Acute myeloid leukemia (AML) is a heterogeneous malignancy of hematopoietic progenitors and the most common acute leukemia in adults. Adhesion molecules involved in leukocyte trafficking are believed to contribute to extramedullary AML (EM-AML), a manifestation associated with aggressive phenotype, poor prognosis, and low remission rates. Despite its clinical impact, the molecular mechanisms driving EM localization remain largely unclear. AML blasts from seven patients were engrafted into NOD SCID/IL2Rγ-null mice to generate patient-derived xenograft (PDX) models. Surface markers involved in leukocyte trafficking (CD56, CXCR4, CCR7) and EM-AML (CD117) were analyzed by flow cytometry and immunohistochemistry. Paired samples from bone marrow/peripheral blood and EM sites were compared.EM infiltration occurred in multiple organs—spleen, liver, meninges, lungs—with each PDX showing a distinct pattern, suggesting blast-intrinsic organ tropism. Blasts from different tissues in the same mouse showed distinct surface marker profiles, stable across passages. For example, in PDX#1, CXCR4+/CCR7– blasts predominated in bone marrow and spleen, whereas double-negative cells localized in the lung (Fig. 1a).These profiles were validated in human samples; in one case of leukemic meningitis, meningeal blasts expressed bright CCR7, unlike circulating blasts, which had low CCR7 expression (Fig. 1b). Similar differences were seen in leukemia cutis versus BM pairs (Fig. 1c).Bioinformatic analysis confirmed CD117, CD56, CXCR4, and CCR7 expression across all ICC 2022 AML categories, with CD56 enriched in t(8;21) AML, a subtype often linked to EM involvement. This translational study shows, from xenograft to patient, that EM-AML is driven by phenotypically distinct leukemic subpopulations with organ-specific marker patterns. These findings support future therapies targeting EM tropism in AML.

Richter’s transformation (RT) is an aggressive lymphoma occurring upon progression from chronic lymphocytic leukemia (CLL). Despite advances in deciphering the RT genetic architecture, the mechanisms driving this disease remain unknown. BCOR disruptive mutations were found in CLL and frequently associated with NOTCH1 aberrations, a common feature in CLL and RT. We engineered mice to knock-out Bcor in B and CLL cells of Eμ-TCL1 mice. Bcor loss resulted in alterations of the B cell compartment and favored CLL transformation into an aggressive lymphoma with reduced survival in Eμ-TCL1 mice. RNA-sequencing demonstrated a molecular signature reminiscent of human RT and implied the involvement of the T cell tumour microenvironment in the disease onset. Bcor deficiency was associated with Notch1 activation in splenic CD19 + CD5+ cells to accelerate Eμ-TCL1 mice lymphoproliferation. Notch1 inhibition progressively reduced circulating CD19+ CD5+ and RT cells infiltrating the spleen of diseased mice with concomitant reduction of PD-1 expressing T cells and improved survival. Our data demonstrated an interplay between the tumour suppressor activity of Bcor and Notch1 in RT pathogenesis with potential for tumour targeting. This model represented a new platform to uncover promising alternatives for this incurable tumour.

Chimeric antigen receptor (CAR) T cells represent a revolutionary immunotherapy that allows specific tumor recognition by a unique single-chain fragment variable (scFv) derived from monoclonal antibodies (mAbs). scFv selection is consequently a fundamental step for CAR construction, to ensure accurate and effective CAR signaling toward tumor antigen binding. However, conventional in vitro and in vivo biological approaches to compare different scFv-derived CARs are expensive and labor-intensive. With the aim to predict the finest scFv binding before CAR-T cell engineering, we performed artificial intelligence (AI)-guided molecular docking and steered molecular dynamics analysis of different anti-CD30 mAb clones. Virtual computational scFv screening showed comparable results to surface plasmon resonance (SPR) and functional CAR-T cell in vitro and in vivo assays, respectively, in terms of binding capacity and anti-tumor efficacy. The proposed fast and low-cost in silico analysis has the potential to advance the development of novel CAR constructs, with a substantial impact on reducing time, costs, and the need for laboratory animal use.

