexity. Diagnostic scoring systems like IAIHG and simplified criteria for AIH were introduced but are inherently limited in diagnosing variant syndromes. The so-called Paris Criteria offer a diagnostic framework with high sensitivity and specificity for variant syndromes, although disagreements among international guidelines persist. Histological findings in AIH and PBC may exhibit overlapping features, rendering histology alone inadequate for a definitive diagnosis. Autoantibody profiles could be helpful, but similarly cannot be considered alone to reach a solid and consistent evaluation. Treatment strategies vary based on the predominant features observed. Individuals with overlapping characteristics favoring AIH ideally benefit from corticosteroids, while patients primarily manifesting PBC features should initially receive treatment with choleretic drugs like ursodeoxycholic acid (UDCA).

Two siblings presenting with novel ADA2 variants, lymphoproliferation, persistence of large granular lymphocytes, and T-cell perturbations.

The presence of large granular lymphocytes has been reported in patients with ADA2 deficiency and T-LGL leukemia. Here we describe two siblings with novel ADA2 variants, expanding the mutational spectrum of ADA2 deficiency. We show that lymphoproliferation, persistence of large granular lymphocytes, T-cell perturbations, and activation of PI3K pathway, measured by means of phosphorylation levels of S6, are detectable in DADA2 patients without T-LGL leukemia.

Idiopathic erythrocytosis: a germline disease?

Polycythemia Vera (PV) is typically caused by V617F or exon 12 JAK2 mutations. Little is known about Polycythemia cases where no JAK2 variants can be detected, and no other causes identified. This condition is defined as idiopathic erythrocytosis (IE). We evaluated clinical-laboratory parameters of a cohort of 56 IE patients and we determined their molecular profile at diagnosis with paired blood/buccal-DNA exome-sequencing coupled with a high-depth targeted OncoPanel to identify a possible underling germline or somatic cause. We demonstrated that most of our cohort (40/56: 71.4%) showed no evidence of clonal hematopoiesis, suggesting that IE is, in large part, a germline disorder. We identified 20 low mutation burden somatic variants (Variant allelic fraction, VAF, < 10%) in only 14 (25%) patients, principally involving DNMT3A and TET2. Only 2 patients presented high mutation burden somatic variants, involving DNMT3A, TET2, ASXL1 and WT1. We identified recurrent germline variants in 42 (75%) patients occurring mainly in JAK/STAT, Hypoxia and Iron metabolism pathways, among them: JAK3-V722I and HIF1A-P582S; a high fraction of patients (48.2%) resulted also mutated in homeostatic iron regulatory gene HFE-H63D or C282Y. By generating cellular models, we showed that JAK3-V722I causes activation of the JAK-STAT5 axis and upregulation of EPAS1/HIF2A, while HIF1A-P582S causes suppression of hepcidin mRNA synthesis, suggesting a major role for these variants in the onset of IE.

The KMT2A recombinome of acute leukemias in 2023.

Chromosomal rearrangements of the human KMT2A/MLL gene are associated with de novo as well as therapy-induced infant, pediatric, and adult acute leukemias. Here, we present the data obtained from 3401 acute leukemia patients that have been analyzed between 2003 and 2022. Genomic breakpoints within the KMT2A gene and the involved translocation partner genes (TPGs) and KMT2A-partial tandem duplications (PTDs) were determined. Including the published data from the literature, a total of 107 in-frame KMT2A gene fusions have been identified so far. Further 16 rearrangements were out-of-frame fusions, 18 patients had no partner gene fused to 5'-KMT2A, two patients had a 5'-KMT2A deletion, and one ETV6::RUNX1 patient had an KMT2A insertion at the breakpoint. The seven most frequent TPGs and PTDs account for more than 90% of all recombinations of the KMT2A, 37 occur recurrently and 63 were identified so far only once. This study provides a comprehensive analysis of the KMT2A recombinome in acute leukemia patients. Besides the scientific gain of information, genomic breakpoint sequences of these patients were used to monitor minimal residual disease (MRD). Thus, this work may be directly translated from the bench to the bedside of patients and meet the clinical needs to improve patient survival.

