Lymphoid Markers Research Articles

IMMU-71. CRANIOENCEPHALIC FUNCTIONAL LYMPHOID UNITS IN GLIOBLASTOMA

Abstract The general accessibility of the brain to immune cells has been a subject of intense debate and reinterpretation within the last decade. While glioblastoma’s brain tumor ecosystem is typically considered immunosuppressed, we report the presence of a tumor-reactive immune cell niche within the cranial bone marrow (CB) situated adjacent to treatment-naive tumors. At time of initial diagnosis, glioblastoma patients underwent clinical and anatomical imaging, which revealed an unexpected co-morbid association between the CB and the tumor. Following this observation, we conducted an extensive experimental study including multiplex immunofluorescence, immunoprofiling, functional assays, and single-cell analysis, which identified an active lymphoid niche within the patient’s CB. CD8+ T cell effector cells residing in this niche exhibited acute and durable antitumor responses, and, compared to cells from the distal bone marrow, showed increased expression of the lymphoid egress marker S1PR1. Single-cell T cell receptor sequencing revealed a rich reservoir of non-exhausted tumor-shared CD8+ T cell clonotypes within the CB lymphoid niche, suggesting re-circulation between the proximal bone and the tumor tissue. These clonotypes were characterized by a high predicted tumor-reactivity, significantly surpassing levels found in blood and distal bone marrow. We discovered the entire developmental spectrum of CD8+ T cells in the CB, indicating that anatomical proximity may play a critical role in the process of early anti-tumor response. Correspondingly, cranial enhancement of CXCR4 radiolabel may serve as a surrogate marker, indicating focal association with improved progression-free survival. Our findings support the preservation and further exploitation of these cranioencephalic units to improve clinical care in glioblastoma patients.

Systemic deficits in lipid homeostasis promote aging-associated impairments in B cell progenitor development.

Organismal aging has been associated with diverse metabolic and functional changes across tissues. Within the immune system, key features of physiological hematopoietic cell aging include increased fat deposition in the bone marrow, impaired hematopoietic stem and progenitor cell (HSPC) function, and a propensity towards myeloid differentiation. This shift in lineage bias can lead to pre-malignant bone marrow conditions such as clonal hematopoiesis of indeterminate potential (CHIP) or clonal cytopenias of undetermined significance (CCUS), frequently setting the stage for subsequent development of age-related cancers in myeloid or lymphoid lineages. At the systemic as well as sub-cellular level, human aging has also been associated with diverse lipid alterations, such as decreased phospholipid membrane fluidity that arises as a result of increased saturated fatty acid (FA) accumulation and a decay in n-3 polyunsaturated fatty acid (PUFA) species by the age of 80 years, however the extent to which impaired FA metabolism contributes to hematopoietic aging is less clear. Here, we performed comprehensive multi-omics analyses and uncovered a role for a key PUFA biosynthesis gene, ELOVL2 , in mouse and human immune cell aging. Whole transcriptome RNA-sequencing studies of bone marrow from aged Elovl2 mutant (enzyme-deficient) mice compared with age-matched controls revealed global down-regulation in lymphoid cell markers and expression of genes involved specifically in B cell development. Flow cytometric analyses of immune cell markers confirmed an aging-associated loss of B cell markers that was exacerbated in the bone marrow of Elovl2 mutant mice and unveiled CD79B, a vital molecular regulator of lymphoid progenitor development from the pro-B to pre-B cell stage, as a putative surface biomarker of accelerated immune aging. Complementary lipidomic studies extended these findings to reveal select alterations in lipid species in aged and Elovl2 mutant mouse bone marrow samples, suggesting significant changes in the biophysical properties of cellular membranes. Furthermore, single cell RNA-seq analysis of human HSPCs across the spectrum of human development and aging uncovered a rare subpopulation (<7%) of CD34 + HSPCs that expresses ELOVL2 in healthy adult bone marrow. This HSPC subset, along with CD79B -expressing lymphoid-committed cells, were almost completely absent in CD34 + cells isolated from elderly (>60 years old) bone marrow samples. Together, these findings uncover new roles for lipid metabolism enzymes in the molecular regulation of cellular aging and immune cell function in mouse and human hematopoiesis. In addition, because systemic loss of ELOVL2 enzymatic activity resulted in down-regulation of B cell genes that are also associated with lymphoproliferative neoplasms, this study sheds light on an intriguing metabolic pathway that could be leveraged in future studies as a novel therapeutic modality to target blood cancers or other age-related conditions involving the B cell lineage.

Abstract B043: Synergy of Subasumstat and anti-CD40 improves survival by augmenting tumor macrophage infiltration

