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SETBP1 mutations are found in various clonal myeloid disorders. However, it is unclear whether they can initiate leukemia, because SETBP1 mutations typically appear as later events during oncogenesis. To answer this question, we generated a mouse model expressing mutated SETBP1 in hematopoietic tissue: this model showed profound alterations in the differentiation program of hematopoietic progenitors and developed a myeloid neoplasm with megakaryocytic dysplasia, splenomegaly, and bone marrow fibrosis, prompting us to investigate SETBP1 mutations in a cohort of 36 triple-negative primary myelofibrosis (TN-PMF) cases. We identified 2 distinct subgroups, one carrying SETBP1 mutations and the other completely devoid of somatic variants. Clinically, a striking difference in disease aggressiveness was noted, with patients with SETBP1 mutation showing a much worse clinical course. In contrast to myelodysplastic/myeloproliferative neoplasms, in which SETBP1 mutations are mostly found as a late clonal event, single-cell clonal hierarchy reconstruction in 3 patients with TN-PMF from our cohort revealed SETBP1 to be a very early event, suggesting that the phenotype of the different SETBP1+ disorders may be shaped by the opposite hierarchy of the same clonal SETBP1 variants.

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.

A group of 27 patients diagnosed with metastatic triple-negative breast cancer (mTNBC) was randomly distributed into two groups and underwent different lines of metronomic treatment (mCHT). The former group (N 14) received first-line mCHT and showed a higher overall survival rate than the second group (N 13), which underwent second-line mCHT. Analysis of one patient still alive from the first group, diagnosed with mTNBC in 2019, showed a complete metabolic response (CMR) after a composite approach implicating first-line mCHT followed by second-line epirubicin and third-line nab-paclitaxel, and was chosen for subsequent molecular characterization. We found altered expression in the cancer stemness-associated gene NOTCH-1 and its corresponding protein. Additionally, we found changes in the expression of oncogenes, such as MYC and AKT, along with their respective proteins. Overall, our data suggest that a first-line treatment with mCHT followed by MTD might be effective by negatively regulating stemness traits usually associated with the emergence of drug resistance.

Kidney transplantation is the best treatment for end-stage renal disease since it offers the greatest survival benefit compared to dialysis. The gap between the number of renal transplants performed and the number of patients awaiting renal transplants leads to a steadily increasing pressure on the scientific community. Kidney preimplantation biopsy is used as a component of the evaluation of organ quality before acceptance for transplantation. However, the reliability and predictive value of biopsy data are controversial. Most of the previously proposed predictive models were not associated with graft survival, but what has to be reaffirmed is that histologic examination of kidney tissue can provide an objective window on the state of the organ that cannot be deduced from clinical records and renal functional studies. The balance of evidence indicates that reliable decisions about donor suitability must be made based on the overall picture. This work discusses recent trends that can reduce diagnostic timing and variability among players in the decision-making process that lead to kidney transplants, from the pathologist's perspective.

The assessment of programmed death-ligand 1 (PD-L1) combined positive scoring (CPS) in head and neck squamous cell carcinoma (HNSCC) is challenged by pre-analytical and inter-observer variabilities. An educational program to compare the diagnostic performances between local pathologists and a board of pathologists on 11 challenging cases from different Italian pathology centers stained with PD-L1 immunohistochemistry on a digital pathology platform is reported. A laboratory-developed test (LDT) using both 22C3 (Dako) and SP263 (Ventana) clones on Dako or Ventana platforms was compared with the companion diagnostic (CDx) Dako 22C3 pharm Dx assay. A computational approach was performed to assess possible correlations between stain features and pathologists’ visual assessments. Technical discordances were noted in five cases (LDT vs. CDx, 45%), due to an abnormal nuclear/cytoplasmic diaminobenzidine (DAB) stain in LDT (n = 2, 18%) and due to variation in terms of intensity, dirty background, and DAB droplets (n = 3, 27%). Interpretative discordances were noted in six cases (LDT vs. CDx, 54%). CPS remained unchanged, increased, or decreased from LDT to CDx in three (27%) cases, two (18%) cases, and one (9%) case, respectively, around relevant cutoffs (1 and 20, k = 0.63). Differences noted in DAB intensity/distribution using computational pathology partly explained the LDT vs. CDx differences in two cases (18%). Digital pathology may help in PD-L1 scoring, serving as a second opinion consultation platform in challenging cases. Computational and artificial intelligence tools will improve clinical decision-making and patient outcomes.