Minimal residual disease and outcome characteristics in infant KMT2A-germline acute lymphoblastic leukaemia treated on the Interfant-06 protocol.

BACKGROUND: The outcome of infants with KMT2A-germline acute lymphoblastic leukaemia (ALL) is superior to that of infants with KMT2A-rearranged ALL but has been inferior to non-infant ALL patients. Here, we describe the outcome and prognostic factors for 167 infants with KMT2A-germline ALL enrolled in the Interfant-06 study. METHODS: Univariate analysis on prognostic factors (age, white blood cell count at diagnosis, prednisolone response and CD10 expression) was performed on KMT2A-germline infants in complete remission at the end of induction (EOI; n = 163). Bone marrow minimal residual disease (MRD) was measured in 73 patients by real-time quantitative polymerase chain reaction at various time points (EOI, n = 68; end of consolidation, n = 56; and before OCTADAD, n = 57). MRD results were classified as negative, intermediate (<5∗10-4), and high (≥5∗10-4). RESULTS: The 6-year event-free and overall survival was 73.9% (standard error [SE] = 3.6) and 87.2% (SE = 2.7). Relapses occurred early, within 36 months from diagnosis in 28 of 31 (90%) infants. Treatment-related mortality was 3.6%. Age <6 months was a favourable prognostic factor with a 6-year disease-free survival (DFS) of 91% (SE = 9.0) compared with 71.7% (SE = 4.2) in infants >6 months of age (P = 0.04). Patients with high EOI MRD ≥5 × 10-4 had a worse outcome (6-year DFS 61.4% [SE = 12.4], n = 16), compared with patients with undetectable EOI MRD (6-year DFS 87.9% [SE = 6.6], n = 28) or intermediate EOI MRD <5 × 10-4 (6-year DFS 76.4% [SE = 11.3], n = 24; P = 0.02). CONCLUSION: We conclude that young age at diagnosis and low EOI MRD seem favourable prognostic factors in infants with KMT2A-germline ALL and should be considered for risk stratification in future clinical trials.

Correction: Standardisation and consensus guidelines for minimal residual disease assessment in Philadelphia-positive acute lymphoblastic leukemia (Ph+ALL) by real-time quantitative reverse transcriptase PCR of e1a2 BCR-ABL1.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Standardisation and consensus guidelines for minimal residual disease assessment in Philadelphia-positive acute lymphoblastic leukemia (Ph + ALL) by real-time quantitative reverse transcriptase PCR of e1a2 BCR-ABL1.

Minimal residual disease (MRD) is a powerful prognostic factor in acute lymphoblastic leukemia (ALL) and is used for patient stratification and treatment decisions, but its precise role in Philadelphia chromosome positive ALL is less clear. This uncertainty results largely from methodological differences relating to the use of real-time quantitative PCR (qRT-PCR) to measure BCR-ABL1 transcript levels for MRD analysis. We here describe the first results by the EURO-MRD consortium on standardization of qRT-PCR for the e1a2 BCR-ABL1 transcript in Ph + ALL, designed to overcome the lack of standardisation of laboratory procedures and data interpretation. Standardised use of EAC primer/probe sets and of centrally prepared plasmid standards had the greatest impact on reducing interlaboratory variability. In QC1 the proportion of analyses with BCR-ABL1/ABL1 ratios within half a log difference were 40/67 (60%) and 52/67 (78%) at 10-3 and 36/67 (53%) and 53/67 (79%) at 10-4BCR-ABL1/ABL1. Standardized RNA extraction, cDNA synthesis and cycler platforms did not improve results further, whereas stringent application of technical criteria for assay quality and uniform criteria for data interpretation and reporting were essential. We provide detailed laboratory recommendations for the standardized MRD analysis in routine diagnostic settings and in multicenter clinical trials for Ph + ALL.

