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We encountered a 34-year-old man with a significant vaping history who presented with acute hypoxic respiratory failure, headaches and gastrointestinal symptoms during the COVID-19 pandemic. Based on his laboratory and chest CT findings, we had a high clinical suspicion for COVID-19 and EVALI, yet distinguishing the diagnoses proved challenging. His SARS-CoV-2 nasopharyngeal PCR swabs were negative. However, this test has a low sensitivity. COVID-19 serology testing was negative, and the patient was ultimately diagnosed with EVALI based on subtle chest CT findings. To our knowledge, this is the first paper highlighting the similarities and distinguishing features of EVALI and COVID-19 acute lung injury.

INTRODUCTION: Reported gastrointestinal manifestations from COVID-19 include transaminitis, acute hepatitis, and mild pancreatic injury;but biliary manifestations have not been reported In this case report, we describe two cases of acute acalculous cholecystitis (AAC) in patients with prolonged hospitalizations for COVID-19 CASE DESCRIPTION/METHODS: A 60-year-old woman with hypertension, type 2 diabetes and hypothyroidism and separately, a 68-year-old man with hypertension, hyperlipidemia, asthma, and obstructive sleep apnea were both admitted during the peak of the SARS-CoV2 pandemic in New York City for acute hypoxic respiratory failure secondary to acute respiratory distress syndrome (ARDS) from COVID-19 pneumonia Both patients were diagnosed with acute acalculous cholecystitis on a general medicine service after a prolonged hospital course including mechanical ventilation (Table 1) Both patients improved clinically after placement of a cholecystostomy tube and antibiotics DISCUSSION: Acute acalculous cholecystitis accounts for 2-15% of all cases of acute cholecystitis and results from ischemia and stasis of the gallbladder, often from critical illness Whereas AAC has been described in a variety of clinical settings including trauma, recent surgery, sepsis and enteral fasting, it has not been described in the context of severe COVID-19 infection requiring prolonged hospitalization It is unclear whether AAC is a direct manifestation of the SARS-CoV2 virus directly or a consequence of prolonged illness from the virus Notably, both of our patients had long ICU stays for COVID-19 but were diagnosed with AAC when they were more clinically stable on a non- ICU medicine service, suggesting that this manifestation may not be merely from severe critical illness alone In patients with a strong clinical suspicion, including new leukocytosis and right upper quadrant pain or bacteremia with gastrointestinal pathogens, providers should not be reassured by non-diagnostic CT or ultrasound imaging because none of these imaging modalities have good sensitivity for AAC COVID-19 is proving to have multiorgan manifestations during its disease course and providers should not fail to recognize biliary complications such as acute acalculous cholecystitis Further studies are necessary to assess the incidence of AAC in patients with prolonged hospitalization from COVID-19 (Figure Presented)

Leukocytosis is a common feature of malignancies. While controversial, there appears to be an association between the degree of tumor-related leukocytosis and prognosis. In this paper, we provide evidence supporting an untapped clinical paradigm linking G-CSF secretion to the induction of leukocytosis and expansion of myeloid-derived suppressor cells, providing an explanation for the association between leukocytosis, elevated neutrophil-to-lymphocyte ratios and prognosis in non-small cell lung cancer. Clinically validating this mechanism may identify MDSCs and G-CSF as dynamic markers of early disease progression and therapeutic response, and shed light onto novel therapeutic avenues for the treatment of patients with non-small cell lung cancer.

Introduction: Acute myeloid leukemia (AML) and the myelodysplastic syndromes (MDS) are clonal hematopoietic neoplasms that arise from leukemia stem cells (LSCs) and hematopoietic stem cells (HSCs), respectively. Standard chemotherapy can efficiently eliminate the bulk of neoplastic cells, however, LSCs and MDS HSCs are relatively resistant to these therapies and can reinitiate and maintain disease. CD99 is a 32-kDa transmembrane polypeptide that is highly expressed on disease stem cells in the vast majority of AML and MDS.Areas covered: In this editorial, we focus on the current literature surrounding the identification of CD99 as a marker of MDS and AML stem cells and preclinical studies revealing the therapeutic efficacy of targeting CD99 in these diseases.Expert opinion/commentary: Cytotoxic CD99 monoclonal antibodies represent promising stem cell-directed therapies that have the potential to markedly improve clinical outcomes for these difficult-to-treat hematologic malignancies.