The estimation of tumor cellular fraction (TCF) is a crucial step in predictive molecular pathology, representing an entry adequacy criterion also in the next-generation sequencing (NGS) era. However, heterogeneity of quantification practices and inter-pathologist variability hamper the robustness of its evaluation, stressing the need for more reliable results. Here, 121 routine histological samples from non-small cell lung cancer (NSCLC) cases with complete NGS profiling were used to evaluate TCF interobserver variability among three different pathologists (pTCF), developing a computational tool (cTCF) and assessing its reliability vs ground truth (GT) tumor cellularity and potential impact on the final molecular results. Inter-pathologist reproducibility was fair to good, with overall Wk ranging between 0.46 and 0.83 (avg. 0.59). The obtained cTCF was comparable to the GT (p = 0.129, 0.502, and 0.130 for surgical, biopsies, and cell block, respectively) and demonstrated good reliability if elaborated by different pathologists (Wk = 0.9). Overall cTCF was lower as compared to pTCF (30 ± 10 vs 52 ± 19, p < 0.001), with more cases < 20% (17, 14%, p = 0.690), but none containing < 100 cells for the algorithm. Similarities were noted between tumor area estimation and pTCF (36 ± 29, p < 0.001), partly explaining variability in the human assessment of tumor cellularity. Finally, the cTCF allowed a reduction of the copy number variations (CNVs) called (27 vs 29, − 6.9%) with an increase of effective CNVs detection (13 vs 7, + 85.7%), some with potential clinical impact previously undetected with pTCF. An automated computational pipeline (Qupath Analysis of Nuclei from Tumor to Uniform Molecular tests, QuANTUM) has been created and is freely available as a QuPath extension. The computational method used in this study has the potential to improve efficacy and reliability of TCF estimation in NSCLC, with demonstrated impact on the final molecular results.

Children with Down syndrome have an augmented risk for B-cell acute lymphoblastic leukemia (DS-ALL), which is associated with lower survival than in non-DS-ALL. It is known that cytogenetic abnormalities common in childhood ALL are less frequent in DS-ALL, while other genetic aberrancies (ie, CRLF2 overexpression and IKZF1 deletions) are increased. A possible cause for the lower survival of DS-ALL that we herewith evaluated for the first time was the incidence and prognostic value of the Philadelphia-like (Ph-like) profile and the IKZF1plus pattern. These features have been associated with poor outcome in non-DS ALL and therefore introduced in current therapeutic protocols. Forty-six out of 70 DS-ALL patients treated in Italy from 2000 to 2014 displayed Ph-like signature, mostly characterized by CRLF2 (n = 33) and IKZF1 (n = 16) alterations; only 2 cases were positive for ABL-class or PAX5-fusion genes. Moreover, in an Italian and German joint cohort of 134 DS-ALL patients, we observed 18% patients positive for IKZF1plus feature. Ph-like signature and IKZF1 deletion were associated with poor outcome (cumulative incidence of relapse: 27.7 ± 6.8% versus 13 ± 7%; P = 0.04 and 35.2 ± 8.6% versus 17 ± 3.9%; P = 0.007, respectively), which further worsens when IKZF1 deletion was co-occurring with P2RY8::CRLF2, qualifying for the IKZF1plus definition (13/15 patients had an event of relapse or treatment-related death). Notably, ex vivo drug screening revealed sensitivity of IKZF1plus blasts for drugs active against Ph-like ALL such as Birinapant and histone deacetylase inhibitors. We provided data in a large setting of a rare condition (DS-ALL) supporting that these patients, not associated with other high-risk features, need tailored therapeutic strategies.

Introduction: Artificial intelligence (AI) integration in nephropathology has been growing rapidly in recent years, facing several challenges including the wide range of histological techniques used, the low occurrence of certain diseases, and the need for data sharing. This narrative review retraces the history of AI in nephropathology and provides insights into potential future developments. Methods: Electronic searches in PubMed-MEDLINE and Embase were made to extract pertinent articles from the literature. Works about automated image analysis or the application of an AI algorithm on non-neoplastic kidney histological samples were included and analyzed to extract information such as publication year, AI task, and learning type. Prepublication servers and reviews were not included. Results: Seventy-six (76) original research articles were selected. Most of the studies were conducted in the United States in the last 7 years. To date, research has been mainly conducted on relatively easy tasks, like single-stain glomerular segmentation. However, there is a trend towards developing more complex tasks such as glomerular multi-stain classification. Conclusion: Deep learning has been used to identify patterns in complex histopathology data and looks promising for the comprehensive assessment of renal biopsy, through the use of multiple stains and virtual staining techniques. Hybrid and collaborative learning approaches have also been explored to utilize large amounts of unlabeled data. A diverse team of experts, including nephropathologists, computer scientists, and clinicians, is crucial for the development of AI systems for nephropathology. Collaborative efforts among multidisciplinary experts result in clinically relevant and effective AI tools.