Background: BCL6 co-repressor (BCOR) is a transcription factor involved in various biological processes including lymphoid development. BCOR disruptive mutations were found in up to 2% of CLL and frequently associated with aberrations of NOTCH1, one of the most common genetic alterations with poor prognosis in CLL and Richter transformation (RT). Recent evidence also indicates that BCOR is involved in NOTCH signalling suppression during embryogenesis. These data indicate the need for further investigation on the role of BCOR mutation and its interplay with NOTCH1 in CLL pathogenesis. Aims: We aim to elucidate the impact of Bcor deficiency in CLL and RT, focusing on the role of active NOTCH1 signalling. Methods: We used a conditional knockout mouse of Bcor (Sportoletti et al, Leukemia 2021) crossed with CD19-Cre mice, to specifically delete Bcor in B-cells, and with the Eμ-TCL1 mouse model of CLL. Mice were characterized for disease phenotype (using flow-cytometry and histological analyses), overall survival and drug response. NOTCH1 activity was assessed by western blot (WB) for the NOTCH1-intracellular domain (NOTCH1-IC) and HES1 expression. Results: B-cell restricted loss of Bcor in Eµ-TCL1 mice significantly expanded leukemic CD5+CD19+ cells in the peripheral blood (PB; p<0.05), spleen (p<0.01), bone marrow (BM; p<0.001), compared to Eµ-TCL1 control mice. No leukemic cells were found in BcorCD19Cre+ mice. Cellular changes resulted in a poorer survival of double mutant mice compared to single-mutant and wild-type (WT) controls (median survival of 334 days vs unreached in BcorCD19Cre+;Eμ-TCL1 vs the other groups, respectively). Adoptive transfer of spleen cells (CD5+CD19+ leukemic burden >50%) resulted in a more rapidly lethal disease in recipient of BcorCD19Cre+;Eμ-TCL1 compared to Eμ-TCL1 cells (median survival of 37.5 vs 74 days, respectively, p<0.05; Figure1A). At necropsy, double mutant mice presented massive splenomegaly, whose histopathological examination revealed a diffuse infiltration by high-mitotic blastoid cells, significantly increased in size, defining a high-grade lymphoid malignancy distinct from the leukemia of Eμ-TCL1 counterparts (Fig.1B). In order to gain mechanistic insight relating to the phenotypic observations, we measured NOTCH1 activity. BcorCD19Cre+;Eμ-TCL1 splenic CD5+CD19+ cells showed significantly increased levels of the active NOTCH1-IC and the up-regulation of its direct target HES1, compared to leukemic cells from Eμ-TCL1 mice and CD19+ cells from non-leukemic BcorCD19Cre+ and WT mice used as control (Fig.1C). In vivo treatment with the NOTCH1 inhibitor Bepridil resulted in a significant growth delay of CD5+CD19+ cells in the PB of mice transplanted with BcorCD19Cre+;Eμ-TCL1 leukemic cells compared to vehicle. At sacrifice, Bepridil caused a significant reduction of spleen dimensions and CD5+CD19+ cellularity, associated with reduced levels of active NOTCH1-IC, c-MYC and HES1 compared to vehicle (28%, 54% and 38% reduction, respectively). NOTCH1 inhibition significantly improved the survival of diseased mice (median survival of 39 vs 33 days in Bepridil vs vehicle, respectively; N=4, p<0.05). Image:Summary/Conclusion: We showed for the first time the tumour suppressor activity of Bcor in a CLL mouse model, ultimately leading to transformation towards a high-grade lymphoma, mimicking human RT. Mechanistically, we implied NOTCH1 signalling activation in Bcor loss mediated tumorigenesis with potential for targeted treatment for high-risk CLL and RT patients.

NPM1 is the most frequently mutated gene in adults with acute myeloid leukemia (AML). The interaction between mutant NPM1 (NPM1c) and exportin-1 (XPO1) causes aberrant cytoplasmic dislocation of NPM1c and promotes the high expression of homeobox (HOX) genes, which is critical for maintaining the leukemic state of NPM1-mutated cells. Although there is a rationale for using XPO1 inhibitors in NPM1-mutated AML, selinexor administered once or twice per week did not translate into clinical benefit in patients with NPM1 mutations. Here, we show that this dosing strategy results in only a temporary disruption of the XPO1-NPM1c interaction, limiting the efficacy of selinexor. Because the second-generation XPO1 inhibitor eltanexor can be administered more frequently, we tested the antileukemic activity of prolonged XPO1 inhibition in NPM1-mutated AML models. Eltanexor caused irreversible HOX downregulation, induced terminal AML differentiation, and prolonged the survival of leukemic mice. This study provides essential information for the appropriate design of clinical trials with XPO1 inhibitors in NPM1-mutated AML.