Acute lymphoblastic leukemia with t(4;11) in children 1 year and older: The 'big sister' of the infant disease?

The t(4;11)-positive acute lymphoblastic leukemia (ALL) is a rare disease in children above the age of 1 year. We studied the clinical and biological characteristics in 32 consecutively diagnosed childhood cases (median age 10.0 years, range 1.0-17.1 years). Immunophenotyping revealed a pro-B and a pre-B stage in 24 and eight cases, respectively. IGH genes were rearranged in 84% of leukemias with a predominance of incomplete DJ(H) joints. Whereas IGK-Kde and TCRD rearrangements were rare, TCRG rearrangements were present in 50% of cases and involved mainly Vgamma11 or Vgamma9 together with a Jgamma1.3./2.3 gene segment, an unusual combination among t(4;11)-negative B-cell precursor ALL. Oligoclonality was found in about 30% as assessed by heterogeneous IGH and TCRG rearrangements. Our data are in line with transformation of a precursor cell at an early stage of B-cell development but retaining the potential to differentiate to the pre-B cell stage in vivo. Although a distinct difference between infant and older childhood cases with t(4;11) became evident, no age-related biological features were found within the childhood age group. In contrast to infants with t(4;11)-positive ALL, childhood cases had a relatively low cumulative incidence of relapse of 25% at 3.5 years with BFM-based high-risk protocols.

Optimization of PCR-based minimal residual disease diagnostics for childhood acute lymphoblastic leukemia in a multi-center setting.

Minimal residual disease (MRD) diagnostics is used for treatment stratification in childhood acute lymphoblastic leukemia. We aimed to identify and solve potential problems in multicenter MRD studies to achieve and maintain consistent results between the AIEOP/BFM ALL-2000 MRD laboratories. As the dot-blot hybridization method was replaced by the real-time quantitative polymerase chain reaction (RQ-PCR) method during the treatment protocol, special attention was given to the comparison of MRD data obtained by both methods and to the reproducibility of RQ-PCR data. Evaluation of all key steps in molecular MRD diagnostics identified several pitfalls that resulted in discordant MRD results. In particular, guidelines for RQ-PCR data interpretation appeared to be crucial for obtaining concordant MRD results. The experimental variation of the RQ-PCR was generally less than three-fold, but logically became larger at low MRD levels below the reproducible sensitivity of the assay (<10(-4)). Finally, MRD data obtained by dot-blot hybridization were comparable to those obtained by RQ-PCR analysis (r(2)=0.74). In conclusion, MRD diagnostics using RQ-PCR analysis of immunoglobulin/T-cell receptor gene rearrangements is feasible in multicenter studies but requires standardization; particularly strict guidelines for interpretation of RQ-PCR data are required. We further recommend regular quality control for laboratories performing MRD diagnostics in international treatment protocols.

Analysis of minimal residual disease by Ig/TCR gene rearrangements: guidelines for interpretation of real-time quantitative PCR data.

Most modern treatment protocols for acute lymphoblastic leukaemia (ALL) include the analysis of minimal residual disease (MRD). To ensure comparable MRD results between different MRD-polymerase chain reaction (PCR) laboratories, standardization and quality control are essential. The European Study Group on MRD detection in ALL (ESG-MRD-ALL), consisting of 30 MRD-PCR laboratories worldwide, has developed guidelines for the interpretation of real-time quantitative PCR-based MRD data. The application of these guidelines ensures identical interpretation of MRD data between different laboratories of the same MRD-based clinical protocol. Furthermore, the ESG-MRD-ALL guidelines will facilitate the comparison of MRD data obtained in different treatment protocols, including those with new drugs.

Characterization of a reference material for BCR-ABL (M-BCR) mRNA quantitation by real-time amplification assays: towards new standards for gene expression measurements.