The shoot apical meristem (SAM) acts as a reservoir for stem cells. The central zone (CZ) harbors stem cells. The stem cell progenitors differentiate in the adjacent peripheral zone and in the rib meristem located just beneath the CZ. The SAM is further divided into distinct clonal layers: the L1 epidermal, L2 sub-epidermal and L3 layers. Collectively, SAMs are complex structures that consist of cells of different clonal origins that are organized into functional domains. By employing fluorescence-activated cell sorting, we have generated gene expression profiles of ten cell populations that belong to different clonal layers as well as domains along the central and peripheral axis. Our work reveals that cells in distinct clonal layers exhibit greater diversity in gene expression and greater transcriptional complexity than clonally related cell types in the central and peripheral axis. Assessment of molecular functions and biological processes reveals that epidermal cells express genes involved in pathogen defense: the L2 layer cells express genes involved in DNA repair pathways and telomere maintenance, and the L3 layers express transcripts involved in ion balance and salt tolerance besides photosynthesis. Strikingly, the stem cell-enriched transcriptome comprises very few hormone-responsive transcripts. In addition to providing insights into the expression profiles of hundreds of transcripts, the data presented here will act as a resource for reverse genetic analysis and will be useful in deciphering molecular pathways involved in cell type specification and their functions.

PTPN11 mutations occur in roughly 5-10% of AML patients. There is no defined impact on prognosis in the literature, although a single center analysis from our group suggested an association with poor outcomes. The pathogenesis of activating PTPN11 mutations is hypothesized to result in increased signaling through RAS resulting in uncontrolled cellular proliferation. PU-H71 is an epichaperome inhibitor that has shown pre-clinical promise in ex-vivo and PDX models of AML (Cell Reports 2015). The efficacy of the drug is dependent on the tumor possessing epichaperomes, hyperconnected networks of chaperones and co-chaperones facilitating tumor survival (Nature 2016, Nature Medicine 2018). Prior studies have found an association between epichaperome abundance and overactivity of the transcription factor MYC. Activating PTPN11 mutations have also been shown to result in MYC overactivity through increased SHP2 signaling (protein product of PTPN11 gene) and thus we hypothesized that through increased activity of MYC, activating PTPN11 mutations in AML lead to an abundant epichaperome network, and in turn render these tumors sensitive to the epichaperome inhibitor, PU-H71. 15 PTPN11 mutated AML peripheral blood and bone marrow samples from two academic medical centers (Weill-Cornell Medical Center and University of Colorado) were collected to be analyzed for epichaperome abundance and sensitivity to the epichaperome inhibitor PU-H71. To detect epichaperome abundance, PTPN11 mutated AML samples were incubated with FITC-bound PU-H71 (labeled F2) as well as a chemically altered FITC- bound PU-H71 "control" (labeled F9) which does not bind the epichaperome (Bioorg Med Chem Lett 2011). The AML cell line MV411 was used as a positive control (given known epichaperome abundance and sensitivity to PU-H71) and intra-sample T-cells were used as negative controls. These samples were analyzed using multi-parameter flow cytometry including markers to distinguish blasts, progenitors and lymphocytes. For analysis of sensitivity to PU-H71, samples were incubated with two different concentrations of PU-H71, 1uM and 0.5uM, for 48 hours and subsequently evaluated for viability using multi-parameter flow cytometry (including markers to distinguish blasts, progenitors and lymphocytes). Leukemic blasts and T cells were gated based on immunophenotype, and the ratio of blast F2 to F9 was combined with the F2-F9 blast to T-cell ratio to calculate epichaperome abundance, using a cut-off value of 4.5 to determine medium to high or low levels of the epichaperome (medium to high, >/=4.5, low, Of the 15 samples analyzed for epichaperome abundance. Seven out of 15 (46.7%) samples showed an abundant