Nucleophosmin (NPM1) mutations in acute myeloid leukemia (AML) affect exon 12, but also sporadically affect exons 9 and 11, causing changes at the protein C-terminal end (tryptophan loss, nuclear export signal [NES] motif creation) that lead to aberrant cytoplasmic NPM1 (NPM1c(+)), detectable by immunohistochemistry. Combining immunohistochemistry and molecular analyses in 929 patients with AML, we found non-exon 12 NPM1 mutations in 5 (1.3%) of 387 NPM1c(+) cases. Besides mutations in exons 9 (n = 1) and 11 (n = 1), novel exon 5 mutations were discovered (n 5 3). Another exon 5 mutation was identified in an additional 141 patients with AML selected for wild-type NPM1 exon 12. Three NPM1 rearrangements (NPM1/RPP30, NPM1/SETBP1, NPM1/CCDC28A) were detected and characterized among 13979 AML samples screened by cytogenetic/fluorescence in situ hybridization and RNA sequencing. Functional studies demonstrated that in AML cases, new NPM1 proteins harbored an efficient extra NES, either newly created or already present in the fusion partner, ensuring its cytoplasmic accumulation. Our findings support NPM1 cytoplasmic relocation as critical for leukemogenesis and reinforce the role of immunohistochemistry in predicting AML-associated NPM1 genetic lesions. This study highlights the need to develop new assays for molecular diagnosis and monitoring of NPM1-mutated AML.

A 69-year-old woman was admitted to the intensive care cardiology unit of the Ospedale Santa Maria della Misericordia (Perugia, Italy), in September, 2017, for tachycardia (heart rate 130 bpm) associated with dyspnoea and palpitations. ECG showed sinus tachycardia and non-specific repolarisation changes. Physical examination was normal. High-sensitivity troponin was within the normal reference range and blood counts were normal, except for moderate normochromic normocytic anaemia. Erythrocyte sedimentation rate was slightly elevated and C-reactive protein was normal. Blood biochemistry revealed normal serum electrolytes and lactate dehydrogenase levels; serum electrophoresis showed notable hypogammaglobulinaemia. Cardiac ultrasound and CT scan revealed a voluminous rounded hypodense mass (41 × 58 mm in size) at the interatrial septum which invaded the right atrium, and had intense fluorodeoxyglucose (FDG) uptake on PET scan (figure, A). No other FDG-positive pathological lesions were observed outside the heart tissue.

Background: Adoptive transfer of CD4+CD25+FOXP3+ regulatory T cells (Tregs) effectively prevents conventional T cell (Tcons) mediated Graft versus Host Disease (GvHD) while it does not impair graft-versus-leukemia effect in haploidentical hematopoietic cell transplantation (haplo-HCT). Moreover Treg immunotherapy promotes fast donor T cell recovery after transplant. Recent studies showed that mouse bone marrow (BM) Tregs localize in the hematopoietic stem cell (HSC) niche, where they contribute to HSCs maintenance and promote donor engraftment and B cell lymphopoiesis. We are investigating if human Tregs promote B cell reconstitution and immunity in preclinical models and in haplo-HCT patients. Methods: Human sample analysis: B cell reconstitution was analysed monthly by FACS in BM and peripheral blood (PB) samples from 66 patients who underwent either Treg/Tcon haplo-HCT (45 patients), or T-cell depleted haplo-HCT (8 patients) or haplo-HCT with post-transplant cyclophosphamide (PTCy, 13 patients). Diagnosis was acute leukemia in 52 patients, lymphoma in 11 and multiple myeloma in 3. PB total immunoglobulin (Ig), anti-Cytomegalovirus (CMV) IgM and CMV viremia were monitored. Humanized mouse model: donor derived human Tregs and purified CD34+ HSCs were co-infused in sublethally irradiated (2 Gy) immune-deficient NSG mice. Donor engraftment and B cell reconstitution were analysed by FACS and histology. Results: B cell reconstitution was faster after Treg/Tcon haplo-HCT when compared to other haplo-HCT protocols. B cell counts were higher in PB of patients that received Treg/Tcon haplo-HCT (p<.05) and were comparable to those of healthy subjects by 4 months after transplant (117±148 cells/mm, Fig.1A). We could detect early frequencies of CD34+CD38+CD10+CD127+ common lymphoid progenitors, CD45+CD10+CD38+CD19- Pre/Pro-B, CD45+CD10+CD38+CD19+ Pre-B, and Pro-B cells in the BM of these patients, that resulted in an increased production of CD38+CD19+CD5-IgM+ immature B