Monitoring of BCR-ABL transcripts has become established practice in the management of chronic myeloid leukemia. However, nucleic acid amplification techniques are prone to variations which limit the reliability of real-time quantitative PCR (RQ-PCR) for clinical decision making, highlighting the need for standardization of assays and reporting of minimal residual disease (MRD) data. We evaluated a lyophilized preparation of a leukemic cell line (K562) as a potential quality control reagent. This was found to be relatively stable, yielding comparable respective levels of ABL, GUS and BCR-ABL transcripts as determined by RQ-PCR before and after accelerated degradation experiments as well as following 5 years storage at -20 degrees C. Vials of freeze-dried cells were sent at ambient temperature to 22 laboratories on four continents, with RQ-PCR analyses detecting BCR-ABL transcripts at levels comparable to those observed in primary patient samples. Our results suggest that freeze-dried cells can be used as quality control reagents with a range of analytical instrumentations and could enable the development of urgently needed international standards simulating clinically relevant levels of MRD.

The MLL recombinome of acute leukemias in 2017.

Chromosomal rearrangements of the human MLL/KMT2A gene are associated with infant, pediatric, adult and therapy-induced acute leukemias. Here we present the data obtained from 2345 acute leukemia patients. Genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) were determined and 11 novel TPGs were identified. Thus, a total of 135 different MLL rearrangements have been identified so far, of which 94 TPGs are now characterized at the molecular level. In all, 35 out of these 94 TPGs occur recurrently, but only 9 specific gene fusions account for more than 90% of all illegitimate recombinations of the MLL gene. We observed an age-dependent breakpoint shift with breakpoints localizing within MLL intron 11 associated with acute lymphoblastic leukemia and younger patients, while breakpoints in MLL intron 9 predominate in AML or older patients. The molecular characterization of MLL breakpoints suggests different etiologies in the different age groups and allows the correlation of functional domains of the MLL gene with clinical outcome. This study provides a comprehensive analysis of the MLL recombinome in acute leukemia and demonstrates that the establishment of patient-specific chromosomal fusion sites allows the design of specific PCR primers for minimal residual disease analyses for all patients.

Quantitative assessment of minimal residual disease in acute myeloid leukemia carrying nucleophosmin (NPM1) gene mutations.

Mutations in exon 12 of the nucleophosmin (NPM1) gene occur in about 60% of adult AML with normal karyotype. By exploiting a specific feature of NPM1 mutants, that is insertion at residue 956 or deletion/insertion at residue 960, we developed highly sensitive, real-time quantitative (RQ) polymerase chain reaction (PCR) assays, either in DNA or RNA, that are specific for various NPM1 mutations. In all 13 AML patients carrying NPM1 mutations at diagnosis, cDNA RQ-PCR showed >30 000 copies of NPM1-mutated transcript. A small or no decrease in copies was observed in three patients showing partial or no response to induction therapy. The number of NPM1-mutated copies was markedly reduced in 10 patients achieving complete hematological remission (five cases: <100 copies; five cases: 580-5046 copies). In four patients studied at different time intervals, the number of NPM1 copies closely correlated with clinical status and predicted impending hematological relapse in two. Thus, reliable, sensitive RQ-PCR assays for NPM1 mutations can now monitor and quantify MRD in AML patients with normal karyotype and NPM1 gene mutations.

Consensus guidelines for microarray gene expression analyses in leukemia from three European leukemia networks.

A plethora of studies have documented that gene expression profiling using DNA microarrays for various types of hematological malignancies provides novel information, which may have diagnostic and prognostic implications. However, to successfully use microarrays for this purpose, the quality and reproducibility of the whole procedure need to be guaranteed. Critical steps of the method are handling, processing and storage of the leukemic sample, purification of tumor cells (or lack thereof), RNA extraction methods, quality control of RNA, labeling techniques, hybridization, washing, scanning, spot filtering, normalization and initial interpretation, and finally the biostatistical analysis. These items have been extensively discussed and evaluated in different multi-center quality rounds within the three networks, that is, I-BFM-SG, the German Competence Network 'Acute and Chronic Leukemias' and the European LeukemiaNet. Based on the exchange of knowledge and experience between the three networks over the last few years, we have formulated guidelines for performing microarray experiments in leukemia. We confine ourselves to leukemias, but many of these requirements also apply to lymphomas or other clinical samples, including solid tumors.