epichaperome. Nine of these samples had complete viability data, of which, five out of nine (55.5%) showed >60% cell death after 48 hour incubation with PU-H71. For these nine samples with data on sensitivity to PU-H71, the presence or absence of epichaperome abundance was predictive of ex vivo sensitivity (5 sensitive, 4 resistant). We have established PDX-mouse models to evaluate in vivo sensitivity to PU-H71, using epichaperome abundant samples. We have confirmed the epichaperome levels in AML blast cells from the peripheral blood of the animals. Currently the treatment and analysis is ongoing. These data suggest that PTPN11 mutated AML represents a subset of AML that could be particularly sensitive to epichaperome inhibition. Additionally, the findings from our studies suggest this assay has promise as a means to accurately predict sensitivity to PU-H71, a useful precision medicine tool for a difficult to treat disease. All samples are currently being analyzed for MYC transcriptional activity and other signaling pathways. Disclosures Chiosis: Samus Therapeutics: Equity Ownership, Patents & Royalties: Intellectual rights to the PU-FITC assay. Ritchie:agios: Other: Advisory board; Pfizer: Other: Advisory board, travel support; Celgene: Other: Advisory board; Celgene, Novartis: Other: travel support; Celgene, Incyte, Novartis, Pfizer: Consultancy; Ariad, Celgene, Incyte, Novartis: Speakers Bureau; AStella, Bristol-Myers Squibb, Novartis, NS Pharma, Pfizer: Research Funding; Tolero: Other: Advisory board; Genentech: Other: Advisory board; Jazz Pharmaceuticals: Research Funding. Desai:Astellas: Honoraria; Celgene: Consultancy; Cellerant: Consultancy; Astex: Research Funding; Sanofi: Consultancy. Lee:Ai Therapeutics: Research Funding; Karyopharm Therapeutics: Consultancy; AstraZeneca Pharmaceuticals: Consultancy; Roche Molecular Systems: Consultancy; Helsinn: Consultancy; Jazz Pharmaceuticals, Inc: Consultancy. Roboz:Astex: Consultancy, Membership on an entity's Board of Directors or advisory committees; Argenx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amphivena: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Actinium: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Trovagene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sandoz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Otsuka: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Eisai: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celltrion: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bayer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees. Guzman:Cellectis: Research Funding; SeqRx: Consultancy; Samus Therapeutics: Patents & Royalties: intellectual rights to the PU-FITC assay.

The epichaperome, which was previously described by our team (Rodina et al. Nature 2016, Joshi et al. Nature Reviews Cancer 2018), is a complex hyper-connected network of chaperones and co-chaperones that are present in a subset of tumors, and its role is to facilitate the survival of malignant cells. We have previously shown abundant epichaperome networks in different tumor types, including AML. PU-H71 is a novel epichaperome inhibitor that selectively targets cells expressing the epichaperome. We hypothesize that personalizing PU-H71 in AML based on epichaperome abundance will significantly improve the clinical efficacy of PU-H71. To investigate this, we assessed the frequency of epichaperome abundance in primary AML samples and sought to develop a method with clinical application to predict response to PU-H71. We screened 55 de novo and relapsed/refractory primary AML bone marrow and peripheral blood samples for epichaperome abundance and cytotoxicity. Epichaperome abundance was assessed by incubating cells with FITC-bound PU-H71 (labeled F2) for 4 hours followed by analysis by multi-parameter flow cytometry. A chemically similar FITC-bound PU-H71, which does not bind the epichaperome, termed F9, was used as a control (Taldone et al. Bioorg Med Chem Lett 2011). MV411 cell lines were also used as positive controls (given known epichaperome abundance and high sensitivity to PU-H71), and intra-sample T-cells were used as negative controls. Furthermore, we assessed cell survival by incubating cells with 0.5uM PU-H71 for 48-hours followed by evaluation by multi-parameter flow cytometry. All analyses were performed in duplicates or triplicates. The ratio of blast F2 to F9 was combined with the F2-F9 blast to T cell ratio to calculate epichaperome abundance. Data on epichaperome abundance was available in 50 patient samples. Cytotoxicity data was available in 41 patients. 