cells, CD38+CD19+CD5+IgM+ transitional B cells and CD19+CD20+ mature B cells. We used a mouse model of xenotransplantation to understand whether donor B cell reconstitution in Treg/Tcon haplo-HCT is boosted by a Treg-mediated effect on donor human HSCs. We found that infusion of human Tregs facilitated donor HSC engraftment. BM and PB human chimerism was increased (p<.05) in mice that received Treg infusion. BM histology revealed the presence of in vivo expanded human CXCR4+ Tregs in the femurs of Treg receiving mice 1 month after transplant. In the same samples we observed a lower number of CD34+ HSCs, suggesting BM Tregs facilitate donor HSC differentiation. Moreover, Treg treatment allowed human HSCs to localize preferentially in the epiphyseal areas of the femurs, where donor cells started to engraft (p<.04). When looking at donor B cell reconstitution, we found HSC-derived mature B cells rapidly abundant and easily detectable starting 30 days after HSC infusion in PB of Treg-treated animals (p<.05). To further assess whether HSC-derived B cells were functional in patients that received Treg/Tcon haplo-HCT, we analysed total Ig production and specific responses to CMV reactivation. Post-transplant hypogammaglobulinemia was rapidly corrected in Treg/Tcon haplo-HCT patients. Total IgM were higher compared to other haplo-HCT protocols and reached normal levels by 3 months after transplant (96±155 mg/dL, Fig.1B). CMV reactivation rate was lower (47% vs 79%) and it occurred later after Treg/Tcon immunotherapy (51±19 vs 40±48 days). New production of anti-CMV specific IgM was documented in 43% of CMV seropositive patients 98±48 days after Treg/Tcon haplo-HCT, while anti-CMV specific IgM were almost undetectable after other haplo-HCT protocols within the first 6 months after transplant (Fig.1C). Such potent B cell responses coupled with T cell reconstitution resulted in a reduction of CMV second reactivations (Fig.1D p<.05) with no patient that died with CMV disease. Conclusions: Adoptive transfer of human Tregs boosts donor HSC engraftment and facilitates the reconstitution of functional HSC-derived donor B cells. Such results suggest that Treg/Tcon haplo-HCT patients could be early vaccinated after transplant. Treg/Tcon immunotherapy promotes control of infections and B cell immunity in patients undergoing haplo-HCT. Disclosures No relevant conflicts of interest to declare.

NPM1 is one of the most frequently mutated genes in acute myeloid leukaemia (AML), with one third of AML patients carrying NPM1 mutations (Falini et al., 2005). NPM1 is a multifunctional nucleolar chaperone, involved in key biological processes such as maintenance of genome stability and ribosome biogenesis (Brunetti et al., 2019). NPM1 mutations are typically heterozygous four base-pair insertions in the last exon of the gene (Falini et al., 2007) that result in the generation of a novel C-terminal nuclear export signal (Bolli et al., 2007). Therefore, in contrast with the nuclear localization of the wild-type protein, mutant NPM1 (NPM1c) localizes to the cytoplasm of leukaemic cells. Data from patients, cell lines and murine models indicate that NPM1 mutations are AML drivers (Heath et al., 2017). Although NPM1c is necessary for AML maintenance (Brunetti et al., 2018), the mechanisms through which it promotes and maintains leukaemia are still unclear. One hypothesis is that NPM1c would relocate nuclear proteins involved in myeloid differentiation (e.g. transcription factors) to the cytoplasm, blocking their normal function. In agreement, a recent study has shown that NPM1 interacts with the myeloid transcription factor PU.1, relocating it to the cytoplasm of NPM1-mutated AML models (Gu et al., 2018). Based on these observations, we sought to confirm the cytoplasmic localization of PU.1 in primary NPM1-mutated AML samples and to determine whether PU.1 localization, studied by immunohistochemistry (IHC) in bone marrow biopsies, could be used as a surrogate for predicting NPM1 mutational status. To expand our analysis, we also explored PU.1 localization in two NPM1 wild-type and two NPM1-mutated AML cell lines through western blotting (WB) of nuclear and cytoplasmic fractions and immunofluorescence (IF). The study was approved by the Local Institutional Board and all patients signed a written informed consent prior to the bone marrow biopsy. For IHC studies, bones were fixed