Complete elimination of B-cell acute lymphoblastic leukemia (B-ALL) by a risk-adapted primary treatment approach remains a clinical key objective, which fails in up to a third of patients. Recent evidence has implicated subpopulations of B-ALL cells with stem-like features in disease persistence. We hypothesized that microRNA-126, a core regulator of hematopoietic and leukemic stem cells, may resolve intratumor heterogeneity in B-ALL and uncover therapy-resistant subpopulations. We exploited patient-derived xenograft (PDX) models with B-ALL cells transduced with a miR-126 reporter allowing the prospective isolation of miR-126(high) cells for their functional and transcriptional characterization. Discrete miR-126(high) populations, often characterized by MIR126 locus demethylation, were identified in 8/9 PDX models and showed increased repopulation potential, in vivo chemotherapy resistance and hallmarks of quiescence, inflammation and stress-response pathway activation. Cells with a miR-126(high) transcriptional profile were identified as distinct disease subpopulations by single-cell RNA sequencing in diagnosis samples from adult and pediatric B-ALL. Expression of miR-126 and locus methylation were tested in several pediatric and adult B-ALL cohorts, which received standardized treatment. High microRNA-126 levels and locus demethylation at diagnosis associate with suboptimal response to induction chemotherapy (MRD > 0.05% at day +33 or MRD+ at day +78). [Figure not available: see fulltext.].

Biallelic KARS1 mutations cause KARS-related diseases, a rare syndromic condition encompassing central and peripheral nervous system impairment, heart and liver disease, and deafness. KARS1 encodes the t-RNA synthase of lysine, an aminoacyl-tRNA synthetase, involved in different physiological mechanisms (such as angiogenesis, post-translational modifications, translation initiation, autophagy and mitochondrial function). Although patients with immune-hematological abnormalities have been individually described, results have not been collectively discussed and functional studies investigating how KARS1 mutations affect B cells have not been performed. Here, we describe one patient with severe developmental delay, sensoneurinal deafness, acute disseminated encephalomyelitis, hypogammaglobulinemia and recurrent infections. Pathogenic biallelic KARS1 variants (Phe291Val/ Pro499Leu) were associated with impaired B cell metabolism (decreased mitochondrial numbers and activity). All published cases of KARS-related diseases were identified. The corresponding authors and researchers involved in the diagnosis of inborn errors of immunity or genetic syndromes were contacted to obtain up-to-date clinical and immunological information. Seventeen patients with KARS-related diseases were identified. Recurrent/severe infections (9/17) and B cell abnormalities (either B cell lymphopenia [3/9], hypogammaglobulinemia [either IgG, IgA or IgM; 6/15] or impaired vaccine responses [4/7]) were frequently reported. Immunoglobulin replacement therapy was given in five patients. Full immunological assessment is warranted in these patients, who may require detailed investigation and specific supportive treatment.

Background: The outcome of children with Philadelphia chromosome-positive (Ph-positive) acute lymphoblastic leukaemia significantly improved with the combination of imatinib and intensive chemotherapy. We aimed to investigate the efficacy of dasatinib, a second-generation ABL-class inhibitor, with intensive chemotherapy in children with newly diagnosed Ph-positive acute lymphoblastic leukaemia. Methods: CA180-372/COG AALL1122 was a joint Children's Oncology Group (COG) and European intergroup study of post-induction treatment of Ph-positive acute lymphoblastic leukaemia (EsPhALL) open-label, single-arm, phase 2 study. Eligible patients (aged >1 year to <18 years) with newly diagnosed Ph-positive acute lymphoblastic leukaemia and performance status of at least 60% received EsPhALL chemotherapy plus dasatinib 60 mg/m2 orally once daily from day 15 of induction. Patients with minimal residual disease of at least 0·05% after induction 1B or who were positive for minimal residual disease after the three consolidation blocks were classified as high risk and allocated to receive haematopoietic stem-cell transplantation (HSCT) in first complete remission. The remaining patients were considered standard risk and received chemotherapy plus dasatinib for 2 years. The primary endpoint was the 3-year event-free survival of dasatinib plus chemotherapy compared with external historical controls. The trial was considered positive if one of the following conditions was met: superiority over chemotherapy alone in the AIEOP-BFM 2000 high-risk group; or non-inferiority (with a margin of –5%) or superiority to imatinib plus chemotherapy in the EsPhALL 2010 cohort. All participants who received at least one dose of dasatinib were included in the safety and efficacy analyses. This trial was registered with ClinicalTrials.gov, NCT01460160, and recruitment is closed. Findings: Between March 13, 2012, and May 27, 2014, 109 patients were enrolled at 69 sites (including 51 COG sites in t

About 10% of children with cancer carry a pathogenic mutation in genes predisposing to tumourigenesis. These mutations are being identified more and more frequently thanks to the use of next-generation sequencing techniques in the diagnostic work-up of cancer, and their integration into clinical practice is a new challenge for oncogenetics. The diagnosis of a cancer predisposing syndrome (CPS) has important implications for the management of the patient, as well as for the patient's family, who may also include potential carriers of the identified predisposing mutation. Clinical criteria have been developed to help clinicians suspect a predisposing condition: attention should be given to the family and patient’s medical history, to specific clinical signs and to specific histopathological tumour subtype. A multidisciplinary team with specific training is essential for the complex clinical management of these conditions.