Characterization of children with FLT3-ITD acute myeloid leukemia: a report from the AIEOP AML-2002 study group.

Recurrent molecular markers have been routinely used in acute myeloid leukemia (AML) for risk assessment at diagnosis, whereas their post-induction monitoring still represents a debated issue. We evaluated the prognostic value and biological impact of minimal residual disease (MRD) and of the allelic ratio (AR) of FLT3-internal-tandem duplication (ITD) in childhood AML. We retrospectively screened 494 children with de novo AML for FLT3-ITD mutation, identifying 54 harboring the mutation; 51% of them presented high ITD-AR at diagnosis and had worse event-free survival (EFS, 19.2 versus 63.5% for low ITD-AR, <0.05). Forty-one percent of children with high levels of MRD after the 1st induction course, measured by a patient-specific real-time-PCR, had worse EFS (22.2 versus 59.4% in low-MRD patients, P<0.05). Next, we correlated these parameters with gene expression, showing that patients with high ITD-AR or persistent MRD had characteristic expression profiles with deregulated genes involved in methylation and acetylation. Moreover, patients with high CyclinA1 expression presented an unfavorable EFS (20.3 versus 51.2% in low CyclinA1 group, P<0.01). Our results suggest that ITD-AR levels and molecular MRD should be considered in planning clinical management of FLT3-ITD patients. Different transcriptional activation of epigenetic and oncogenic profiles may explain variability in outcome among these patients, for whom novel therapeutic approaches are desirable.

The histone deacetylase inhibitor givinostat (ITF2357) exhibits potent anti-tumor activity against CRLF2-rearranged BCP-ALL.

Leukemias bearing CRLF2 and JAK2 gene alterations are characterized by aberrant JAK/STAT signaling and poor prognosis. The HDAC inhibitor givinostat/ITF2357 has been shown to exert anti-neoplastic activity against both systemic juvenile idiopathic arthritis and myeloproliferative neoplasms through inhibition of the JAK/STAT pathway. These findings led us to hypothesize that givinostat might also act against CRLF2-rearranged BCP-ALL, which lack effective therapies. Here, we found that givinostat inhibited proliferation and induced apoptosis of BCP-ALL CRLF2-rearranged cell lines, positive for exon 16 JAK2 mutations. Likewise, givinostat killed primary cells, but not their normal hematopoietic counterparts, from patients carrying CRLF2 rearrangements. At low doses, givinostat downregulated the expression of genes belonging to the JAK/STAT pathway and inhibited STAT5 phosphorylation. In vivo, givinostat significantly reduced engraftment of human blasts in patient-derived xenograft models of CRLF2-positive BCP-ALL. Importantly, givinostat killed ruxolitinib-resistant cells and potentiated the effect of current chemotherapy. Thus, givinostat in combination with conventional chemotherapy may represent an effective therapeutic option for these difficult-to-treat subsets of ALL. Lastly, the selective killing of cancer cells by givinostat may allow the design of reduced intensity regimens in CRLF2-rearranged Down syndrome-associated BCP-ALL patients with an overall benefit in terms of both toxicity and related complications.

Drug-induced immunophenotypic modulation in childhood ALL: implications for minimal residual disease detection.