22/41 (54%) of patient samples underwent ≥60% cell death, respectively, and 7/41 (17%) were resistant to PU-H71, as defined by < 5% cytotoxicity. An ROC curve using epichaperome cut-offs of 1, 2.5, 4.5, 6, 8 and 15 provided an AUC of 0.97 (95% CI 0.889-1.056, p=0.006). Using a cut-off of 4.5 for epichaperome abundance, 22/50 (44%) of samples expressed high levels of the epichaperome, and sensitivity and specificity for predicting >60% in vitro cytotoxicity to PU-H71 were 81.8% (95% CI 59.7-94.8%) and 94.7% (95% CI 74-100%) (Figure 1). This method's diagnostic accuracy was 87.8%, with positive and negative likelihood ratios of 15.5 and 0.19. Collectively, our data suggests that this flow cytometry-based evaluation of epichaperome abundance is a promising tool for detecting epichaperome abundance and personalizing the treatment of PU-H71 in patients with AML. Disclosures Chiosis: Samus Therapeutics: Equity Ownership, Patents & Royalties: Intellectual rights to the PU-FITC assay. Roboz:Trovagene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sandoz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Otsuka: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celltrion: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Actinium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amphivena: Consultancy, Membership on an entity's Board of Directors or advisory committees; Argenx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astex: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bayer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Eisai: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees. Guzman:Samus Therapeutics: Patents & Royalties: intellectual rights to the PU-FITC assay; Cellectis: Research Funding; SeqRx: Consultancy.

Acute myeloid leukemia (AML) and the myelodysplastic syndromes (MDS) are initiated and sustained by self-renewing malignant stem cells; thus, eradication of AML and MDS stem cells is required for cure. We identified CD99 as a cell surface protein frequently overexpressed on AML and MDS stem cells. Expression of CD99 allows for prospective separation of leukemic stem cells (LSCs) from functionally normal hematopoietic stem cells (HSCs) in AML, and high CD99 expression on AML blasts enriches for functional LSCs as demonstrated by limiting dilution xenotransplant studies. Monoclonal antibodies (mAbs) targeting CD99 induce the death of AML and MDS cells in a SRC-family kinase dependent manner in the absence of immune effector cells or complement, and administration of anti-CD99 mAbs exhibit anti-leukemic activity in AML xenografts. These data establish CD99 as a novel marker of AML and MDS stem cells, as well as a promising therapeutic target in these disorders.

In clinical practice, leukocytosis is often overlooked after infectious and hematologic disease are ruled out, particularly in patients with solid tumors. This is unfortunate, as the mechanisms that mediate paraneoplastic leukocytosis may play a significant role in the underlying pathophysiology of cancer progression and prognosis. The relatively new discovery of neutrophilic and monocytic myeloid-derived suppressor cells (MDSCs) and their role in mediating tumor metastasis has particularly shed light into this process [Annu Rev Med, 66:97-110 (2015)]. Here, we present the case of a 58-year old gentleman with non-small cell lung cancer complicated by brain metastasis, status post resection who presented with sepsis and acute kidney injury (AKI) requiring ICU care for worsening AKI, hypoxic respiratory failure and leukocytosis. His peak WBC count, absolute neutrophilia and monocytosis were: 178.1, 172.7 and 4.2k/µL, respectively. His peripheral blood smear revealed mature neutrophils with left-shift and no blast forms. The underlying etiology of his leukocytosis was initially attributed to steroids administration and infection (Figure 1). His leukocytosis progressed, however, despite improvement in his sepsis and tapering of