During the submission of the accepted manuscript, first names and surnames had been inverted. The list of authors has been corrected above. The original version has been corrected.

The most frequent genetic aberration leading to infant ALL (iALL) is the chromosomal translocation t(4;11), generating the fusion oncogenes KMT2A:AFF1 and AFF1:KMT2A, respectively. KMT2A-r iALL displays a dismal prognosis through high relapse rates and relapse-associated mortality. Relapse occurs frequently despite ongoing chemotherapy and without the accumulation of secondary mutations. A rational explanation for the observed chemo-resistance and satisfactory treatment options remain to be elucidated. We found that elevated ICOSLG expression level at diagnosis was associated with inferior event free survival (EFS) in a cohort of 43 patients with t(4;-11) iALL and that a cohort of 18 patients with iALL at relapse displayed strongly increased ICOSLG expression. Furthermore, co-culturing t(4;11) ALL cells (ICOSLGhi) with primary T-cells resulted in the development of Tregs. This was impaired through treatment with a neutralizing ICOSLG antibody. These findings imply ICOSLG (1) as a relapse-predicting biomarker, and (2) as a therapeutic target involved in a potential immune evasion relapse-mechanism of infant t(4;11) ALL.

Somatic loss of function mutations in cohesin genes are frequently associated with various cancer types, while cohesin disruption in the germline causes cohesinopathies such as Cornelia‐de‐ Lange syndrome (CdLS). Here, we present the discovery of a recurrent heterozygous RAD21 germline aberration at amino acid position 298 (p.P298S/A) identified in three children with lymphoblastic leukemia or lymphoma in a total dataset of 482 pediatric cancer patients. While RAD21 p.P298S/A did not disrupt the formation of the cohesin complex, it altered RAD21 gene expression, DNA damage response and primary patient fibroblasts showed increased G2/M arrest after irradiation and Mitomycin‐C treatment. Subsequent single‐cell RNA‐sequencing analysis of healthy human bone marrow confirmed the upregulation of distinct cohesin gene patterns during hematopoiesis, highlighting the importance of RAD21 expression within proliferating B‐ and T‐cells. Our clinical and functional data therefore suggest that RAD21 germline variants can predispose to childhood lymphoblastic leukemia or lymphoma without displaying a CdLS phenotype.

B-cell acute lymphoblastic leukemia (B-ALL) is the commonest childhood cancer. High hyperdiploidy (HHD) identifies the most frequent cytogenetic subgroup in childhood B-ALL. Although hyperdiploidy represents an important prognostic factor in childhood B-ALL, the specific chromosome gains with prognostic value in HHD-B-ALL remain controversial, and the current knowledge about the hierarchy of chromosome gains, clonal heterogeneity and chromosomal instability in HHD-B-ALL remains very limited. We applied automated sequential-iFISH coupled with single-cell computational modeling to identify the specific chromosomal gains of the eight typically gained chromosomes in a large cohort of 72 primary diagnostic (DX, n = 62) and matched relapse (REL, n = 10) samples from HHD-B-ALL patients with either favorable or unfavorable clinical outcome in order to characterize the clonal heterogeneity, specific chromosome gains and clonal evolution. Our data show a high degree of clonal heterogeneity and a hierarchical order of chromosome gains in DX samples of HHD-B-ALL. The rates of specific chromosome gains and clonal heterogeneity found in DX samples differ between HHD-B-ALL patients with favorable or unfavorable clinical outcome. In fact, our comprehensive analyses at DX using a computationally defined risk predictor revealed low levels of trisomies +18+10 and low levels of clonal heterogeneity as robust relapse risk factors in minimal residual disease (MRD)-negative childhood HHD-B-ALL patients: relapse-free survival beyond 5 years: 22.1% versus 87.9%, P < 0.0001 and 33.3% versus 80%, P < 0.0001, respectively. Moreover, longitudinal analysis of matched DX-REL HHD-B-ALL samples revealed distinct patterns of clonal evolution at relapse. Our study offers a reliable prognostic sub-stratification of pediatric MRD-negative HHD-B-ALL patients.

The Rubinstein–Taybi syndrome (RSTS) is a rare developmental disorder characterized by craniofacial dysmorphisms, broad thumbs and toes, intellectual disability, growth deficiency, and recurrent infections. Mutations in the cyclic adenosine monophosphate response element-binding protein (CREB)-binding protein (CREBBP) or in the E1A-associated protein p300 (EP300) genes have been demonstrated in 55% (RSTS1) and up to 8% of the patients (RSTS2), respectively. Dysfunction of immune response has been reported in a subgroup of individuals with RSTS. Here we characterize two patients carrying the same EP300 variant and distinctive RSTS features (including congenital heart abnormalities, short stature, feeding problems, and gastroesophageal reflux). Whole exome sequencing did not support a dual molecular diagnosis hypothesis. Nonetheless, patients showed distinct clinical manifestations and immunological features. The most severe phenotype was associated with reduced T-cell production and diversity. This latter feature was confirmed in a control group of four RSTS patients.