Abstract Background: Identification of effective immunomodulatory strategies remains a major unmet need in pancreatic ductal adenocarcinoma (PDAC). Agonistic anti-CD40 antibody (aCD40) exhibits direct cytotoxicity on tumor cells and augments antigen presentation. Subasumstat(SUB), a sumoylation inhibitor, augments innate and adaptive immune responses by increasing interferon signaling. We hypothesized that SUB+aCD40 combination would promote an effective antitumor immune response in an aggressive orthotopic model of PDAC. Methods: 2000 KPC 46 cells were implanted in the pancreas of four groups of F1 hybrid (initial survival, CD8+ T-cell depletion, and clodronate liposome depletion) and nude mice, enrolled when tumors had reached 3-5mm. The vehicle (veh) group received 80ul SUB vehicle (q48 hrs) and 100 ug blank IgG (qwk); SUB only group received 15mg/kg q48h; aCD40 only group received 100ug qwk; SUB+aCD40 group received 15mg/kg SUB q48h and 100 ug aCD40 qwk. Single cell RNA sequencing (scRNASeq) was conducted on n=2 tumors from each group. Immunohistochemistry (IHC) was performed on veh and SUB+aCD40 tumors for macrophage infiltration using F4/80. Flow cytometry (FC) was employed for the presence of lymphoid and myeloid markers in veh, SUB+aCD40, and clodronate depleted SUB+aCD40 (SUB+aCD40+clod) groups. Results: Mice receiving SUB+aCD40 had a significantly improved median survival over those receiving monotherapy or vehicle (24.5d vs. SUB:15.5d, aCD40:20d, veh: 19d). scRNASeq revealed increased tumor infiltrating CD8+ T-cell and myeloid populations when comparing veh and monotherapy to SUB+aCD40. F4/80 IHC confirmed increased macrophage infiltration in SUB+aCD40 vs veh (p = 0.01). The survival advantage conferred by SUB+aCD40 was preserved in both CD8+ T-cell depleted mice (p=0.025) and nude mice (p=0.037), while clodronate eliminated the treatment survival advantage (p=0.39). FC showed increased tumor macrophage/monocyte infiltration in SUB+aCD40 vs veh (veh:11.9%, SUB+aCD40:22.5%, p=0.03) and reduction in SUB+aCD40+clod (12.6%, p=0.01). CD8+ T-cell CD69 expression decreased in the SUB+aCD40+clod group (SUB+aCD40:19%, SUB+aCD40+clod:6%, p&amp;lt; 0.01). CD8 T-cell infiltration (SUB+aCD40:4.95%, SUB+aCD40+clod:3.85%, p=0.27) and CD4/CD8 T-cell ratio (veh: 1.26, SUB+aCD40:0.52, SUB+aCD40+clod:0.69, p=0.09) were not affected by clodronate. Conclusion: SUB+aCD40 therapy confers a survival advantage in the KPC46 model of PDAC in a T-cell independent manner by increasing macrophage/monocyte infiltration. SUB+aCD40 promotes CD8+ T-cell infiltration and activation, but they do not contribute to survival advantage, possibly due to exhaustion. Future work will include addition of therapeutics to mitigate/reverse T-cell exhaustion to harness both innate and adaptive immunity in treating PDAC. Citation Format: Asimina Courelli, Kevin Li, James Lee, Herve Tiriac, Yuan Chen, Andrew Lowy. Synergy of Subasumstat and anti-CD40 improves survival by augmenting tumor macrophage infiltration [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B043.

AML-683 Lymphoid Markers as Predictors of Adult Acute Myeloid Leukemia Prognosis

AML-683 Lymphoid Markers as Predictors of Adult Acute Myeloid Leukemia Prognosis

Understanding the Puzzle of Primary Breast Lymphoma: An Experience in a Tertiary Care Center in North India.

Background Breast carcinoma is the most common cause of death in women arising primarily from either epithelial or stromal components. Primary breast lymphoma (PBL) is a less common type of neoplasm arising from the lymphoid tissue and is classified as non-Hodgkin's lymphoma (NHL). PBL is a rare tumor that accounts for less than 1% of total primary malignant neoplasms of the breast. The majority of PBLs are of B-cell origin; the most common histological type is diffuse large B-cell lymphoma (DLBCL) followed by follicular lymphoma (FL) and other T-cell and B-cell lymphomas. Material and methods We conducted this five-year retrospective descriptive PBL study from July 2019 to May 2024 at a tertiary care center in North India. We included 11 cases of diagnosed PBL based on morphology and confirmation by at least one lymphoid differentiation marker, that is, leukocyte common antigen (LCA). We retrieved paraffin-embedded blocks from the archive and conducted routine hematoxylin and eosin (H&E) staining and Leishman's stain. We applied an extended immunohistochemistry (IHC) panel to confirm the microscopic findings. We used the Dako® (Agilent Diagnostics & Genomics Group) antibody with a dilution of 1:100. Results In this study, patients' mean age was 46.8 years (32 to 84 years). Radiological findings indicated that the tumor was indistinguishable from carcinoma. Microscopic findings showed that cells were medium to large lymphoid cells displaying moderate pleomorphism, centrally placed nuclei, vesicular to coarsely clumped chromatin, and scant cytoplasm. IHC showed one case of T-cell origin (ALCL) and 10 of B-cell origin. Among the B-cell cases, four were DLBCL, four were high-grade NHL likely DLBCL and two were high-grade lymphoma. Conclusion Primary breast lymphoma is a rare tumor that closely resembles breast carcinoma clinically as well as radiologically. This study is an effort to highlight the importance of clinical, radiological, and histopathological parameters as well as immunohistochemical features in confirming diagnosis. We also emphasize a treatment protocol and survival outcome in hopes that this knowledge will provide an understanding of early diagnosis and proper management of disease.

Cranioencephalic functional lymphoid units in glioblastoma.

The ecosystem of brain tumors is considered immunosuppressed, but our current knowledge may be incomplete. Here we analyzed clinical cell and tissue specimens derived from patients presenting with glioblastoma or nonmalignant intracranial disease to report that the cranial bone (CB) marrow, in juxtaposition to treatment-naive glioblastoma tumors, harbors active lymphoid populations at the time of initial diagnosis. Clinical and anatomical imaging, single-cell molecular and immune cell profiling and quantification of tumor reactivity identified CD8+ T cell clonotypes in the CB that were also found in the tumor. These were characterized by acute and durable antitumor response rooted in the entire T cell developmental spectrum. In contrast to distal bone marrow, the CB niche proximal to the tumor showed increased frequencies of tumor-reactive CD8+ effector types expressing the lymphoid egress marker S1PR1. In line with this, cranial enhancement of CXCR4 radiolabel may serve as a surrogate marker indicating focal association with improved progression-free survival. The data of this study advocate preservation and further exploitation of these cranioencephalic units for the clinical care of glioblastoma.