in B5 solution, decalcified in ethylenediaminetetraacetic acid and embedded into paraffin. Antigens were unmasked by incubating paraffin sections in EnVision FLEX Target Retrieval Solution High pH (Dako-Agilent, Santa Clara, CA, USA). Cell lines were cultured in complete RPMI 1640 and MEM Alpha media. For subcellular fractionation, we used the NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Fisher Scientific, Waltham, MA, USA) starting from 5 × 106 cells and following the manufacturer's instructions. Whole cell lysates as well as nuclear and cytoplasmic fractions were run on 4–15% precast gels and transferred onto polyvinylidene fluoride membranes. Two gels were loaded simultaneously for parallel blotting. For IF, 5 × 104 cells were cytospinned onto slides, fixed with 4% paraformaldehyde (PFA), permeabilized with 0·1% Triton X-100 and blocked with 1% bovine serum albumin. Primary antibodies, dilutions and incubation times used for each experiment are summarized in Table SI. To determine PU.1 localization in primary AML cells, we performed IHC on sections from 39 bone marrow biopsies characterized by a high blast count, either at diagnosis or at relapse. We studied 17 NPM1 wild-type and 22 NPM1-mutated samples using a commercially available PU.1 antibody (referred to as ab1), together with an antibody directed against the N-terminus of NPM1 (recognizing both wild-type and mutant forms, referred to as tNPM1). Surprisingly, PU.1 was localized to the nucleus of all NPM1-mutated samples (Fig 1A and Figure S1), with only 4 of 22 cases displaying weak cytoplasmic staining. Similarly, PU.1 localization was nuclear in all NPM1 wild-type samples (Fig 1A and Figure S1), with 3 of 17 cases also showing weak cytoplasmic staining. As expected, IHC with tNPM1 confirmed nuclear NPM1 localization in all NPM1 wild-type samples and simultaneous nuclear and cytoplasmic localization of NPM1 in all NPM1-mutated cases (Fig 1A and Figure S1). To confirm these results, we repeated IHC on 12 of 39 biopsies (six NPM1 wild-type and six NPM1-mutated) using a different commercially available PU.1 antibody (referred to as ab2). Again, PU.1 localized to the nucleus in all cases (Fig 1A and Figure S1) with only faint cytoplasmic staining in two NPM1 wild-type and one NPM1-mutated sample (Figure S1 – patients 5, 6 and 8 respectively). In summary, IHC of primary bone marrow biopsies did not reveal clear differences in PU.1 localization between NPM1 wild-type and NPM1-mutated samples and was not able to demonstrate cytoplasmic localization of PU.1 in mutated cases. To validate our observations, we analyzed PU.1 localization in two NPM1-mutated (OCI-AML3 and IMS-M2) and two NPM1 wild-type (OCI-AML2 and HNT34) AML cell lines. IF with either ab1 or ab2 revealed strong staining for PU.1 in the nuclei, but no cytoplasmic localization in any cell line (Fig 1B and Figure S2). Staining with tNPM1 confirmed simultaneous nuclear and cytoplasmic localization of NPM1 in NPM1-mutated cells and nuclear staining in NPM1 wild-type cells (Fig 1B). To validate IF results, we evaluated PU.1 expression in whole-cell lysates from AML cells (Figure S3) and then performed nuclear and cytoplasmic fractionation of the same cell lines, followed by WB analysis. Immunoblotting with either ab1 or ab2 confirmed nuclear localization of PU.1, with no clear signal in any of the cytoplasmic fractions (Fig 1C). Staining with an anti-NPM1c antibody confirmed the presence of mutant NPM1 in the cytoplasmic fractions of NPM1-mutated cells (Fig 1C). Altogether, IF and WB data in AML cell lines corroborate our observation in primary AML samples, indicating that PU.1 localization is not altered by the presence of NPM1c in leukaemic cells. AML with mutated NPM1 is one of the most frequent subtypes of leukaemia in adults. About 50% of adult patients diagnosed with NPM1-mutated AML eventually die of leukaemia and the prognosis is even worse in elderly patients (Brunetti et al., 2019). A better understanding of the molecular mechanisms downstream of NPM1c is necessary to open new therapeutic avenues for this frequent AML subtype. Here, in contrast with previous results, we failed to show cytoplasmic PU.1 localization in primary AML samples and AML cell lines with mutated NPM1. The discrepancy between previously published data and our results may be partially explained by some technical differences. First, our