Inborn errors of immunity (IEIs) are caused by deleterious variants in immune-related genes. ASXL1 is an epigenetic modifier not previously linked to an IEI. Clonal hematopoiesis and hematologic neoplasms often feature somatic ASXL1 variants, and Bohring-Opitz syndrome, a neurodevelopmental disorder, is caused by heterozygous truncating ASXL1 variants. We present an IEI caused by biallelic germline missense variants in ASXL1. The patient had a history of hematologic abnormalities and viral-associated complications, including chronic macrocytosis, persistent vaccine-strain rubella granulomas, and EBV-associated Hodgkin lymphoma. Immunophenotyping revealed loss of B cells, hypogammaglobulinemia, and impairments in cytotoxic T and NK cell populations. T cells exhibited skewing toward an exhausted memory phenotype, global DNA methylation loss, and increased epigenetic aging. These aberrations were ameliorated by wild-type ASXL1 transduction, confirming the patient variants' pathogenicity. This study defines a novel human IEI caused by ASXL1 deficiency, a diagnosis that should be considered in individuals with chronic viral infections, viral-associated malignancies, and combined immune deficiency.

Multiparameter flow cytometry is widely used for acute myeloid leukemia minimal residual disease testing (AML MRD) but is time consuming and demands substantial expertise. Machine learning offers potential advancements in accuracy and efficiency, but has yet to be widely adopted for this application. To explore this, we trained single cell XGBoost classifiers from 98 diagnostic AML cell populations and 30 MRD negative samples. Performance was assessed by cross-validation. Predictions were integrated with UMAP as a heatmap parameter for an augmented/interactive AML MRD analysis framework, which was benchmarked against traditional MRD analysis for 25 test cases. The results showed that XGBoost achieved a median AUC of 0.97, effectively distinguishing diverse AML cell populations from normal cells. When integrated with UMAP, the classifiers highlighted MRD populations against the background of normal events. Our pipeline, MAGIC-DR, incorporated classifier predictions and UMAP into flow cytometry standard (FCS) files. This enabled a human-in-the-loop machine learning guided MRD workflow. Validation against conventional analysis for 25 MRD samples showed 100% concordance in myeloid blast detection, with MAGIC-DR also identifying several immature monocytic populations not readily found by conventional analysis. In conclusion, Integrating a supervised classifier with unsupervised dimension reduction offers a robust method for AML MRD analysis that can be seamlessly integrated into conventional workflows. Our approach can support and augment human analysis by highlighting abnormal populations that can be gated on for quantification and further assessment. This has the potential to speed up MRD analysis, and potentially improve detection sensitivity for certain AML immunophenotypes.