Clinicohematological profile and immunophenotypic patterns of childhood acute leukemia: Prognostic correlation

Acute leukemia (AL) presents a heterogeneous molecular profile, requiring precise diagnostic categorization and subcategorization. The present study aims to estimate the clinicohematological profile and immunophenotypic pattern of childhood AL while conducting prognostic assessments. This cross-sectional study analyzed a total of 68 samples of AL collected from January 2019 to June 2021. The male-to-female ratio was 4.6:1, with a mean age of 6.6 &amp;plusmn; 3.4 years. Total leukocyte count (TLC) was significantly increased in all types of AL (P = 0.03). The median value (interquartile range) of TLC (&amp;times;106/dL) was 8,450 (4,100 &amp;ndash; 27,950), with blast counts in peripheral smears at 59 (24 &amp;ndash; 80), and in bone marrow aspirates (BMAs) at 95 (75 &amp;ndash; 98). There was a significant association (P &lt; 0.001) and a strong association (C = 0.9110) between the morphology of BMA with immunophenotype. Based on immunophenotype, AL was categorized into four groups: B-cell acute lymphoblastic leukemia (B-ALL) (51.5%), T-cell acute lymphoblastic leukemia (T-ALL) (10.3%), AML (22%), and mixed phenotype AL (MPAL) (16.2%). Furthermore, eight subgroups were identified: B lineage, Ia-Common-B-ALL (88.6%) and Ib-Pre-B-ALL (11.4%); T-lineage, IIa-Cortical T-ALL (71.4%) and IIb-Pre-T-ALL (28.6%); AML subgroups, IIIa-M2 (73.93%) and III-M4 (26.7%); and MPAL subgroups, IVa-aberrant expression of myeloid antigens in B-ALL (90.9%), and IVb-aberrant expression of lymphoid markers in AML (9.1%). A poor prognostic immunophenotype (T-ALL, AML) was significantly (P = 0.023) more prevalent in deceased patients with AL. The highest mortality rate was observed in AML (86.4%), followed by T-ALL (57.2%). The most common immunophenotype observed was Common-B-ALL in childhood AL, and a poor prognostic immunophenotype (T-ALL and AML) with the highest mortality rate was found in AML. Thus, knowledge about clinicohematological and immunophenotypic patterns will aid in patient management.

Granulocytic neoplasm suggestive of primary myeloid sarcoma in 3 dogs.

Myeloid sarcoma (MS) is a solid tumor of granulocytic origin with extramedullary localization. This tumor is rare in humans and animals. The diagnostic approach is heterogeneous, and the definitive diagnosis may be difficult to achieve. Primary MS has never been described as a spontaneous neoplasm in companion dogs. Two purebred and 1 mixed-breed dogs, 6- to 11-year-old, developed round cell tumors in the mediastinum, lymph nodes (LNs) and tonsils, and LNs, respectively. Granulocytic origin and exclusion of lymphoid lineage were confirmed by flow cytometry, supported by immunohistochemistry or immunocytochemistry. Pivotal to the diagnosis were positive labeling for myeloid (CD11b, CD14) and hematopoietic precursors (CD34) markers, along with negative labeling for lymphoid markers. Blood and bone marrow infiltration were not detected at initial diagnosis, excluding acute myeloid leukemia. The behavior of these tumors was aggressive, resulting in poor clinical outcomes, even when chemotherapy was attempted.

Hematopoietic stem cell transplant for adults with mixed phenotypic acute leukemia.

6517 Background: Mixed phenotype acute leukemia (MPAL) is a rare acute leukemia subtype characterized by expression of lymphoid and myeloid lineage markers on blasts. Consequently, the role of hematopoietic stem cell transplant (HCT) consolidation is unclear. We conducted a retrospective study of MPAL patients treated at our institution integrating the impact of transplant status, chemotherapy, and initial response on survival. Methods: We evaluated patients with MPAL at Fred Hutchinson Cancer Center from 1/2005 – 9/2022, some of whom were previously reported (Huang et al, ASH 2023). Independent pathology review using 2022 WHO criteria verified MPAL diagnosis. Potentially transplant eligible patients were defined as those &lt;75 years of age, diagnosed at least 6 months prior to data retrieval date, and alive 100 days after diagnosis with an evaluable induction response. Log-rank and Cox proportional hazard models were used to compare survival and adjust for baseline covariates. Survival was landmarked at 100 days. Results: Of 55 total patients, 42 were potentially HCT eligible and 30 received HCT (71.4%; 20/30 myeloablative). Patients who did not complete HCT were similar in age (median 46.2, range 18-70 vs median 50.3, range 24 – 74, p = ns) and had similar ECOG (ECOG 0-1 86.6% vs 75.0%, p = ns) at diagnosis. Nine of 12 non-transplanted patients had age-adjusted HCT-CI score ≤ 3. Patients receiving a HCT had better progression free survival (PFS) (p = 0.025) and were more likely to be alive at 48 months (61.6% vs. 32.4%, p = 0.011) with a median overall survival (mOS) of not reached (NR) (95% CI, 43 months – NR) compared to a mOS of 13 months (95% CI, 9 months – NR) for those not transplanted. Given that our prior work showed that the type of induction chemotherapy did not influence OS (p = 0.40) or remission status (p = 0.16) (Huang et al. ASH 2023), we investigated impact of remission status at the time of transplant. Differences in PFS and OS persisted when stratifying patients by induction response status with HCT status (PFS p = 0.0048; OS p &lt; 0.0001). Patients who had a complete response (CR) followed by HCT had the longest mOS = NR (17 of 24 patients alive after 48 mo). Patients who had resistant disease (RD) and completed HCT had mOS = 43 months (95% CI, 43 months– NR). Patients who achieved CR but did not undergo HCT had mOS = 19 months (95% CI, 11 months – NR). Patients who had RD and did not undergo HCT had median OS of 6 months (95% CI, 6 – NR). The hazard ratio for death among patients who underwent HCT was 0.28 (95% CI 0.077 – 0.99, p = 0.049). Conclusions: HCT consolidation was significantly correlated with improved survival (p = 0.011). While potentially biased by selection and limited by small sample size, patients benefitted from undergoing a HCT regardless of response to prior therapy (p &lt; 0.0001). This study suggests that patients with MPAL in CR after induction chemotherapy as well as those with less than complete responses should be considered for HCT consolidation.

A Comprehensive Flow Cytometry Panel for Analysis of Idiopathic Subglottic Stenosis.