his steroids. Thus, we suspected either an evolving hematologic neoplasm or exogenous secretion of G-CSF by his tumor. Nonetheless, given his worsening clinical status, we initiated empiric hydroxyurea and leukapheresis. His FISH and PCR for BCR-ABL were negative in addition to the absence of leukemia-associated mutations and gene fusions and a normal phenotype by flow cytometry. However, we detected the highest documented level of G-CSF secreted by any tumor in the literature at 41,108.6pg/mL (normal On evaluation of this patient's clinical history, his malignancy and white count were stable until day 388 of his diagnosis when he developed new onset leukocytosis and neutrophilia associated with disease progression and metastasis. Furthermore, he died within 23 days of developing peak leukocytosis, neutrophilia and monocytosis and the discovery of his profoundly elevated G-CSF level. The association between the onset of his leukocytosis with the discovery of his disease progression and metastasis suggest that G-CSF secretion and leukocytosis may have been linked to his poor prognosis. We performed an extensive literature review and found that neutrophilic and monocytic MDSCs may provide a potential explanation for this phenomenon. We believe that leukocytosis in the setting of solid neoplasms may be driven by increased G-CSF secretion by tumors or tumor microenvironments. Additionally, G-CSF secretion fosters the expansion of neutrophilic and monocytic MDSCs, which play a significant role in tumor progression and metastasis and may contribute to poor prognosis [PNAS 106(16): 6742-7 (2009), PNAS 107(50): 21248-55 (2010)]. Consequently, we believe that the significance of leukocytosis in patients with solid tumors should not be overlooked and that there may exist a novel, untapped paradigm between paraneoplastic G-CSF secretion, leukocytosis, neutrophilic and monocytic MDSCs and prognosis. Disclosures Mauro: Novartis: Consultancy, Research Funding; Pfizer: Consultancy; Takeda: Consultancy; Bristol-Myers Squibb: Consultancy.

In the USA, approximately 26,000 adolescents and young adults (AYAs) aged 15-29 years are diagnosed with cancer every year. The cure rate among this population exceeds 80%, resulting in a growing number of AYA cancer survivors. AYA cancer survivors suffer from a wide range of long-term treatment-related toxicities that adversely affect quality of life and increase the risk of premature death. Therefore, it is important to recognize the unique medical needs of the AYA cancer survivors and develop a cost-effective and systemic approach to screen and prevent cancer treatment-related sequelae and the adverse health outcomes.

CD99 is a 32-kDa glycoprotein involved in leukocyte migration and homotypic cell aggregation. Since its initial discovery as a marker on acute lymphoblastic leukemia (ALL), few studies have investigated its potential targeting and biological role in this disease. We have shown that CD99 is up-regulated in malignant stem cells in acute myeloid leukemia (AML) and the myelodysplastic syndromes (MDS), and that monoclonal antibodies (mAbs) targeting CD99 induce cell death. Given that targeting CD99 holds promise in AML/MDS, we sought to determine whether it is an effective target in other hematologic malignancies. We began by screening 15 T-, B-, and plasma cell lines as well as normal peripheral blood and umbilical cord CD34+ cells for CD99 expression by flow cytometry. CD99 expression was 7- and 10-fold higher on 1/1 T-ALL and 1/2 anaplastic large cell lymphoma (ALCL) cell lines compared with CD34+ cells, and 2- and 3-fold higher relative to normal peripheral blood T cells, respectively. However, it was minimally expressed in 11/12 B cell lymphomas, plasma cell dyscrasias, and peripheral T cell neoplasms. CD99 expression (degree, localization) was also assessed on 264 lymphoma patient samples by immunohistochemistry (IHC) using the CD99 mAb, 12E7. We found that 11/20 (55%) T-lymphoblastic lymphomas, 7/16 (44%) angioimmunoblastic T-cell lymphomas, 4/13 (31%) ALCLs, 10/63 (16%) peripheral T-cell lymphomas, and 0/3 (0%) of NK/T cell lymphomas express CD99 by IHC, while only 1/70 (1.4%) diffuse large B cell lymphomas, 2/24 (8%) mantle cell lymphomas, 2/17 (12%) follicular lymphomas, 4/22 (18%) chronic lymphocytic leukemias, and 3/16 (19%) marginal zone lymphomas express CD99. Staining was predominately moderate and cytoplasmic. Using a BioGPS dataset from T-ALL patient bone marrow samples, CD99 transcript was found to be up-regulated in T-ALL bone marrow (n=117) relative to normal bone marrow (n=7) (p To test whether CD99 mAbs are cytotoxic to T-ALL and ALCL cell lines, cells were incubated with 5µg/ml CD99 mAb in the presence of 7µg/ml anti-IgG antibody, and cell survival was assessed by flow cytometry following 72-hours relative to IgG isotype control. 