Introduction: One-quarter of the relapses in children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) occur very early (within 18 months, before completion of treatment), and prognosis in these patients is worse compared to cases that relapse after treatment has ended. Methods: In this study, we performed a genomic analysis of diagnosis–relapse pairs of 12 children who relapsed very early, followed by a deep-sequencing validation of all identified mutations. In addition, we included one case with a good initial treatment response and on-treatment relapse at the end of upfront therapy. Results: We observed a dynamic clonal evolution in all cases, with relapse almost exclusively originating from a subclone at diagnosis. We identified several driver mutations that may have influenced the outgrowth of a minor clone at diagnosis to become the major clone at relapse. For example, a minimal residual disease (MRD)-based standard-risk patient with ETV6-RUNX1-positive leukemia developed a relapse from a TP53-mutated subclone after loss of the wildtype allele. Furthermore, two patients with TCF3-PBX1-positive leukemia that developed a very early relapse carried E1099K WHSC1 mutations at diagnosis, a hotspot mutation that was recurrently encountered in other very early TCF3-PBX1-positive leukemia relapses as well. In addition to alterations in known relapse drivers, we found two cases with truncating mutations in the cohesin gene RAD21. Conclusion: Comprehensive genomic characterization of diagnosis–relapse pairs shows that very early relapses in BCP-ALL frequently arise from minor subclones at diagnosis. A detailed understanding of the therapeutic pressure driving these events may aid the development of improved therapies.

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.

Comparison of treatment strategies in de novo pediatric acute lymphoblastic leukemia (ALL) requires standardized measures of efficacy. Key parameters that define disease-related events, including complete remission (CR), treatment failure (TF; not achieving CR), and relapse (loss of CR) require an updated consensus incorporating modern diagnostics. We collected the definitions of CR, TF, and relapse from recent and current pediatric clinical trials for the treatment of ALL, including the key components of response evaluation (timing, anatomic sites, detection methods, and thresholds) and found significant heterogeneity, most notably in the definition of TF. Representatives of the major international ALL clinical trial groups convened to establish consensus definitions. CR should be defined at a time point no earlier than at the end of induction and should include the reduction of blasts below a specific threshold in bone marrow and extramedullary sites, incorporating minimal residual disease (MRD) techniques for marrow evaluations. TF should be defined as failure to achieve CR by a prespecified time point in therapy. Relapse can only be defined in patients who have achieved CR and must include a specific threshold of leukemic cells in the bone marrow confirmed by MRD, the detection of central nervous system leukemia, or documentation of extramedullary disease. Definitions of TF and relapse should harmonize with eligibility criteria for clinical trials in relapsed/refractory ALL. These consensus definitions will enhance the ability to compare outcomes across pediatric ALL trials and facilitate development of future international collaborative trials.

Objective. The use of standardized structured reports (SSR) and suitable terminologies like SNOMED-CT can enhance data retrieval and analysis, fostering large-scale studies and collaboration. However, the still large prevalence of narrative reports in our laboratories warrants alternative and automated labeling approaches. In this project, natural language processing (NLP) methods were used to associate SNOMED-CT codes to structured and unstructured reports from an Italian Digital Pathology Department. Methods. Two NLP-based automatic coding systems (support vector machine, SVM, and long-short term memory, LSTM) were trained and applied to a series of narrative reports. Results. The 1163 cases were tested with both algorithms, showing good performances in terms of accuracy, precision, recall, and F1 score, with SVM showing slightly better performances as compared to LSTM (0.84, 0.87, 0.83, 0.82 vs 0.83, 0.85, 0.83, 0.82, respectively). The integration of an explainability allowed identification of terms and groups of words of importance, enabling fine-tuning, balancing semantic meaning and model performance. Conclusions. AI tools allow the automatic SNOMED-CT labeling of the pathology archives, providing a retrospective fix to the large lack of organization of narrative reports.

Although a great cure rate has been achieved for pediatric BCP-ALL, approximately 15% of patients do not respond to conventional chemotherapy and experience disease relapse. A major effort to improve the cure rates by treatment intensification would result in an undesirable increase in treatment-related toxicity and mortality, raising the need to identify novel therapeutic approaches. High-throughput (HTP) drug screening enables the profiling of patients’ responses in vitro and allows the repurposing of compounds currently used for other diseases, which can be immediately available for clinical application. The aim of this study was to apply HTP drug screening to identify potentially effective compounds for the treatment of pediatric BCP-ALL patients with poor prognosis, such as patients with Down Syndrome (DS) or carrying rearrangements involving PAX5 or KMT2A/MLL genes. Patient-derived Xenografts (PDX) samples from 34 BCP-ALL patients (9 DS CRLF2r, 15 PAX5r, 10 MLLr), 7 human BCP-ALL cell lines and 14 hematopoietic healthy donor samples were screened on a semi-automated HTP drug screening platform using a 174 compound library (FDA/EMA-approved or in preclinical studies). We identified 9 compounds active against BCP-ALL (ABT-199/venetoclax, AUY922/luminespib, dexamethasone, EC144, JQ1, NVP-HSP990, paclitaxel, PF-04929113 and vincristine), but sparing normal cells. Ex vivo validations confirmed that the BCL2 inhibitor venetoclax exerts an anti-leukemic effect against all three ALL subgroups at nanomolar concentrations. Overall, this study points out the benefit of HTP screening application for drug repurposing to allow the identification of effective and clinically translatable therapeutic agents for difficult-to-treat childhood BCP-ALL subgroups.