To present a comprehensive flow cytometry panel for idiopathic subglottic stenosis (iSGS). Controlled ex vivo cohort study. Tertiary care academic hospital in a metropolitan area. Flow cytometry and single-cell RNA sequencing were performed on 9 paired normal and scar tissue samples from iSGS patients. Flow cytometry was used to assess the presence of myeloid (CD11b, CD14, CD15, Siglec8), lymphoid (CD3, CD4, CD8, gamma delta [γδ], FOXP3), endothelial (CD31), fibroblast (CD90, SMA), and epithelial (CD326, CK5) markers. On flow cytometry, iSGS scar is characterized by an increased presence of myeloid, lymphoid, endothelial, and fibroblast cell types, but a decreased presence of epithelial cells. In the myeloid lineage, iSGS scar samples demonstrated increased CD11b+ monocytes (P < .001), Siglec8+ eosinophils (P = .03), and CD14+ monocytes (P = .02). In the lymphoid lineage, iSGS scar demonstrated increased CD3+ T-cells (P < .001), CD4+ helper T-cells (P < .001), γδ+ T-cells (P < .001), and FOXP3+ regulatory T-cells (P = .002). iSGS scar exhibited specific increases in CD90+ (P = .04) and SMA+ (P < .001) fibroblasts but decreased CD326+ (E-cadherin) epithelial cells (P = .01) relative to normal samples. We present a comprehensive flow cytometry panel for iSGS. This flow panel may serve as a common platform among airway scientists to elucidate the cellular mechanisms underpinning iSGS and other upper airway pathologies. Scar iSGS samples demonstrate a distinct cellular profile relative to normal iSGS specimens, exhibiting increased fibroblast, endothelial, and inflammatory cell types but decreased epithelium.

Lymphoid markers as predictors of adult acute myeloid leukemia prognosis

Background Acute myeloid leukaemia (AML) constitutes the most prevalent aggressive leukaemia in adults, with uncontrolled proliferation of immature myeloid cells and a variety of clinical manifestations. Whereas most AML patients respond to chemotherapy initially, half of all patients relapse within 5 years of being diagnosed. Aim and objectives The present study aimed to investigate the prognostic value of co expression of lymphocytes markers in patients with acute myeloid leukemia as regarding clinical outcome and survival. Patients and methods The present study was enrolled at Hematology Unit at Nasser Institute Hospital. This is a retrospective study done on (50) patients aged from 18 to 65 years, completion of treatment (or part of treatment undertaken), and follow-up duration of at least six months after diagnosis. Results The present study results showed a statistically significant difference between patients who achieved complete remission and those who relapsed or were refractory to treatment in the lymphoid markers CD7 and CD19. CD7 was the most frequent lymphoid marker. The lymphoid marker CD4 was associated with a higher complete remission rate. CD19 was associated with the highest relapse rate. Conclusion CD7 was the most frequent lymphoid marker. The lymphoid marker CD4 was associated with a higher complete remission rate compared to CD7, CD5, CD2 and CD19; While CD19 was associated with the highest relapse rate

Abstract 4169: A deep learning approach to guide acquisition region selection for imaging mass cytometry

Abstract The growth in cancer immunotherapy agents requires an understanding of the immune contexture of the tumor microenvironment. This can be aided by high-plex imaging and analysis to obtain phenotypes of specific cells and study their biodistribution and interactions. Imaging Mass Cytometry™ (IMC) is the method of choice for single-step staining and high-plex imaging of tissues, avoiding the complications of autofluorescence and cyclic imaging. IMC has expanded its capabilities with three distinct imaging modes: Preview, Cell, and Tissue. The Preview Mode is a rapid scanning system that captures a comprehensive overview of the stained tissue, mapping out the distribution of over 40 markers and revealing tissue heterogeneity. This enables researchers to make informed decisions about which areas warrant closer examination on the same. Building on this, Cell Mode offers high-resolution imaging for detailed analysis of the Regions of Interest (ROI) identified during Preview, all using the same slide. Tissue Mode complements these by providing a fast acquisition of the entire tissue at a lower resolution, which is optimal for quantitative pixel-based analysis of tissue biology. These modes support automated, continuous imaging of more than 40 large tissue samples (400 mm2) weekly. Following Preview Mode, the selection of ROIs for high-resolution imaging is a critical step, enhanced by automated AI algorithms to ensure it is informed by biomarker expression. Tissue sections of colon adenocarcinoma were stained with a 30-marker IMC panel of structural, tumor, stroma, immune cell, and immune activation markers. Images were acquired on Hyperion XTi™ (Standard BioTools), first in Preview Mode, then in Cell Mode with automatic selection of ROIs using Phenoplex™ (Visiopharm). ROIs were automatically selected based on two criteria: 1) actively proliferating and non-proliferating tumor regions; 2) cold and hot tumor regions as identified by hotspots of lymphoid and myeloid immune markers within stromal or epithelial tumor regions. Single cell analysis of the high-resolution images obtained in Cell Mode, was performed as a multi-step workflow using Phenoplex. Tissue segmentation was used to divide the tissue into epithelial tumor (E-Cadherin/panCK), stromal, and tumor margin regions; cell segmentation was based on Iridium DNA channels; and phenotyping was performed using the guided highplex workflow. These data were used to compare the immune contexture through a series of t-SNE plots partitioned by spatial region and clinical variables. This work demonstrates that the multi-modal features of Hyperion XTi can greatly accelerate the ability of IMC users to gain useful insights from complex biological samples. Phenoplex enables a comprehensive workflow for the analysis of this complex data, providing automated ROI selection, and phenotyping and spatial analyses of high-resolution IMC images for biological assessment. Citation Format: Fabian Schneider, Smriti Kala, Sam Lim, Clinton Hupple, Nina Lane, James Robert Mansfield. A deep learning approach to guide acquisition region selection for imaging mass cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4169.