4/5 T-ALL cell lines (KOPTK1, Loucy, CCRF HSB-2, PF283) were sensitive to the cytotoxicity of CD99 mAb, mediating 30-96% cell death (p≤0.003), with 2/4 cell lines displaying 90-96% cytotoxicity. Remarkably, incubating CD99 mAb with a primary T-ALL patient sample induced 100% cell death within 48 hours of treatment (p To determine whether CD99 mAb cytotoxicity depends on the level of CD99 expression, we stably transduced KOPTK1 cells with an optimized CD99 shRNA (199-fold reduction in CD99 mean fluorescence intensity [MFI]), stably transduced CD99-low Mac2A (ALCL) cells with TetOn CD99 (17-fold increase in CD99 MFI), and analyzed the cells for cytotoxicity following 24-hour incubations with CD99 mAbs. CD99 mAb-induced cell death increased from 4.4% to 88% upon overexpressing CD99, and decreased from 89% to 20% upon knocking down CD99, suggesting that cell death is dependent on the level of CD99 expression. To elucidate the functional role of CD99 in T-cell neoplasms, we xenografted KOPTK1 cells expressing CD99 shRNA into sublethally irradiated NOD/SCID/IL-2Rgc-null (NSG) mice. Animals transplanted with CD99 knockdown showed no improved survival compared to controls (n=4 and 5 in each group, respectively). We further evaluated the potential oncogenic role of CD99 in vitro, and observed no effect of CD99 knock down in KOPTK1 or overexpression in Mac2A on cell cycle status or proliferation by PI staining and cell counting. Our data indicate that CD99 is expressed in a subset of T-lineage neoplasms. While there is no evidence for a functional role of CD99 in the growth or survival of T-ALL and ALCL, CD99 can be targeted by CD99 mAbs to induce apoptosis with rapid kinetics and in a manner that is dependent on levels of CD99 expression and independent of complement. Thus, CD99 is a promising target in the treatment of a subset of T-cell neoplasms. Disclosures No relevant conflicts of interest to declare.

Introduction: CD99 is a 32 kDa transmembrane protein that regulates T cell maturation and the transendothelial migration of leukocytes. CD99 is also routinely used as a biomarker to aid in the diagnosis of T-cell acute lymphoblastic lymphoma/leukemia (T-ALL), Ewing sarcoma, and neuroendocrine tumors. We have previously shown that monoclonal antibodies (mAbs) directed against CD99 effectively induce cytotoxicity in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), however, it remains unclear whether CD99 is a viable therapeutic target in T-cell neoplasms. Design: CD99 protein expression was assessed on 115 lymphoma patient samples by immunohistochemistry (IHC) using tissue microarrays. CD99 expression was also measured by flow cytometry on mature T-cell lymphoma cell lines, T-ALL cell lines, and peripheral blood (PB) T-cells. Cell lines exhibiting the highest levels of CD99 expression were used for in vitro cytotoxicity assays and were incubated with 40 μg/ml of anti-CD99 mAbs (DN16, O13, or 12E7) in the presence of increasing concentrations of an anti-IgG cross-linking antibody (Ab) (50-100 μg/ml). Cell survival and morphology were assessed by flow cytometry and light microscopy 72 hours following incubation with anti-CD99 mAbs. Results: Various T-cell neoplasms showed CD99 expression by IHC based on a 5% cut-off: 11/20 (55%) T lymphoblastic lymphomas, 7/16 (44%) angioimmunoblastic T-cell lymphomas, 4/13 (31%) anaplastic large cell lymphomas (ALCLs), 10/63 (16%) peripheral T-cell lymphomas, and 0/3 (0%) NK/T cell lymphomas. Flow cytometric analysis revealed that CD99 is up-regulated on Karpas-299 (ALCL) and Kopt-K1 (T-ALL) cell lines relative to PB T-cells (normalized MFI’s: 56.1, 39.5, 20.5), while low levels of CD99 were expressed on Mac2A (ALCL), Hut78 (sezary syndrome), and