Renal amyloidosis is a rare condition caused by the progressive accumulation of misfolded proteins within glomeruli, vessels, and interstitium, causing functional decline and requiring prompt treatment due to its significant morbidity and mortality. Congo red (CR) stain on renal biopsy samples is the gold standard for diagnosis, but the need for polarized light is limiting the digitization of this nephropathology field. This study explores the feasibility and reliability of CR fluorescence on virtual slides (CRFvs) in evaluating the diagnostic accuracy and proposing an automated digital pipeline for its assessment. Whole-slide images from 154 renal biopsies with CR were scanned through a Texas red fluorescence filter (NanoZoomer S60, Hamamatsu) at the digital Nephropathology Center of the Istituto di Ricovero e Cura a Carattere Scientifico San Gerardo, Monza, Italy, and evaluated double-blinded for the detection and quantification through the amyloid score and a custom ImageJ pipeline was built to automatically detect amyloid-containing regions. Interobserver agreement for CRFvs was optimal (k = 0.90; 95% CI, 0.81-0.98), with even better concordance when consensus-based CRFvs evaluation was compared to the standard CR birefringence (BR) (k = 0.98; 95% CI, 0.93-1). Excellent performance was achieved in the assessment of amyloid score overall by CRFvs (weighted k = 0.70; 95% CI, 0.08-1), especially within the interstitium (weighted k = 0.60; 95% CI, 0.35-0.84), overcoming the misinterpretation of interstitial and capsular collagen BR. The application of an automated digital pathology pipeline (Streamlined Pipeline for Amyloid detection through CR fluorescence Digital Analysis, SPADA) further increased the performance of pathologists, leading to a complete concordance with the standard BR. This study represents an initial step in the validation of CRFvs, demonstrating its general reliability in a digital nephropathology center. The computational method used in this stu

Infant acute myeloid leukemia (AML) is a heterogeneous disease, genetically distinct from its adult counterpart. Chromosomal translocations involving the KMT2A gene (MLL) are especially common in affected infants of less than 1 year of age, and are associated with a dismal prognosis. While these rearrangements are likely to arise in utero, the cell of origin has not been conclusively identified. This knowledge could lead to a better understanding of the biology of the disease and support the identification of new therapeutic vulnerabilities. Over the last few years, important progress in understanding the dynamics of fetal hematopoiesis has been made. Several reports have highlighted how hematopoietic stem cells (HSC) provide little contribution to fetal hematopoiesis, which is instead largely sustained by HSC-independent progenitors. Here, we used conditional Cre-Lox transgenic mouse models to engineer the Mll-Af9 translocation in defined subsets of embryonic hematopoietic progenitors. We show that embryonic hematopoiesis is generally permissive for Mll-Af9-induced leukemic transformation. Surprisingly, the selective introduction of Mll-Af9 in HSC-independent progenitors generated a transplantable myeloid leukemia, whereas it did not when introduced in embryonic HSC-derived cells. Ex vivo engineering of the Mll-Af9 rearrangement in HSC-independent progenitors using a CRISPR/Cas9-based approach resulted in the activation of an aberrant myeloid-biased self-renewal program. Overall, our results demonstrate that HSC-independent hematopoietic progenitors represent a permissive environment for Mll-Af9-induced leukemic transformation, and can likely act as cells of origin of infant AML.

Introduction: Digital pathology can improve the technical and interpretative workflows in nephropathology by creating hub-spoke networks and virtuous collaboration projects among centers in different geographical regions. New high-resolution fast-scanning instruments combined with currently existing equipment were tested in a nephropathology hub to evaluate possible upgrading in the routine processing phases. Methods: The scanning performance of two different instruments (Aperio vs hybrid MIDI II) was evaluated and a comparative quality control check was performed on obtained whole slide images. Results: Both with default and custom settings for light microscopy, MIDI II proved to be faster, with only slightly more time required to prepare the scan and larger final file size as compared to Aperio (p < 0.001). No differences were noted in the number of out-of-focus slides per case (p = 0.75). Regarding immunofluorescence, the new scanner required longer preparation time (p = 0.001) with comparable scanning times and final file size (p = 0.169 and p = 0.177, respectively). Quality control showed differences in 3 quality features related to white background and blurriness (p < 0.001). No major discordances in the final diagnosis were recorded after comparing the report obtained with slides scanned using the two instruments, with only one case (4%) showing minor disagreement. Conclusion: The present report describes the experience of a hub nephropathology center adopting next generation digital pathology tools for the routine assessment of renal biopsies, highlighting the need for a complementary approach towards a philosophy of interoperability.