Flow cytometric detection of leukemic blasts in Libyan pediatric patients with acute lymphoblastic leukemia

ABSTRACT The diagnosis of acute lymphoblastic leukemia (ALL), which is the most common type of cancer in children, has become more accurate with the use of flow cytometry. Here, this technology was used to immunophenotype leukemic cells in peripheral blood samples from Libyan pediatric ALL patients. We recruited 152 newly diagnosed patients at Tripoli Medical Center (Tripoli, Libya) by morphological examination of blood and bone marrow. Twenty-three surface and cytoplasmic antigen markers were used to characterize B and T cells in circulating blood cells by four-color flow cytometry. Six children (3.9%) turned out to have biphenotypic acute leukemia, 88 (57.9%) had B ALL, and 58 (38.1%) had T ALL. There were 68 cases of pro-B ALL CD10-positive (44.7%), 8 cases of pro-B ALL CD10-negative (5.2%), 6 cases of pre-B ALL (3.9%), and 6 of mature-B ALL (3.9%). CD13 was the most commonly expressed myeloid antigen in ALL. We present immunophenotypic data for the first time describing ALL cases in Libya. The reported results indicate that the most common subtype was pro-B ALL, and the frequency of T-ALL subtype was higher compared to previous studies. Six cases were positive for both myeloid and B lymphoid markers. Our findings may provide the basis for future studies to correlate immunophenotypic profile and genetic characteristics with treatment response among ALL patients.

Flow cytometric-based detection of CD80 is a useful diagnostic marker of acute myeloid leukemia in dogs.

CD80, a co-stimulatory molecule required for optimal T cell activation, is expressed on antigen-presenting cells, including monocytes and dendritic cells, in dogs and humans. We hypothesized that CD80 would be expressed on tumor cells in dogs from acute myeloid leukemia (AML) but not dogs with lymphoid neoplasms. We first evaluated the cellular staining pattern of a hamster anti-murine CD80 antibody (clone 16-10A1, ThermoFisher Scientific Cat# 17-0801-82, RRID: AB_469417) in blood and bone marrow aspirates from healthy dogs. Using flow cytometric analysis and examination of modified Wright's-stained cytologic smears of unsorted and flow cytometric or immunomagnetic bead-sorted leukocytes, we show that the antibody binds to mature and immature neutrophils and monocytes, but not lymphocytes or eosinophils, in blood and bone marrow. We then added the antibody to routine flow cytometric panels for immunophenotyping hematopoietic neoplasms in dogs. We found that the antibody labeled tumor cells in 72% of 39 dogs with AML and 36% of 11 dogs with acute leukemia expressing lymphoid and myeloid markers ("mixed lineage") but none of the dogs with B (n = 37) or T (n = 35) lymphoid neoplasms. A higher proportion of tumor cells in dogs with AML were labeled with the anti-CD80 antibody vs antibodies against other myeloid-associated antigens, including CD4 (36%, p = 0.003), CD11b (44%), CD11c (46%), CD14 (38%, p = 0.006) and CD18 (59%, clone YFC118). In contrast, antibodies against CD11b and CD11c bound to tumor cells in 8-32% of the lymphoid neoplasms. We show that CD80, as detected by antibody clone 16-10A1, is a sensitive and specific marker for AML and would be useful to include in flow cytometric immunophenotyping panels in dogs.

Not all hematological malignancies in the gastrointestinal tract are lymphoma

Abstract Introduction/Objective Myeloid sarcoma (MS) is a rare entity that may develop in patients with acute myeloid leukemia (AML). Presentation of MS in the gastrointestinal (GI) tract is uncommon. We describe two cases of MS involving unusual sites: the stomach and appendix. Methods/Case Report A 64-year-old female consulted for fatigue and abdominal pain. A CT scan of abdomen/pelvis revealed appendicitis and CBC showed 76% blasts. An appendectomy was performed. Histologic examination demonstrated the appendiceal wall infiltrated by malignant cells, ultimately demonstrated to be positive for MPO and weak/variable CD117. In the second case, a 50-year-old male presented to the emergency department for nausea, vomiting, and elevated amylase. Esophagogastroduodenoscopy with endoscopic ultrasound demonstrated pancreatic inflammatory changes and a subepithelial nodule in the gastric fundus. Biopsy of the nodule revealed gastric epithelium infiltrated by sheets of malignant cells, ultimately demonstrated to be positive for CD34, myeloperoxidase and CD43. Results (if a Case Study enter NA) NA Conclusion MS is defined as a tumor of myeloid blasts forming outside the bone marrow. Overall, it is an uncommon entity that can occur in patients with known AML, and rarely, as the first sign of AML. Presentation of MS in the GI tract is infrequent (10%), involving primarily the small intestine (63%). The differential diagnosis includes lymphoma, poorly differentiated carcinomas, and melanomas. A review of the clinical history for AML or other myeloproliferative neoplasms should raise suspicion of MS. Expression of lymphoid markers and scant tissue for IHC studies can be diagnostic challenges, rendering B cell lymphoma as the most common misdiagnosis of MS in up to 40-47% of cases. Our two MS cases presented exclusively with GI symptoms without previously known AML and involved unusual GI sites. IHC for myeloid lineage, monocytic or myelomonocytic differentiation should be considered in specific cases. In patients without prior AML diagnosis, especially without hematologic findings, MS is a diagnostic challenge.

Stag2a or Stag2b Knockout Zebrafish Are Viable, Yet Loss of Stag2a Is Associated with Abnormal Expression of Early and Late Myeloid and Lymphoid Markers

Introduction:Mutations in Stromal antigen 2 ( STAG2) are frequently found in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). In mice, loss of Stag2 is embryonic lethal. Conditional knockout of Stag2 in adult mice results in MDS-like hematopoietic anomalies. Zebrafish harbor two paralogues of STAG2 ( stag2a and stag2b). To date, a complete stag2-null zebrafish model organism has not been developed. Recently reported, a stag2b-/- zebrafish demonstrated downregulation in primitive erythropoiesis. However, any hematopoiesis beyond 2 days post fertilization (2 dpf) was not detailed. Furthermore, a stag2a mutant zebrafish has not been reported. Methods and Results: Using CRISPR/Cas9 genome editing, we created a stag2a mutant anda stag2b mutant zebrafish. Both mutations result in a frame shift and premature protein truncations. Unlike the murine Stag2 -/- mutant, zebrafish stag2a -/- mutants and stag2b -/-mutants survive to adulthood, with no obvious morphological phenotypes. Detection of hemoglobin, using O-dianisidine staining, reveals anemia at 31 hours post fertilization (31 hpf) in stag2a +/- mutants (10/29), stag2a -/-mutants (10/15), stag2b +/-mutants (6/11), and stag2b -/-mutants (5/7); wildtype siblings appear unaffected. Intriguingly, at 4 dpf, stag2a maternal-zygotic mutants show increased expression levels of early and late myeloid and lymphoid markers. ( Figure 1) Work is ongoing to analyze blood cell lineages from zebrafish whole kidney marrow, the equivalent of mammalian bone marrow, in adult stag2a and adult stag2b mutants. In addition, we are characterizing the zebrafish stag2a;stag2b double mutants, which we will present. Conclusions: In zebrafish, stag2a and stag2b contribute to proper primitive and definitive hematopoiesis. Our organismal model of Stag2 deletion in the zebrafish will yield new insight into how loss of STAG2 may lead to MDS/AML.