Hut102 (mycosis fungoides) cell lines (normalized MFI’s: 0.19, 10.9, 0.5, respectively). Incubations of Kopt-K1 cells with DN16 or 12E7 induced significant cell death in the presence of increasing concentrations of anti-IgG Ab relative to IgG isotype control (DN16: 70-81% cell death, p≤.0002; 12E7: 42-78% cell death, p≤.0003). Karpas-299 incubations with DN16 or O13 also led to significant reductions in cell numbers in the presence of anti-IgG Ab (O13: 25% cell death with 75 μg/ml anti-IgG Ab, p Conclusions: Our studies show that CD99 is expressed on T-ALL blasts and T-cell lymphomas, particularly in ALCL. Anti-CD99 mAbs are cytotoxic to T-ALL and ALCL cell lines in the presence of anti-IgG Ab, suggesting that cross-linking or multimerization of CD99 is required to induce cell death. Collectively, these studies indicate that targeting CD99 with therapeutic mAbs may represent a novel strategy in the treatment of multiple T-cell neoplasms. Citation Format: Montreh Tavakkoli*, Dong H. Lee, Stephen S. Chung, Christopher Y. Park. Targeting CD99 in T-cell neoplasms. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3566. doi:10.1158/1538-7445.AM2015-3566

Historically, acute promyelocytic leukemia (APL) was considered to be one of the most fatal forms of acute leukemia with poor outcomes before the introduction of the vitamin A derivative all-trans retinoic acid (ATRA). With considerable advances in therapy, including the introduction of ATRA initially as a single agent and then in combination with anthracyclines, and more recently by development of arsenic trioxide (ATO)-containing regimens, APL is now characterized by complete remission rates of 90% and cure rates of ∼80%, even higher among low-risk patients. Furthermore, with ATRA–ATO combinations, chemotherapy may safely be omitted in low-risk patients. The disease is now considered to be the most curable subtype of acute myeloid leukemia (AML) in adults. Nevertheless, APL remains associated with a significant incidence of early death related to the characteristic bleeding diathesis. Early death, rather than resistant disease so common in all other subtypes of AML, has emerged as the major cause of treatment failure.

The myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are initiated and sustained by self-renewing stem cells. We performed transcriptomal profiling of purified MDS hematopoietic stem cells (HSCs) and identified CD99 as a highly expressed cell surface transcript. Flow cytometry of MDS patient bone marrow (BM) samples (n=63) confirmed that CD99 is frequently increased on MDS HSCs (85%). Assessment of 78 paired diagnosis/relapse AML specimens revealed that CD99 is also frequently overexpressed in AML at diagnosis (81%) and relapse (83%). CD99 is a potential leukemic stem cell (LSC) marker, as CD99 high cells exhibit a CD34+CD38-CD90-CD45RA+ LMPP-like immunophenotype, previously shown to be enriched for LSC activity, whereas CD99 low cells exhibit a CD34+CD38-CD45RA- phenotype resembling normal HSCs and MPPs. Providing additional support that CD99 is a LSC marker in AML, CD99 surface expression is significantly higher in the LSC-enriched CD34+CD38- fraction compared to bulk blasts (p=0.003), as well as in relapsed as compared to diagnostic specimens (p=0.007). To determine if CD99 is preferentially expressed on functional LSCs, we purified the top 10% and bottom 10% of CD99 expressing leukemic blasts from the CD34+CD38-CD90-CD45RA+ fraction of a primary AML specimen. Limiting dilution xenotransplantation experiments demonstrated an estimated LSC frequency of 1 in 24,401 in the top 10% of CD99 expressors, whereas transplantation of up to 360,000 of the bottom 10% of CD99 expressors did not lead to any engraftment. Thus, within the LMPP-like LSC-enriched compartment, LSC activity appears to be restricted to high CD99 expressors. To determine the function of CD99 in AML, we stably transduced MOLM13 AML cells with a CD99 shRNA (8.0-fold knockdown) and xenografted them into NSG mice. Animals transplanted with these cells showed improved survival compared to vector controls (58d vs. 34d, p=0.02). Consistent with its described role in leukocyte trafficking, overexpression of CD99 in AML cell lines promoted transendothelial migration in transwell assays. In primary AML