work is founded on IHC in primary patient samples, while previous studies were focused on AML models only. Second, while previously published work used methanol-based fixation (Gu et al., 2018), in our study IF was performed on PFA-fixed cells, which may have limited the detection of PU.1 in the cytoplasm. However, the overlap between IHC results in primary samples and IF and WB results in cell lines supports our conclusions. Although we cannot exclude that sporadic AML cases may show partial cytoplasmic localization of PU.1, we conclude that subcellular PU.1 localization should not be used to predict NPM1 mutations in AML patients. Further studies are needed to clarify the proportion and the biological relevance of PU.1 delocalization in AML with mutated NPM1. This work has been supported by the University of Perugia. GP and CB designed the experiments. GP performed the experiments and analyzed the data. BB collected patient samples and performed IHC. RR acquired IHC and IF pictures. LB supervised the study and wrote the letter with GP and CB. All authors approved the manuscript. The authors declare no competing interests. Fig S1. NPM1 and PU.1 immunohistochemical staining in patients' bone marrow biopsies. Fig S2. NPM1 and PU.1 immunofluorescence in AML cell lines. Fig S3. Western blot of whole cell lysates of AML cell lines. Table SI. Primary antibodies used in the experiments. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Background:NPM1 is among the most frequently mutated genes in acute myeloid leukemia (AML), with one third of newly diagnosed cases carrying NPM1 mutations. These mutations are heterozygous small insertions that result in the generation of a novel C‐terminal nuclear export signal. In contrast with the nucleolar localization of the wild type NPM1, mutant NPM1 (NPM1c) is aberrantly localized to the cytoplasm of leukemic cells. It has been recently proposed that NPM1c directly binds the myeloid transcription factor PU.1, relocating it from the nucleus to the cytoplasm (Gu et al., JCI 2018). Therefore, we sought to investigate whether PU.1 subcellular localization could be used to predict NPM1 mutations in AML.Aims:This study aimed to determine the impact of PU.1 subcellular localization in the diagnosis of AML with mutated NPM1.Methods:We analysed 39 bone marrow biopsies from patients with AML either at diagnosis or at relapse (22 NPM1‐mutated and 17 NPM1‐wild type). Samples were studied by immunohistochemistry (IHC) using two different commercially available monoclonal antibodies against PU.1 (ab1 and ab2) and total NPM1 (tNPM1). We also evaluated PU.1 localization in 4 AML cell lines (2 NPM1‐mutated ‐ i.e. OCI‐AML3 and IMS‐M2 ‐ and 2 NPM1‐wild type ‐ i.e. OCI‐AML2 and HNT34) through western blot (WB) of nuclear and cytoplasmic fractions and immunofluorescence (IF). The mutational status of the NPM1 gene was determined by PCR and capillary electrophoresis in all patients and cell lines. An homemade antibody specific for NPM1c (Martelli et al., Leukemia 2008) was used to confirm the presence of NPM1 mutation in the cell lines in WB.Results:As expected, all NPM1‐wild type cases showed nuclear localization of NPM1, while all NPM1‐mutated cases displayed simultaneous nuclear and cytoplasmic localization. In all NPM1‐wild type cases PU.1 localization was nuclear (Figure 1A), with 3/17 cases showing also weak cytoplasmic staining. Surprisingly, all NPM1‐mutated cases showed nuclear localization of PU.1 (Figure 1A), with 4/22 cases displaying also weak cytoplasmic staining. To validate these results, we stained 6 NPM1‐wild type and 6 NPM1‐mutated biopsies also with a second anti‐PU.1 antibody. All NPM1‐wild type and NPM1‐mutated cases had nuclear localization of PU.1, with respectively 2/6 and 1/6 cases with faint cytoplasmic staining, supporting our previous observastions. To further investigate PU.1 localization in NPM1‐mutated AML, we performed IF in two NPM1‐mutated and 2 NPM1‐wild type AML cell lines. All cell lines, studied with two different PU.1 antibodies showed strong PU.1 staining in the nuclei, but no cytoplasmic PU.1 was detected in any of the four cell lines. Finally, to confirm IF data, we performed WB analysis in the same AML cell lines. PU.1 was clearly detected in the nuclear fraction of all cell lines, independently of NPM1 mutational status, while no band was clearly visible in any of the cytoplasmic