Inflammatory Signatures Define a New High-Risk T-Lineage ALL Subtype

T-lineage acute lymphoblastic leukemia (ALL) is an aggressive cancer comprising of diverse subtypes that are challenging to stratify using conventional immunophenotyping and have historically exhibited poor treatment outcomes in response to cytotoxic chemotherapy ( Nguyen, K et al. Leukemia 2008). Early T-cell Precursor ALL (ETP-ALL) represent a distinct subtype that displays a developmental block at the earliest stages of T-cell commitment, accompanied by the aberrant expression of myeloid and stem cell markers ( Coustan-Smith, E. et al. Lancet Oncol. 2009). The classification can be further complicated by T/Myeloid Mixed phenotype acute leukemia (T/My-MPAL), a rare and aggressive malignancy characterized by blasts with both T-cell lymphoid and myeloid markers ( George, B. S. et al. Biomedicines 2022). Accurate clinical diagnosis of T-lineage ALL is hindered by phenotypic variations among its subsets. Nevertheless, the precise diagnosis holds critical importance as drug sensitivity in preclinical models of T-lineage ALL is closely linked to the differentiation state, such as the sensitivity of ETP-ALL to the BCL-2 inhibitor venetoclax ( Chonghaile, T. N. et al. Nat Cancer 2021). This highlights the critical need to identify consistent phenotypic traits associated with unique therapeutic vulnerabilities to effectively tailor therapy to individual patients. To gain insights into subset-specific therapeutic vulnerabilities, we performed an integrative multiomic analysis of bone marrow (BM) samples (n=21) from newly diagnosed T-lineage ALL patients, including T-cell ALL (T-ALL), ETP-ALL, and T/My-MPAL. Leveraging cellular indexing of transcriptomes and epitopes in conjunction with T-cell receptor sequencing, we identified a distinct subset of patients spanning all three subtypes characterized by the high frequency of leukemic cells that most closely resemble healthy hematopoietic stem and progenitor cells (HSPC) by Symphony ( Kang, J. B. et al. Nat Commun 2021) query mapping, indicative of an earlier stage of differentiation arrest ( Figure 1A bottom). Pathway analysis revealed an inflammatory signature in patients with high frequencies of stem-like cells suggestive of an inflammatory microenvironment akin to high-risk AML ( Lasry, A. et al. Nat Cancer 2023), which we refer to as inflammatory T-lineage ALL. Next, we mined our multiomic data to generate a comprehensive geneset for inflammatory T-lineage ALL scoring in single cells and bulk samples ( Figure 1A top). Interestingly, Olink cytokine array analysis uncovered upregulation of production of cytokines IL-1β, IL-18, and/or OSM in inflammatory T-lineage ALL samples. Thus, this comprehensive multiomic analysis identified a subset of T-lineage ALL patients characterized by early differentiation arrest and inflammatory signatures. To explore the relationship between inflammatory T-lineage status and clinical response, we utilized the ssGSEA algorithm to score activity of our inflammatory signature in bulk RNA-sequencing data from the TARGET T-lineage ALL dataset (n=265, 74/265 inflammatory) ( Liu, Y. et al. Nat Genet 2017) and the Tran et al. study (n=27, 17/27 inflammatory) ( Tran, T. H. et al. Blood Adv 2022). We found a significant association between high inflammatory ssGSEA score and elevated MRD. To uncover the inflammatory T-lineage ALL mutational landscape, we performed whole exome sequencing on our study cohort, and compiled the reported mutations in the TARGET dataset. Interestingly, inflammatory T-lineage ALL patients, as predicted by inflammatory signature activity, had a significantly higher prevalence of mutations affecting cytokine signaling ( NRAS, JAK3), chromatin remodeling genes ( EZH2), and the WT1 oncogene. These analyses demonstrate a significant relationship between inflammatory status, genomic lesions, and MRD. Finally, we demonstrate that inflammatory, but not non-inflammatory, T-lineage ALL cells are significantly more sensitive to BCL-2 inhibitor venetoclax ex vivo (Figure 1B).Overall, our study has identified a new subtype of T-lineage ALL defined by an early arrest in T-cell development, inflammatory signaling, a unique mutational landscape, sensitivity to venetoclax, and an association with adverse clinical parameters.

Molecular Subtypes in BCR::ABL1-Positive ALL Are Defined By Cellular Origins and Cooperating Genomic Events