specimens, CD99 expression was higher on PB as compared with BM specimens (p=0.03). Together, these findings suggest that CD99 may promote AML aggressiveness by enhancing transendothelial migration and mobilization. Unexpectedly, CD99 transcript expression in the ECOG 1900 AML patient cohort (n=308) positively correlated with survival (p=0.001). We propose that CD99 may improve survival in the context of chemotherapy by promoting mobilization and thus chemosensitivity of AML cells, and AML cells with low levels of CD99 may exhibit increased retention in chemoprotective BM niches. In line with this hypothesis, the poor prognosis of low CD99 expression appeared to be mitigated by intensification of chemotherapy. To determine whether CD99 may be a relevant therapeutic target, we tested the ability of monoclonal antibodies (mAb) against CD99 to induce direct cytotoxicity in vitro. Anti-CD99 mAbs induced significant cell death in 11 AML and two MDS-derived cell lines, as well as in primary AML blasts (n=7) and MDS HSCs/CD34+ cells (n=3). These mAbs induced cytotoxicity in a CDC and ADCC independent manner, with relative sparing of normal cells expressing low levels of CD99 such as primary human HSCs and endothelial cells, suggesting a mechanism of cytotoxicity unique to AML and MDS. Pre-coating of primary human LSCs with an anti-CD99 mAb prior to transplantation into NSG mice led to impaired engraftment at eight weeks (20% vs. 67%, p=0.009) and improved survival (p=0.05). Immunofluorescence imaging demonstrated that mAb ligation promotes clustering of CD99, and this clustering appears to be critical for cytotoxicity. Accordingly, cross-linking of IgG isotype anti-CD99 mAbs with an anti-IgG secondary antibody enhanced cytotoxicity 3.4-fold. Anti-CD99 mAb treatment was also associated with activation of Src-family kinases, and we propose that anti-CD99 mAbs may promote cytotoxicity by inducing oncogenic stress via dysregulated Src-family kinase activation. Our results establish CD99 as a cell surface marker expressed in AML and MDS stem cells, as a mediator of transendothelial migration, and as a promising therapeutic target for direct targeting by mAbs. Further studies are needed to evaluate the potential of targeting CD99 in AML/MDS in vivo and to further characterize the mechanisms of CD99 mediated cell death. Disclosures: No relevant conflicts of interest to declare.

Plant stem cell populations, unlike their animal counterparts, do not use cell migration and oriented cell divisions to maintain their size, and therefore require a precise coordination between self-renewing divisions of stem cells, and rates of cell division and differentiation among stem cell progenitors. Shoot apical meristems (SAMs) of higher plants harbor a set of stem cells within the central zone (CZ) that divide infrequently. Stem cell daughters that are displaced towards the surrounding peripheral zone (PZ) divide at a faster rate and enter into differentiation at specific locations to form leaves or flowers. The relative ratios of cells in the CZ and the PZ are maintained, despite a constant displacement of cells from the CZ into the PZ, and subsequent allocation of cells within the PZ to form organ primordia. The mechanisms that mediate this homeostatic balance are not well understood. A homeodomain transcription factor WUSCHEL, expressed in the rib meristem (RM), located beneath the CZ, has been shown to provide nonautonomous cues for stem cell specification. By employing transient spatial manipulation and live imaging, we show that an elevated level of WUS not only induces expansion of the CZ, but also results in increased cell division rates in cells of the PZ; conversely, decreases in WUS level lead to a smaller CZ and are associated with a reduction in cell division rate. Moreover, low levels of WUS lead to enlarged organ primordia, by elevating the responsiveness of the PZ cells to the plant hormone auxin. This reveals a function of WUS in mediating the balance between differentiating and non-differentiating cells of the PZ. Regulation of stem cell numbers, growth and differentiation patterns by a single transcription factor forms a interconnected and self-correcting feedback loop to provide robustness to stem cell homeostasis in a dynamic cellular environment.