Rearrangements leading to constitutive PDGFRB activity are a rare cause of myeloid/lymphoid neoplasms, usually presenting as myelodysplastic/myeloproliferative neoplasms with prominent eosinophilia. These rearrangements are generally detectable by conventional karyotyping and are important to identify as they typically confer sensitivity to imatinib. Here we report a unique case of a myeloid/lymphoid neoplasm with cryptic PDGFRB rearrangement that presented as an unusual combination of T-lymphoblastic lymphoma (T-LBL) and systemic mastocytosis (SM). Notably, a novel PDGFRB fusion partner (JAKMIP2) was identified in this case by optical genome mapping (OGM). The patient presented to medical attention with an enlarging neck mass and was diagnosed with T-LBL. The staging bone marrow biopsy was negative for T-LBL but unexpectedly revealed SM positive for KIT D816V mutation. At the time of assessment the patient had mild monocytosis without eosinophilia. Bone marrow cytogenetic analysis showed a normal karyotype; however, interphase FISH detected a cryptic PDGFRB rearrangement with metaphase FISH suggesting an intrachromosomal chromosome 5 rearrangement. The same PDGFRB rearrangement was subsequently identified on the lymph node, demonstrating a clonal relationship between the patient's T-LBL and SM. OGM revealed a tandem duplication event at chromosome 5q32 spanning chr5:147,629,580-150,098,238 (hg38) with breakpoints disrupting PDGFRB and JAKMIP2, resulting in a previously undescribed JAKMIP2::PDGFRB fusion. To date, the patient has shown a sustained cytogenetic and clinical response to imatinib therapy. This case highlights how OGM enables characterization of cryptic and novel genomic rearrangements, with great potential for improving classification and enabling appropriately targeted treatment of patients with diverse hematologic malignancies. Rearrangements leading to constitutive PDGFRB activity are a rare cause of myeloid/lymphoid neoplasms, usually presenting as myelodysplastic/myeloproliferative neoplasms with prominent eosinophilia. These rearrangements are generally detectable by conventional karyotyping and are important to identify as they typically confer sensitivity to imatinib. Here we report a unique case of a myeloid/lymphoid neoplasm with cryptic PDGFRB rearrangement that presented as an unusual combination of T-lymphoblastic lymphoma (T-LBL) and systemic mastocytosis (SM). Notably, a novel PDGFRB fusion partner (JAKMIP2) was identified in this case by optical genome mapping (OGM). The patient presented to medical attention with an enlarging neck mass and was diagnosed with T-LBL. The staging bone marrow biopsy was negative for T-LBL but unexpectedly revealed SM positive for KIT D816V mutation. At the time of assessment the patient had mild monocytosis without eosinophilia. Bone marrow cytogenetic analysis showed a normal karyotype; however, interphase FISH detected a cryptic PDGFRB rearrangement with metaphase FISH suggesting an intrachromosomal chromosome 5 rearrangement. The same PDGFRB rearrangement was subsequently identified on the lymph node, demonstrating a clonal relationship between the patient's T-LBL and SM. OGM revealed a tandem duplication event at chromosome 5q32 spanning chr5:147,629,580-150,098,238 (hg38) with breakpoints disrupting PDGFRB and JAKMIP2, resulting in a previously undescribed JAKMIP2::PDGFRB fusion. To date, the patient has shown a sustained cytogenetic and clinical response to imatinib therapy. This case highlights how OGM enables characterization of cryptic and novel genomic rearrangements, with great potential for improving classification and enabling appropriately targeted treatment of patients with diverse hematologic malignancies.

Fig. S1. Drug-interaction matrix plotting cell killing observed in excess of the additive Bliss expectation between MEK and PI3K inhibition in Pt-resistant epithelial ovarian carcinoma (OVCAR3; 0.20-100 microM, 48 h).Ï‘ Fig. S2. Changes in serum cytokine levels following a single i.v. bolus of LbL nanoparticles in immuno-competent BALC/c mice at t=0 h. Error represents SD of 4 technical replicates from n=3 mice.

ABSTRACT Inborn errors of immunity (IEI) are a group of disorders caused by deleterious variants in immune-related genes, including some that function as epigenetic regulators. Additional sex combs-like 1 (ASXL1) is an epigenetic modifier that has not previously been linked to an IEI. Somatic ASXL1 variants are found in clonal hematopoiesis and hematologic neoplasms, while heterozygous germline variants cause Bohring–Opitz syndrome. We present a new IEI caused by biallelic germline variants in ASXL1 . The patient had a complex and unusual history of disease progression notable for persistent cutaneous vaccine-strain rubella granulomas initially manifesting in early childhood, chronic macrocytosis and mild bone marrow cellular hypoplasia, and Epstein Barr virus– associated Hodgkin lymphoma in adolescence. Detailed immunophenotyping revealed progressive loss of B-cells, hypogammaglobinemia, and T-cell lymphopenia with severe skewing toward a memory phenotype and elevated expression of T-cell exhaustion and senescence markers. Molecular investigations confirmed ASXL1 protein deficiency in the patient’s T-cells and fibroblasts. The T-cells exhibited marked loss of DNA methylation, increased epigenetic aging, and CD8 T-cell dysfunction. These aberrations were ameliorated by lentivirus-mediated transduction with wild-type ASXL1 , confirming the pathogenicity of ASXL1 variants. This study defines a novel human IEI caused by ASXL1 deficiency, a diagnosis that should be considered in individuals with chronic viral infections, virus-associated hematologic malignancies, and combined immunodeficiency. Furthermore, our findings provide fresh insights into the mechanisms underlying the roles of human ASXL1 in T-cell function as well as in the development and maintenance of lymphomas.