Two developmental trajectories are acknowledged in BCR::ABL1-positive acute lymphoblastic leukemia (ALL) as distinct diagnostic entities by the ICC classification: ‘lymphoid-only’ with BCR::ABL1 restricted to the leukemic B precursor compartment vs. ‘multilineage’ with BCR::ABL1 involvement also in other hematopoietic lineages. Diagnostic standards for establishing this distinction are lacking and associated biological and clinical features are insufficiently characterized. To establish developmental trajectories, biological phenotypes, and clinical impact we analyzed n=277 BCR::ABL1-positive ALL patients (age: 2-84 years, median: 46) including our GMALL reference data set (n=113) and two independent validation cohorts (MLL: n=61; St Jude's: n=103; Gu Z, et al. Nat Genet. 2019). Unsupervised gene expression analysis (RNA-Seq: n=277) identified two major gene expression clusters with two further sub-clusters each (Figure A). This clustering was confirmed by a machine learning classifier trained on the GMALL data set which achieved similar grouping of samples in the external validation cohorts. BCR:: ABL1-FISH on FACS-sorted bone marrow/peripheral blood samples revealed the presence of BCR::ABL1 in leukemic as well as myeloid cells in n=18/18 samples from the 1 st major cluster (termed 'multilineage'). In the 2 nd major cluster ('lymphoid'), BCR::ABL1 was restricted to the lymphoid lineage in n=13/16 samples (p&amp;lt;0.001), with n=3/16 cases harboring BCR::ABL1-positive myeloid cells at lower frequencies compared to the multilineage cluster. In each cluster, two patients also harbored BCR::ABL1-positive mature B cells. T cells remained BCR::ABL1-negative. Diagnostic FACS data and analysis of proximity to normal human lymphoid gene expression confirmed more frequent myeloid co-expression and higher proximity to normal pro-B cells in the multilineage cluster, whereas lymphoid cases had increased lymphoid surface marker expression and a stronger proximity to normal pre-B I cells. Genomic profiling using WGS/SNParrays (n=160) revealed significant enrichment for genomic events in the four gene expression sub-clusters: focal deletions comprising exons 1 and 2 of HBS1-like translational GTPase ( HBS1L) or monosomy 7 were strongly enriched in the two multilineage sub-clusters ('delHBS1L'; n=20/27 vs. n=3/133 in remaining cohort or 'del7'; n=16/25 vs. n=10/135; p&amp;lt;0.001). Remarkably, a novel alternative HBS1L transcript with a putative TSS in intron 3 was highly expressed in both multilineage sub-clusters but not in BCR::ABL1 negative ALL cases or healthy B lymphoid progenitors, suggesting this transcript as novel cooperating event in multilineage BCR::ABL1-positive ALL. One lymphoid sub-cluster was enriched for homozygous deletions in IKZF1 ('IKZF1'; n=11/55 vs. n=4/105; p=0.008), whereas the other was enriched for homozygous CDKN2A/B deletions (n=21/53 vs. n=3/107; p&amp;lt;0.001) and PAX5 deletions (n=33/53 vs n=26/107; p&amp;lt;0.001; 'CDKN2A/PAX5'). Hyperdiploid karyotypes were exclusive to the lymphoid main cluster, mostly in CDKN2A/PAX5. We analyzed the clinical implications of these newly established BCR::ABL1-positive ALL subtypes in our homogenously treated GMALL adult patient cohort (n=98, first diagnosis 2014-2021) including TKI treatment combined with age-adapted chemotherapy, MRD monitoring and allo-SCT in 1 st CR (n=84/98, 86%). Overall survival (OS) at 3 years was uniformly high in multilineage and lymphoid cases (70%±6% vs. 70%±8%; p=0.890; Figure B). However, analysis of the sub-clusters revealed an inferior 3-years OS for the IKZF1 -/- enriched cluster in contrast to excellent outcome in hyperdiploid cases and intermediate outcomes in the remaining sub-clusters (Figure B). These data highlight that transcriptomic signatures in BCR::ABL1-positive ALL are driven by developmental disease origins (‘lymphoid’ vs. ‘multilineage‘) and specific patterns of corresponding genomic events. Novel molecular subtypes of BCR::ABL1-positive ALL have distinct outcomes in the context of current GMALL treatment protocols. Gene-expression based subtype definitions enable classification of BCR::ABL1-positive ALL according to ICC definitions based on RNA-Seq data alone. These definitions have been implemented in ALLCatchR - our freely available tool for ALL subtype allocation - to facilitate validation and application in clinical diagnostics.

The ELN 2022 Risk Stratification Has No Impact on the Dismal Prognosis of Patients with Acute Undifferentiated Leukemia

Introduction: Acute undifferentiated leukemia (AUL) is a rare subtype of acute leukemia. It is characterized by lack of expression of myeloid or lymphoid lineage markers, frequently expressing CD56 or CD7. The dismal prognosis of AUL was reported in small studies; the largest previously reported group with this disorder included 24 patients (Weinberg, Mod Pathol, 2019). Furthermore, data are lacking regarding cytogenetic and molecular patterns of AUL. In this study, the cytogenetic and molecular changes of AUL patients are described, together with the clinical outcome. Methods: AUL was defined, in accordance with the WHO classification, as an acute leukemia that does not qualify for myeloid, B- or T-cell lineage. Immunophenotyping of BM cells at diagnosis was performed using 8-color flow cytometry. Multi-parameter Cytognos acute orientation tube (ALOT) and the combination of the following antigens were used: CD34/CD117/CD14/CD56/CD36/CD123/HLA-DR/CD45/CD64/CD11c. (Weinberg, Mod Pathol, 2019). Results: Three hundred and eighty-five patients with acute myeloid leukemia (AML) were diagnosed at Rambam Health Care Campus in Haifa, Israel between April 2015 and April 2021, of whom 30 (8%) were defined as AUL. The baseline characteristics are presented in Table 1. The median age was 62 years, none of the patients harbored NPM1 mutation, with 1 patient expressing FLT3-ITD mutation. Cytogenetic analysis was completed for all patients: 9 (30%) had adverse-risk cytogenetics by ELN 2022 criteria. A comprehensive molecular study using molecular inverted probe was performed on all patients, with final results available on 17. Nine of these 17 (53%) had high-risk mutations; RUNX1 was found in 4 patients. Notably, 4 other patients had JAK2 mutation, with or without previous myeloproliferative neoplasm. Only 3 patients had mutations characterizing secondary AML (Lindsley, Blood, 2015). Only 2 patients in this cohort had TP53 mutation or del17p. Fourteen patients were classified as adverse-risk AML by ELN 2022 using cytogenetic and molecular markers, while 15 were classified as intermediate risk. Only 4 patients in this adverse-risk cohort were not allogeneic hematopoietic stem cell transplant (HSCT) candidates due to age or comorbidity limitations. The outcome of the cohort is presented in Table 2. Twenty-two fit patients received 7+3 intensive chemotherapy, of whom 10 achieved complete remission (CR) after induction. Four of the 10 patients needed reinduction chemotherapy due to a high blast count on the bone marrow performed at day 14. Five patients were treated with low-intensity therapies such as hypomethylating agents ± novel agents; 2 achieved CR. One patient received FLAG-Ida chemotherapy and another patient was treated with ECOG1910 protocol due to clinical presentation more compatible with acute lymphoid leukemia; both achieved CR. Of the 14 patients achieving CR, 4 relapsed before HSCT and needed further salvage therapy. Eventually, 13 (50% of the transplant-eligible candidate cohort) patients proceeded to HSCT. Relapse occurred in 14 patients (10 post-HSCT and 4 before HSCT), none of them achieved long-term survival. Four of the 30 patients are alive, all in remission. The median overall survival of the entire cohort is 1 year (Fig 1). The outcome of patients with adverse or intermediate risk according to the ELN risk classification did not differ. The CR rate and relapse rate and median overall survival (OS) were similar in both groups (Table 2; Fig 1). Discussion: These data emphasize the dismal outcome of patients diagnosed with AUL, with the 5-year OS of 7.5%, far lower than the survival of other types of acute leukemia. In this cohort, 15 patients were classified as intermediate risk AML; yet, their CR rate and long-term survival were not different from the whole cohort. The CR rate achieved with the widely used 7+3 protocol, was inferior, raising the question whether this protocol should be the “standard of choice” in such patients. A limitation of this study is that not all patients were evaluated by the NGS panel, bringing about the issue whether more patients had high-risk mutations, who would then be classified as adverse risk. Conclusion: The unique subtype of AUL is associated with a particularly adverse risk genotype, irrespective of known prognostic determinants, as in the 2022 ELN classification.