Assessment of minimal residual disease (MRD) by flow cytometry is considered to be based on the reproducibility of the leukemic immunophenotype detected at diagnosis. However, we previously noticed modulation of surface antigen expression in acute lymphoblastic leukemia (ALL) during the early treatment. Hence, we investigated this in 30 children with B-cell precursor ALL consecutively enrolled in the AIEOP-BFM ALL 2000 protocol. Quantitative expression of seven antigens useful in MRD monitoring was studied at diagnosis and compared to that measured at different time points of remission induction therapy. Downmodulation in the expression of CD10 and CD34 occurred at follow-up. By contrast, upmodulation of CD19, CD20, CD45RA, and CD11a was observed, while the expression of CD58 remained stable. Despite this, we could unambiguously discriminate leukemic cells from normal residual B cells. This holds true when bone marrow (BM) samples from similarly treated T-ALL patients, but not from healthy donors, were used as reference. Our results indicate that immunophenotypic modulation occurs in ALL during the early phases of BFM-type protocols. However, the accuracy of MRD detection by flow cytometry seems not negatively affected if adequate analysis protocols are employed. Investigators should take this phenomenon into account in order to avoid pitfalls in flow cytometric MRD studies.

The paired box domain gene PAX5 is fused to ETV6/TEL in an acute lymphoblastic leukemia case.

The PAX5 gene, encoding the B-cell-specific activator protein, is a critical determinant of commitment to the B-lymphocyte pathway. This gene, mapped at 9p13, is juxtaposed to the immunoglobulin heavy chain (IgH) gene as a result of the t(9;14)(p13;q32), a rare but recurring translocation found in a subset of B-cell non-Hodgkin's lymphoma cases. In all of these, this translocation results in deregulated expression of the gene product because of the proximity of IgH. We present here the molecular characterization of a previously reported acute lymphoblastic leukemia case carrying a t(9;12)(q11;p13) translocation. Using 5' rapid amplification of cDNA ends PCR, a novel chimeric transcript was identified that contained the NH(2)-terminal region of PAX5 and most of the ETV6/TEL gene on 12p13. According to the fusion transcript, the resulting chimeric protein would retain the PAX5 paired-box domain and both the helix-loop-helix and DNA binding domains of TEL. Thus, it is reasonable to hypothesize that this protein could act as an aberrant transcription factor. This is the first report of PAX5 rearrangement in a human malignancy resulting in a chimeric transcript.

Influence of Light-curing Parameters on Biofilm Development and Flexural Strength of a Silorane-based Composite.

OBJECTIVES: The aim of this study was to evaluate the differences in biological and mechanical performances of a silorane-based and a methacrylate-based composite. Another aim was to assess the influence of light-curing time and light-curing intensity on in vitro biofilm formation and flexural strength of the two tested composites. METHODS: Experiment 1: 432 specimens obtained from a silorane-based composite and from a standard methacrylate-based composite were divided into six groups and light-cured for 10, 20, 30, 40, 60, or 80 seconds, using one of two light-curing intensities, 400 mW/cm(2) or 800 mW/cm(2). At 24 hours, a monospecific Streptococcus mutans biofilm adherent to the surfaces of the samples was obtained. Then, a colorimetric technique (MTT assay) was used to evaluate the adherent viable biomass. Two samples per group were observed using confocal laser scanning microscopy. Analysis of variance (ANOVA) and Tukey tests were used to analyze the results (p<0.05). Experiment 2: 192 bar-shaped specimens were obtained and light-cured as in the previous experiment. A three-point bend test using a universal testing machine was performed to obtain flexural strength values. ANOVA and Tukey tests were used to analyze the results (p<0.05). RESULTS: In experiment 1, a highly significant difference (p<0.0001) in biofilm development was shown between silorane-based and methacrylate-based composites. In fact, the silorane-based composite exhibited better biological performance. Significant differences were also found between the two light-curing intensities (p<0.018) and for curing times (p<0.0001): silorane-based composite light-cured for 80 seconds at 800 mW/cm(2) light-curing intensity showed the lowest biofilm development. In experiment 2, a significant difference in flexural strength (p<0.0318) was only found between the different composites. Nevertheless, both resin composites showed flexural strength values in accordance with International Organization for Standardization guidelines even after 10 seconds of light-curing time. CONCLUSIONS: Silorane-based composite was less prone to biofilm development compared with a methacrylate-based composite. Acceptable flexural strength values for both composites were obtained after 10 seconds of light-curing time.