fractions (Figure 1B ‐ ab1). These results were confirmed with a second anti‐PU.1 antibody (Figure 1B ‐ ab2).Summary/Conclusion:In summary, we did not observe differences in PU.1 localization between NPM1‐wild type and NPM1‐mutated AML cases. No case showed strong PU.1 staining in the cytoplasm, with cell line data supporting our observations. We conclude that PU.1 localization should not be used to predict NPM1 mutations in AML. Our data suggest that further studies are needed to determine the amount of PU.1 undergoing NPM1c‐mediated relocation to the cytoplasm and to establish the biological relevance of PU.1 abnormal localization in AML with mutated NPM1.image

Objective A standardization of minor salivary gland (MSG) histopathology in primary Sjögren’s syndrome (pSS) has been recently proposed. Although there is strong agreement that germinal center (GC)-like structures should be routinely identified, due to their prognostic value, a consensus regarding the best protocol is still lacking. Aim of this study was to compare the performance of different histological techniques and operators to identify GC-like structures in pSS MSGs. MSG biopsies from 50 pSS patients were studied. Methods Three blinded operators (one pathologist and two rheumatologists with different years of experience in pSS MSG assessment) assessed 50 MSGs of which one slide was stained with haematoxylin and eosin (H&E) and consecutive slides were processed to investigate CD3/CD20, CD21 and Bcl-6 expression. Results By assessing 225 foci, the best agreement was between H&E-stained sections evaluated by the rheumatologist with more years of experience in pSS MSG assessment and CD3/CD20 segregation. In the foci with CD21 positivity, the agreement further increased. Bcl-6- foci could display a GC, detected with other staining, but not vice versa. Conclusion GC assessment on H&E-stained sections should be performed with caution, being operator-dependent. The combination of H&E with CD3/CD20 and CD21 staining should be recommended as it is reliable, feasible, able to overcome the bias of operator experience and easily transferrable into routine practice.

Background:Richter Syndrome (RS) is defined as the occurrence of a high‐grade lymphoma (usually diffuse large B‐cell lymphoma ‐ DLBCL) in patients with a previous/concomitant diagnosis of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL).RS usually occurs in lymph‐nodes, but sometimes may infiltrate extranodal sites.Isolated cardiac involvement is very unique.Most DLBCL‐type RS does not share lesions with de novo subtypes.NOTCH1 mutation was reported in almost 30% of cases,but NOTCH1 signaling activation independent of its mutational status,as NOTCH ligands mapping in tumor niche, remain scarcely estimated, as opposed to CLL.This has directed our attention to the role of cell non‐autonomous NOTCH1 activation at the cross‐roads between other pathways coming from the niche (i.e. immune checkpoint axis). CLL cells also express the NOTCH ligand JAGGED1 (JAG1), that contributes to CLL pathogenesis by activating its own signaling.Experimental proofs in other settings were made, linking the NOTCH1‐JAG1 signaling to the immunological niche.Strikingly, DLBCL‐type RS exhibits PD‐1/PD‐L1 axis upregulation in up to 80% of cases.Taking advantage of these findings, we decided to perform immunohistochemistry (IHC), using validated antibodies,to dissect the precise anatomy of the NOTCH/ligand interface in RS,in vivo.Aims:1) To investigate context‐dependent, cell‐autonomous/cell non‐autonomous NOTCH1 network activation, using IHC stains. Specifically, we focused on NOTCH1 and its ligand JAG1, a well‐known pivotal player in embryonic heart and cardiac diseases. 2) To zoom into the PD‐1/PDL‐1 axis activation in immune niche, and crosslink the immune‐evasion pathway to tumor oncogenic promotion.Methods:A 69‐year old female was admitted to the Cardiology Intensive Care Unit (ICU), for tachy‐/bradycardia. A cardiac computed tomography identified a voluminous rounded mass at the interatrial septum.The pathologic diagnosis was consistent with non‐GC DLBCL.Bone marrow (BM) biopsy detected CLL/SLL.Molecular analysis did not reveal SF3B1,NOTCH1 and TP53 alterations but evidenced the same IgVH mutation in both mass and BM specimens,proving a clonally‐related RS.Results:IHC stains for the cleaved NOTCH1‐intracellular domain (ICD) protein was