Unraveling Clonal Evolution and Mechanisms of Treatment Resistance in Murine KMT2A-Rearranged Leukemia with Subclonal FLT3 N676K

Our understanding of how individual mutations, whether present in all or just a subset of the leukemia cells, affect cellular response to therapy is limited. Herein, we investigate the impact of different therapies on survival, evolution, and resistance patterns in an acute myeloid leukemia (AML) mouse model driven by KMT2A::MLLT3 and subclonal FLT3 N676K. Leukemic bone marrow expressing KMT2A::MLLT3-mCherry in all cells and FLT3 N676K-GFP in 40% of cells was re-transplanted into sublethally irradiated recipients. Upon engraftment, recipients were subjected to different treatments, including chemotherapy (cytarabine for 5 days+doxorubicin for 3 days), the FLT3 inhibitor AC220, chemotherapy followed by AC220, or AC220+Trametinib, a MEK inhibitor. Targeted treatment was administered for 28 days, while the control group received vehicle ( Fig 1a). Each treatment prolonged survival with a median latency of 30 days for chemotherapy 37.5 days for AC220, 42 days for chemotherapy+AC220, and 45 days for AC220+Trametenib, and 25.5 days for control mice. At sacrifice, all mice displayed splenomegaly and leukocytosis. The treatment impacted the evolution of the KMT2A:: MLLT3+ FLT3 N676K cells and they expanded to clonal dominance in control and chemotherapy-treated mice. However, three distinct patterns of evolution were detected in AC220-treated mice with the FLT3 N676K-containing cells either expanding to clonal dominance (&amp;gt;80% of KMT2A:: MLLT3+ FLT3 N676K), the FLT3 N676K-containing cells being eradicated (&amp;gt;80% of KMT2A::MLLT3-alone), or dual similar sized clones expressing KMT2A:: MLLT3-alone or KMT2A::MLLT3+ FLT3 N676K(Fig1 b).Eradication of the FLT3 N676K-cells was rare, predominantly observed upon AC220+Trametinib treatment and the frequency of dual clones increased when chemotherapy was followed by AC220, in line with treatment affecting the evolution of genetically distinct clones. RNA-sequencing was used to determine altered gene expression patterns upon resistance and unveiled three major clusters including 1) mice expressing KMT2A:: MLLT3-alone, 2) control and chemotherapy-treated mice, and 3) AC220-treated mice. Notably, a subset of AC220-treated mice clustered close to the control and chemotherapy-treated mice suggesting potential resistance mechanisms through activation of the MEK/ERK-pathway. All leukemias had a myeloid Cd11b + phenotype. However, the control and chemotherapy-treated leukemias also displayed an aberrant expression of the lymphoid marker B220 and showed enrichment of gene sets correlating with stemness and oxidative phosphorylation, that suggested a switch in cellular phenotype and metabolic state. In contrast, AC220-treated leukemias lacked or had a low fraction of B220 + cells and showed enrichment of gene sets correlating with granulocyte-monocyte progenitors (GMP) and immune regulatory pathways (Fig 1 c). Acquired AC220-resistance mutations was rare (n=2), suggesting activation of alternative pathways upon resistance. Re-treatment of established cell lines from treated animals with AC220 showed that the cells were resistant. To uncover mechanisms of resistance at the cellular level, single cell RNA-sequencing was performed on four mice and revealed the emergence of distinct cellular subpopulations in AC220-treated mice, with a strong GMP signature and upregulation of the homeobox gene Six1(Fig 1d).Overexpression of Six1 in all AC220-treated leukemias was further confirmed by bulk RNA-sequencing. Interestingly, Six1 has previously been described as a regulator for leukemia stem cell maintenance in AML and under Wnt/ß-catenin signalling control. Subsequent drug assays showed AC220-treated cells exhibited sensitivity to Six1 pharmacological inhibition, suggesting a dependency on Six1, as well as sensitivity to Wnt/ß-catenin inhibition. Taken together, the treatment given affected survival and impacted evolution of the FLT3 N676K-leukemia cells. The general absence of acquired mutations in most mice suggests that target-independent mechanisms may underline acquired resistance in most mice, and we propose the Six1/Wnt/ß-catenin axis as a potential vulnerability upon AC220-resistance.