A 76-year-old man presented to the haematology clinic at Vancouver General Hospital (Vancouver, BC, Canada) in September, 2022, with fatigue. A full-body MRI showed cervical, intrathoracic, and intraabdominal lymphadenopathy, including an anterior mediastinal mass believed to be a lymph node conglomerate, and bony lesions in the spine, ribs, pelvis, and femur. He had a history of prostate cancer and non-invasive bladder cancer. A neck lymph node core biopsy and EBUS-guided lymph node aspirate showed non-necrotising granulomatous inflammation with no evidence of malignancy.

Primary atopic disorders are a group of inborn errors of immunity that skew the immune system toward severe allergic disease. Defining the biology underlying these extreme monogenic phenotypes reveals shared mechanisms underlying common polygenic allergic disease and identifies potential drug targets. Germline gain-of-function (GOF) variants in JAK1 are a cause of severe atopy and eosinophilia. Modeling the JAK1GOF (p.A634D) variant in both zebrafish and human induced pluripotent stem cells (iPSCs) revealed enhanced myelopoiesis. RNA-Seq of JAK1GOF human whole blood, iPSCs, and transgenic zebrafish revealed a shared core set of dysregulated genes involved in IL-4, IL-13, and IFN signaling. Immunophenotypic and transcriptomic analysis of patients carrying a JAK1GOF variant revealed marked Th cell skewing. Moreover, long-term ruxolitinib treatment of 2 children carrying the JAK1GOF (p.A634D) variant remarkably improved their growth, eosinophilia, and clinical features of allergic inflammation. This work highlights the role of JAK1 signaling in atopic immune dysregulation and the clinical impact of JAK1/2 inhibition in treating eosinophilic and allergic disease.

Background and Objectives The coronavirus disease 2019 (COVID-19) pandemic raised concerns about the vulnerability of platelet supply and the uncertain impact of the resumption of elective surgery on utilization. We report the impact of COVID-19 on platelet supply and utilization across a large, integrated healthcare system in the Canadian province of British Columbia (BC). Materials and Methods Historical platelet use in BC by indication was compiled for fiscal year 2010/2011-2019/2020. Platelet collections, initial daily inventory and disposition data were assessed pre-COVID-19 (1 April 2018-15 March 2020) and for two COVID-19 time periods in BC: a shutdown phase with elective surgeries halted (16 March-17 May, 2020) and a renewal phase when elective surgeries resumed (18 May-27 September 2020); comparisons were made provincially and for individual health authorities. Results Historically, elective surgeries accounted for 10% of platelets transfused in BC. Initial daily supplier inventory increased from baseline during both COVID-19 periods (93/90 units vs. 75 units pre-COVID-19). During the shutdown phase, platelet utilization decreased 10.4% (41 units/week; p < 0.0001), and remained significantly decreased during the ensuing renewal period. Decreased platelet utilization was attributed to fewer transfusions during the shutdown phase followed by a decreased discard/expiry rate during the renewal phase compared to pre-COVID-19 (15.2% vs. 18.9% pre-COVID-19; p < 0.0001). Differences in COVID-19 platelet utilization patterns were noted between health authorities. Conclusion Decreased platelet utilization was observed in BC compared to pre-COVID-19, likely due to a transient reduction in elective surgery as well as practice and policy changes triggered by pandemic concerns.