negative on both RS lymphoma cells and the surrounding stroma, as in BM specimen. On the contrary, JAG1 was markedly positive both on large neoplastic lymphocytes and in the stromal compartment (Figure 1A). Conversely, JAG1 staining was moderate‐to‐weak on CLL lymphocytes in the BM, as well as in the stromal scaffold (Figure 1B), indicating differential expressions. Unexpectedly, JAG1 upregulation occurred independently of NOTCH1 receptor co‐activation. According to this, JAG1 may stimulate other pro‐survival cascades in the neoplastic B cells, transducing microenvironmental cues, coming from TME, where JAG1 itself was found highly expressed. In addition, we performed IHC stain for PD‐1, proving strong positivity on tumor‐infiltrating lymphocytes (TILs) (Figure 1C); moreover, PD‐L1 marked both neoplastic cells and stromal meshwork in the cardiac TME (Figure 1D).Summary/Conclusion:Our report proved the importance of IHC in characterizing JAG1‐NOTCH1 ligand‐receptor interactions in the RS pathogenesis, in order to anatomically characterize cell‐extrinsic pathways in vivo. We traced JAG1 upregulation, independently of NOTCH1 signaling activation and described PD‐1/PD‐L1 axis functional switching in the context of a very unique extra‐nodal RS localization, thus identifying novel potential actionable therapeutic targets.image

Dissecting the pathogenesis of classical Hodgkin lymphoma (cHL), a common cancer in young adults, remains challenging because of the rarity of tumor cells in involved tissues (usually <5%). Here, we analyzed the coding genome of cHL by microdissecting tumor and normal cells from 34 patient biopsies for a total of ∼50 000 singly isolated lymphoma cells. We uncovered several recurrently mutated genes, namely, STAT6 (32% of cases), GNA13 (24%), XPO1 (18%), and ITPKB (16%), and document the functional role of mutant STAT6 in sustaining tumor cell viability. Mutations of STAT6 genetically and functionally cooperated with disruption of SOCS1, a JAK-STAT pathway inhibitor, to promote cHL growth. Overall, 87% of cases showed dysregulation of the JAK-STAT pathway by genetic alterations in multiple genes (also including STAT3, STAT5B, JAK1, JAK2, and PTPN1), attesting to the pivotal role of this pathway in cHL pathogenesis and highlighting its potential as a new therapeutic target in this disease.

Background A standardisation of minor salivary gland (MSG) histopathology in primary Sjögren’s syndrome (pSS) has been recently proposed by the EULAR study group on Sjögren’s syndrome. Although there is strong agreement that germinal centres (GCs) should be routinely identified, due to their prognostic value, a consensus regarding the best protocol is still lacking. Objectives Aim of this study was to compare the performance of different histological techniques and operators with variable experience in MSG histopathology to identify GCs in pSS MSGs. Methods MSG biopsies from 50 pSS patients were studied. Three blinded operators (expert rheumatologist, expert pathologist and rheumatologist with scarce experience on MSG histopathology) scored one slide stained with haematoxilin and eosin (H and E). Consecutive slides were processed by immunofluorescence and immunohistochemistry to assess CD3/CD20, CD21 and Bcl-6 expression. Results Overall, the prevalence of GC in MSG specimens (namely the presence of at least one focus positive for at least one operator or histological technique) ranged between 26% and 52%. By separate assessment of 225 foci, the best agreement was between H and E-stained sections evaluated by the expert rheumatologist and CD3/CD20 segregation (Cohen’s kappa=0.72). In the foci with CD21 positivity, the agreement with the expert rheumatologist further increased (Cohen’s kappa=0.75). Among the 3 methods employed, the best agreement was observed between B/T-cell segregation and the positivity for CD21 staining (0.84). The absence of Bcl-6 in a focus does not necessarily rule out the presence of the GC detected with other stainings, Conclusions GC assessment on H and E-stained sections should be performed with caution, as it is dependent on the background and expertise of the operator. The combination of H and E with CD3/CD20 and CD21 staining should be recommended as it is reliable, feasible, able to overcome the bias of operator experience and easily transferrable into routine practice. Disclosure of Interest None declared