Aims: Identification and characterisation of monoclonal gammopathies of renal significance (MGRS) is critical for therapeutic purposes. Amyloidosis represents one of the most common forms of MGRS, and renal biopsy remains the gold standard for their classification, although mass spectrometry has shown greater sensitivity in this area. Methods: In the present study, a new in situ proteomic technique, matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI), is investigated as an alternative to conventional laser capture microdissection MS for the characterisation of amyloids. MALDI-MSI was performed on 16 cases (3 lambda light chain amyloidosis (AL), 3 AL kappa, 3 serum amyloid A amyloidosis (SAA), 2 lambda light chain deposition disease (LCDD), 2 challenging amyloid cases and 3 controls). Analysis began with regions of interest labelled by the pathologist, and then automatic segmentation was performed. Results: MALDI-MSI correctly identified and typed cases with known amyloid type (AL kappa, AL lambda and SAA). A 'restricted fingerprint' for amyloid detection composed of apolipoprotein E, serum amyloid protein and apolipoprotein A1 showed the best automatic segmentation performance (area under the curve >0.7). Conclusions: MALDI-MSI correctly assigned minimal/challenging cases of amyloidosis to the correct type (AL lambda) and identified lambda light chains in LCDD cases, highlighting the promising role of MALDI-MSI for amyloid typing.

Rarely, immunophenotypically immature B-cell precursor acute lymphoblastic leukemia (BCP-ALL) carries an immunoglobulin-MYC rearrangement (IG-MYC-r). This can result in diagnostic confusion with Burkitt lymphoma/leukemia and use of individualized treatment schedules of unproven efficacy. Here we compare the molecular characteristics of these conditions and investigate historic clinical outcome data. We identified 90 cases registered in a national BCP-ALL clinical trial/registry. When present, diagnostic material underwent cytogenetic, exome, methylome and transcriptome analyses. The outcomes analyzed were 3-year event-free survival and overall survival. IG-MYC-r was identified in diverse cytogenetic backgrounds, co-existing with either established BCP-ALL-specific abnormalities (high hyperdiploidy, n=3; KMT2A-rearrangement, n=6; iAMP21, n=1; BCR-ABL1, n=1); BCL2/BCL6-rearrangements (n=15); or, most commonly, as the only defining feature (n=64). Within this final group, precursor-like V(D)J breakpoints predominated (8/9) and KRAS mutations were common (5/11). DNA methylation identified a cluster of V(D)J-rearranged cases, clearly distinct from Burkitt leukemia/lymphoma. Children with IG-MYC-r within that subgroup had a 3-year event-free survival of 47% and overall survival of 60%, representing a high-risk BCP-ALL. To develop effective management strategies this group of patients must be allowed access to contemporary, minimal residual disease-adapted, prospective clinical trial protocols.

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.

Although new techniques (i.e., droplet digital-PCR, next-generation sequencing, advanced flow cytometry) are being developed, DNA-based allele-specific real-time quantitative (RQ)-PCR is still the gold standard for sensitive and accurate immunoglobulin/T cell receptor (IG/TR)-based minimal residual disease (MRD) monitoring, allowing the detection of up to 1 leukemic cell in 100,000 normal lymphoid cells. We herewith describe the standard PCR procedure which has been developed and standardized (with minor modification in single labs) through the last 20 years of activity of the EuroMRD Consortium, a volunteer activity of expert laboratories that is continuously providing education, standardization, quality control rounds, and guidelines for interpretation of RQ-PCR data.

The KMT2A/AFF1 rearrangement is associated with an unfavorable prognosis in infant acute lymphocytic leukemia (ALL). Discordant ALL in monozygotic twins is uncommon and represents an attractive resource to evaluate intrauterine environment–genetic interplay in ALL. Mutational and epigenetic profiles were characterized for a discordant KMT2A/AFF1-rearranged infant monozy-gotic twin pair and their parents, and they were compared to three independent KMT2A/AFF1-positive ALL infants, in which the DNA methylation and gene expression profiles were investigated. A de novo Q61H NRAS mutation was detected in the affected twin at diagnosis and backtracked in both twins at birth. The KMT2A/AFF1 rearrangement was absent at birth in both twins. Genetic analyses conducted at birth gave more insights into the timing of the mutation hit. We identified correlations between DNA methylation and gene expression changes for 32 genes in the three independent affected versus remitted patients. The strongest correlations were observed for the RAB32, PDK4, CXCL3, RANBP17, and MACROD2 genes. This epigenetic signature could be a putative target for the development of novel epigenetic-based therapies and could help in explaining the molecular mechanisms characterizing ALL infants with KMT2A/AFF1 fusions.