BACKGROUND:Flow cytometry is widely used for B-ALL minimal residual disease (MRD) analysis given its speed, availability, and sensitivity; however, distinguishing B-lymphoblasts from regenerative B-cells is not always straightforward. Radar plots, which project multiple markers onto a single plot, have been applied to other MRD analyses. Here we aimed to develop optimized radar plots for B-ALL MRD analysis.METHODS:We compiled Children's Oncology Group (COG) flow data from 20 MRD-positive and 9 MRD-negative B-ALL cases (enriched for hematogones) to create labeled training and test data sets with equal numbers of B-lymphoblasts, hematogones, and mature B-cells. We used an automated approach to create hundreds of radar plots and ranked them based on the ability of support vector machine (SVM) models to separate blasts from normal B-cells in the training data set. Top-performing radar plots were compared with PCA, t-SNE, and UMAP plots, evaluated with the test data set, and integrated into clinical workflows.RESULTS:SVM area under the ROC curve (AUC) for COG tube 1/2 radar plots improved from 0.949/0.921 to 0.989/0.968 after optimization. Performance was superior to PCA plots and comparable to UMAP, but with better generalizability to new data. When integrated into an MRD workflow, optimized radar plots distinguished B-lymphoblasts from other CD19-positive populations. MRD quantified by radar plots and serial gating were strongly correlated.DISCUSSION:Radar plots were successfully optimized to discriminate between diverse B-lymphoblast populations and non-malignant CD19-positive populations in B-ALL MRD analysis. Our novel radar plot optimization strategy could be adapted to other MRD panels and clinical scenarios.