B cell acute lymphoblastic leukemia (B-ALL) is the most common childhood cancer. As predicted by its prenatal origin, infant B-ALL (iB-ALL) shows an exceptionally silent DNA mutational landscape, suggesting that alternative epigenetic mechanisms may substantially contribute to its leukemogenesis. Here, we have integrated genome-wide DNA methylome and transcriptome data from 69 patients with de novo MLL-rearranged leukemia (MLLr) and non-MLLr iB-ALL leukemia uniformly treated according to the Interfant-99/06 protocol. iB-ALL methylome signatures display a plethora of common and specific alterations associated with chromatin states related to enhancer and transcriptional control in normal hematopoietic cells. DNA methylation, gene expression, and gene coexpression network analyses segregated MLLr away from non-MLLr iB-ALL and identified a coordinated and enriched expression of the AP-1 complex members FOS and JUN and RUNX factors in MLLr iB-ALL, consistent with the significant enrichment of hypomethylated CpGs in these genes. Integrative methylome-transcriptome analysis identified consistent cancer cell vulnerabilities, revealed a robust iB-ALL- specific gene expression-correlating dmCpG signature, and confirmed an epigenetic control of AP-1 and RUNX members in reshaping the molecular network of MLLr iB-ALL. Finally, pharmacological inhibition or functional ablation of AP-1 dramatically impaired MLLr-leukemic growth in vitro and in vivo using MLLr-iB-ALL patient-derived xenografts, providing rationale for new therapeutic avenues in MLLr-iB-ALL.

Chimeric antigen receptor T-cells (CAR T-cells) for the treatment of relapsing/refractory B-cell precursor acute lymphoblastic leukemia have led to exciting clinical results. However, CAR T-cell approaches revealed a potential risk of CD19-/CAR+ leukemic relapse due to inadvertent transduction of leukemia cells. BackgroundMethods We evaluated the impact of a high percentage of leukemia blast contamination in patient-derived starting material (SM) on CAR T-cell drug product (DP) manufacturing. In vitro as well as in vivo models were employed to identify characteristics of the construct associated with better profile of safety in case of inadvertent B-cell leukemia transduction during CAR T-cell manufacturing. Results The presence of large amounts of CD19+ cells in SM did not affect the transduction level of DPs, as well as the CAR T-cell rate of expansion at the end of standard production of 14 days. DPs were deeply characterized by flow cytometry and molecular biology for Ig-rearrangements, showing that the level of B-cell contamination in DPs did not correlate with the percentage of CD19+ cells in SM, in the studied patient cohort. Moreover, we investigated whether CAR design may affect the control of CAR+ leukemia cells. We provided evidences that CAR.CD19 short linker (SL) prevents complete epitope masking in CD19+CAR+ leukemia cells and we demonstrated in vitro and in vivo that CD19 +CAR(SL)+leukemic cells are killed by CAR.CD19 T-cells. Conclusions Taken together, these data suggest that a VL-VH SL may result in a safe CAR-T product, even when manufacturing starts from biological materials characterized by heavy contamination of leukemia blasts.

Current diagnostic standards for lymphoproliferative disorders include multiple tests for detection of clonal immunoglobulin (IG) and/or T-cell receptor (TCR) rearrangements, translocations, copy-number alterations (CNAs), and somatic mutations. The EuroClonality-NGS DNA Capture (EuroClonality-NDC) assay was designed as an integrated tool to characterize these alterations by capturing IGH switch regions along with variable, diversity, and joining genes of all IG and TCR loci in addition to clinically relevant genes for CNA and mutation analysis. Diagnostic performance against standard-of-care clinical testing was assessed in a cohort of 280 B-and T-cell malignancies from 10 European laboratories, including 88 formalin-fixed paraffin-embedded samples and 21 reactive lesions. DNA samples were subjected to the EuroClonality-NDC protocol in 7 EuroClonality-NGS laboratories and analyzed using a bespoke bioinformatic pipeline. The EuroClonality-NDC assay detected B-cell clonality in 191 (97%) of 197 B-cell malignancies and T-cell clonality in 71 (97%) of 73 T-cell malignancies. Limit of detection (LOD) for IG/TCR rearrangements was established at 5% using cell line blends. Chromosomal translocations were detected in 145 (95%) of 152 cases known to be positive. CNAs were validated for immunogenetic and oncogenetic regions, highlighting their novel role in confirming clonality in somatically hypermutated cases. Single-nucleotide variant LOD was determined as 4% allele frequency, and an orthogonal validation using 32 samples resulted in 98% concordance. The EuroClonality-NDC assay is a robust tool providing a single end-To-end workflow for simultaneous detection of B-and T-cell clonality, translocations, CNAs, and sequence variants.