BCR/ABL1 rearrangement or t(9;22)(q34.1;q11.2) is the most common recurrent cytogenetic abnormality seen in B/myeloid mixed phenotype acute leukemia (MPAL) but is also present in ~50% of adult B-ALL (Swerdlow et al., 2017). Both entities can demonstrate B lymphoblasts with aberrant myeloid expression; however, an important immunophenotypic finding that distinguishes MPAL from B-ALL with aberrant myeloid expression is reciprocal intensities of myeloid and lymphoid markers. This pattern is emphasized as the most recognizable feature of MPAL in the WHO classification but there are few examples in the literature demonstrating its practical application. Herein, we highlight this signature inverse expression characteristic using a case of MPAL B/myeloid with BCR/ABL1. Detailed methodology and antibody panels have been previously described in Cytometry B (DOI: 10.1002/cyto.b.21979). In brief, sodium heparin anticoagulated bone marrow (BM) was processed within 6 h of collection. BM specimens were washed twice using 37°C phosphate-buffered saline and adjusted to ~10 × 109/L. One hundred microliter were stained using antibody panels per DOI: 10.1002/cyto.b.21979. Specifically, the markers presented here included: CD10-APC-AF700 (ALB1), CD13-PE (SJ1D1) CD19-APC-AF700 (J3-119), CD19-APC-A750 (J3-119), CD19-ECD (J3-119), CD33-PC5.5 (D3HL60.251) CD34-PC7 (581), CD45-KO (J.33) CD117-APC (104D2D1) cCD79a-APC (HM47) and cMPO-PE (CLB-MPO-1), all antibodies from Beckman Coulter (BC). For surface staining, cells were incubated for 15 min in the dark at room temperature, and then lysed using VersaLyse (BC, ref. A09777) and IOTest-3 fixative (BC, ref. A07800). For intracellular staining BC IntraPrep™ reagent kit (BC, ref. A07802) was used according to manufacturer's instructions. After washing, samples were suspended in IOTest-3 Fixative (BC, ref. A07800) and PBS. A minimum of 50 × 103 CD45+ events were acquired on a Navios™ Flow cytometer (BC) within 1 h of preparation. Listmode data files were analyzed using Kaluza analysis software (BC, v1.3). Fluorochrome abbreviations: Phycoerythrin (PE), Phycoerythrin Texas Red-X (ECD), Phycoerythrin Cyanin 7 (PC7), Allophycocyanin (APC), Allophycocyanin Alexa Fluor 700 (APC-AF700), Allophycocyanin Alexa Fluor 750 (APCAF750) and Krome Orange (KO). A previously healthy 25-year-old male presented to medical attention with a 3-week history of progressive exertional dyspnea along with a new rash on his chest and shins. Physical examination was notable for mild tachycardia, pallor, and a petechial rash. Initial blood work revealed marked leukocytosis with a blast count of 134 × 109/L (83% blasts), profound anemia and thrombocytopenia. BM studies revealed 91% blasts by morphology with minimal granulocyte maturation. Blasts showed variable morphology ranging from small blasts with high nuclear/cytoplasmic ratio to medium-sized blasts with prominent nucleoli and granules, but no Auer rods. The trephine biopsy showed almost 100% cellularity with normal hematopoiesis replaced by a dense blast infiltrate. Comprehensive flow cytometry was performed using four 10 color panels, routine at our institution for evaluation of new acute leukemia. Panel 1 showed a large CD34 positive blast population in the CD45-dim low side scatter region with partial expression of CD117 alongside variable expression of CD13 and CD33. Such strong expression of myeloid markers initially suggested a myeloid leukemia (Figure 1a–d). Uniformly positive CD19, however, then broadened the differential diagnosis to B-ALL with aberrant myeloid expression, AML with aberrant CD19 (e.g., AML with t(8;21)(q22;q22.1), or B/myeloid MPAL (Figure 1e). The next sequence of markers demonstrated a large proportion of the blasts expressed CD10 and CD79a (Figure 1f,i), meeting WHO criteria for assigning at least a B lineage with a small proportion of the blasts being MPO positive (2.95%; Figure 1g). Cytoplasmic CD3 was negative (not shown), narrowing the differential to B-ALL with aberrant myeloid expression or B/myeloid MPAL. While MPO was only weakly/partially expressed, importantly MPAL-defining inverse co-expression patterns between myeloid and lymphoid markers were evident. MPO was only expressed in the cells brightest for the myeloid marker CD33 and dimmest for the B lineage associated marker CD79a (Figure 1i,j, black backgating on MPO+). A similar negative inverse correlation could be demonstrated for CD10 and CD33 (Figure 1k,l, black backgating on CD117+); however, CD19 intensity did not vary. Similar to MPO, CD117 was only expressed in cells that were strongly positive for CD33, but negative for CD10. Given these phenotypic associations, we inferred that MPO was expressed in CD19+ cells positive for the myeloid markers CD33 and CD117, but negative for B cell markers CD10 and CD79a. Based on this characteristic inverse B-lymphoid and myeloid expression pattern, a preliminary diagnosis of MPAL, B/myeloid was communicated to the treatment team the day of the BM biopsy. Subsequent cytogenetics results demonstrated t(9;22)(q34.1;q11.2) in 19/20 metaphases and a p210 BCR/ABL1 transcript by molecular. A final WHO classification of MPAL with t(9;22)(q34.1;q11.2) was made. Next generation sequencing results detected a variant of uncertain significance (VUS) in RUNX1 leading to the duplication of a valine residue at position 119 (RUNX1c.356_358dupTGG/p.Val119dup), variant allele frequency 47%. The patient was started on a tyrosine kinase inhibitor based B-ALL regimen and eventually went on to receive an allogeneic stem cell transplant and is currently in remission. Acute leukemias are increasingly classified by recurrent molecular/cytogenetic abnormalities. In this case, t(9;22)(q34.1;q11.2); BCR/ABL1 did not assist with distinguishing MPAL from B-ALL since it a common finding in both entities. In fact, BCR/ABL1 in cases such as this one may create more ambiguity since it is known to be associated with aberrant myeloid expression in B-ALL. The presence of a BCR/ABL1 p210 isoform and mixed phenotype blasts also raises the possibility of chronic myeloid leukemia (CML) presenting in a mixed blast crisis. In our case this was unlikely based on the acute clinical history and absence of other suggestive features of CML such as splenomegaly, peripheral granulocytosis, or residual granulocyte maturation. Given the controversies related to isolated MPO expression in otherwise typical B-ALL presentations, this case helps emphasize the value of additional aberrant myeloid markers, particularly when the pattern of B-lymphoid and myeloid markers are inversely correlated. The fact that lineage defining MPO expression clearly followed this pattern made it highly unlikely that the MPO staining was non-specific. Other phenotypic support for MPAL included CD117 positivity, which in contrast to CD13 and CD33, is rare in B-ALL. It is likely that additional genetic events occur that ultimately determine the dominant blast lineage(s) at presentation since BCR/ABL1 may be detected in de novo ALL, AML, MPAL and blast phase CML. The added finding of a RUNX1 mutation in this case (albeit classified as a VUS) is interesting as emerging evidence suggests that RUNX1 can play a role in the pathogenesis of MPAL, and specifically seems to be enriched in B/myeloid MPAL (Takahashi et al., 2018). In the tyrosine kinase inhibitor era the clinical impact of distinguishing MPAL, B-myeloid with BCR/ABL1, and B-ALL with BCR/ABL1 remains unclear; however, the categorization as MPAL in a young patient may favor allotransplant consolidation where feasible, regardless of measurable residual disease. This highlights the therapeutic importance of accurate flow cytometric interpretation and awareness of the distinguishing features between these two subtypes of acute leukemia.

See article on page 435–448, in this issue See article on page 449–463, in this issue

Case 1: A 35-year-old male with newly diagnosed HIV infection presented with diffuse lymphadenopathy, pancytopenia (hemoglobin 97 g/L, platelets 30 × 109/L, neutrophils 1.9 × 109/L), and a leukoerythroblastic blood film. Bone marrow and lymph node biopsies both showed a diffuse infiltrate of variable medium to large lymphoid cells with a starry sky appearance, prominent nucleoli, and frequent mitoses with large areas of necrosis seen in the marrow (Figure 1A, 40× objective). The infiltrating cells had a germinal center B-cell (GCB) phenotype (CD20/CD10/BCL6 positive), were BCL2 negative, and showed a variable proliferation index of 70%–95%. AlthoughMYCwas uniformly negative by immunohistochemistry (IHC; Figure 1A inset, 40× objective), a MYC translocation was identified by fluorescence in situ hybridization (FISH; Figure 1B), highly suggestive of a false negative IHC result. As BCL2/BCL6 rearrangements by FISH were negative, Burkitt lymphoma (BL) was considered. However, diffuse large B-cell lymphoma (DLBCL) was favored given themorphology and variable proliferation index. Case 2: A 71-year-old male presented with easy bruising and night sweats. He was found to have severe thrombocytopenia (platelets= 5 × 109/L) and a leukoerythroblastic blood film containing abnormal circulating lymphoid cells with vacuolated basophilic cytoplasm. The bone marrow biopsy showed an atypical B-cell infiltrate

Small interfering RNA (siRNA) therapy has significant potential for the treatment of myriad diseases, including cancer. While intravenous routes of delivery have been found to be effective for efficient targeting to the liver, achieving high accumulations selectively in other organs, including lung tissues, can be a challenge. We demonstrate the rational design and engineering of a layer-by-layer (LbL) nanoparticle-containing aerosol that is able to achieve efficient, multistage delivery of siRNA in vitro. For the purpose, LbL nanoparticles were, for the first time, encapsulated in composite porous micro scale particles using a supercritical CO2-assisted spray drying (SASD) apparatus using chitosan as an excipient. Such particles exhibited aerodynamic properties highly favorable for pulmonary administration, and effective silencing of mutant KRAS in lung cancer cells derived from tumors of a non-small cell lung cancer (NSCLC) autochthonous model. Furthermore, efficient alveolar accumulation following inhalation in healthy mice was also observed, corroborating in vitro aerodynamic results, and opening new perspectives for further studies of effective lung therapies These results show that multistage aerosols assembled by supercritical CO2-assisted spray drying can enable efficient RNA interference therapy of pulmonary diseases including lung cancer.

In response to DNA damage, a synthetic lethal relationship exists between the cell cycle checkpoint kinase MK2 and the tumor suppressor p53. Here, we describe the concept of augmented synthetic lethality (ASL): depletion of a third gene product enhances a pre-existing synthetic lethal combination. We show that loss of the DNA repair protein XPA markedly augments the synthetic lethality between MK2 and p53, enhancing anti-tumor responses alone and in combination with cisplatin chemotherapy. Delivery of siRNA-peptide nanoplexes co-targeting MK2 and XPA to pre-existing p53-deficient tumors in a highly aggressive, immunocompetent mouse model of lung adenocarcinoma improves long-term survival and cisplatin response beyond those of the synthetic lethal p53 mutant/MK2 combination alone. These findings establish a mechanism for co-targeting DNA damage-induced cell cycle checkpoints in combination with repair of cisplatin-DNA lesions in vivo using RNAi nanocarriers, and motivate further exploration of ASL as a generalized strategy to improve cancer treatment.