Hematologic Malignancy Cell Research Articles

Evaluating antimicrobial duration for Gram-negative bacteremia in patients with neutropenia due to hematologic malignancy or hematopoietic stem cell transplantation.

In the management of Gram-negative bloodstream infection (GN-BSI), short antimicrobial courses have been increasingly demonstrated to be non-inferior to prolonged therapy, with lower risk of Clostridioides difficile infection (CDI) and emergence of multi-drug resistant (MDR) organisms. However, immunocompromised hosts were excluded from these studies. We investigated outcomes of short (≤10 days), intermediate (11-14 days), and prolonged (≥15 days) antimicrobial durations for GN-BSI in neutropenic patients. A retrospective cohort study was conducted on neutropenic patients with monomicrobial GN-BSI between 2018 and 2022. The primary outcome was a composite of all-cause mortality and microbiologic relapse within 90 days after therapy completion. The secondary outcome was a composite of 90-day CDI and development of MDR-GN bacteria. Cox regression analysis with propensity score (PS) adjustment was used to compare outcomes between the three groups. A total of 206 patients were classified into short (n = 67), intermediate (n = 81), or prolonged (n = 58) duration. Neutropenia was predominantly secondary to hematopoietic stem cell transplantation (48%) or hematologic malignancy (35%). The primary sources of infection included intra-abdominal (51%), vascular catheter (27%), and urinary (8%). Most patients received definitive therapy with cefepime or carbapenem. No significant difference in the primary composite endpoint was observed for intermediate versus short (PS-adjusted hazard ratio [aHR] 0.89; 95% confidence interval [95% CI] 0.39-2.03) or prolonged versus short therapy (PS-aHR 1.20; 95% CI 0.52-2.74). There was no significant difference in the secondary composite endpoint of CDI or MDR-GN emergence. Our data suggest that short antimicrobial courses had comparable 90-day outcomes as intermediate and prolonged regimens for GN-BSI among immunocompromised patients with neutropenia.

NK Cell-Targeted Immunotherapies in Bladder Cancer: Beyond Checkpoint Inhibitors.

For decades, immunotherapies have been integral for the treatment and management of bladder cancer, with immune checkpoint inhibitors (ICIs) transforming patient care in recent years. However, response rates are poor to T cell-targeted ICIs such as programmed cell death protein 1 (PD-1) and programmed cell death-ligand 1 (PD-L1) blocking antibodies, framing a critical need for complementary immunotherapies. Promising strategies involve harnessing the activation potential of natural killer (NK) cells. They quickly exert their antitumor activity via signaling through germline-encoded activating receptors and are rapidly sensitized to new tissue microenvironments via their regulation by polymorphic HLA class I, KIR and NKG2A receptors. In this review, we examined the roles of currently available NK-targeted antitumor treatment strategies such as engineered viral vectors, small-molecule IMiDs, NK agonist antibodies, interleukins, and chimeric antigen receptor (CAR) NK cells, and their potential for improving the efficacy of immunotherapy in the treatment of bladder cancer. Through review of current literature, we summarized our knowledge of NK cells in solid tumors and hematologic malignancies as their roles pertain to novel immunotherapies already being applied to the treatment of bladder cancer or that offer rationale for considering as potential novel immunotherapeutic strategies. NK cells play a critical role in shaping the tumor microenvironment (TME) that can be exploited to improve T cell-targeted immunotherapies. Emerging evidence suggests that NK cells are a prime target for improving antitumor functions in immunotherapies for the treatment of bladder cancer. Further research into profiling NK cells in settings of immunotherapies for bladder cancer could help identify patients who might maximally benefit from NK cell-targeted immunotherapies and the various approaches for exploiting their antitumor properties.

A retrospective analysis on mucormycosis in patients with hematological diseases: a single center experience from Turkey

Aim: Mucormycosis is an acute, invasive, devastating and highly fatal fungal infection, affecting particularly immunocompromised patients; fortunately, it is rare. This study aimed to describe the attitude of mucormycosis in patients with a hematological disease, and to evaluate the risk factors associated with mortality.
 Material and Method: We retrospectively assessed the demographic and clinical data of patients who were diagnosed with mucormycosis in Erciyes University Hematology and Bone Marrow Transplantation Center, between 2010 and 2020. The study was included 34 patients with a history of either hematological malignancy or hematopoietic stem cell transplantation. 
 Results: Twenty-seven patients had proven infection, and the others had possible infection. The most frequent underlying disease was acute leukemia. Seven-teen patients had a history of allogeneic transplantation, and frequency of mucormycosis was 3.5% among allogeneic transplant recipients. The most frequent site of infection was the rhino-orbital region (85.3%). Forty-seven percent of patients presented with acute orbital symptoms. Fifteen patients were on a mucor-active antifungal (posaconazole and liposomal Amphotericin B) prophylaxis or treatment at the time of diagnosis. All patients received liposomal Amphotericin B and seven patients received posaconazole additionally as initial therapy. Surgical debridement was performed in 91.1% of patients. The two-year mucor-related mortality rate was 44.1%. The survival curves were significantly lower in patients with concomitant fungal pneumonia, allogeneic transplantation and also in patients who were receiving mucor-active antifungal drugs at the time of diagnosis.
 Conclusion: Mucormycosis remains a significant problem for hematology clinicians despite the expanding use of antifungal prophylaxis. Moreover, breakthrough infections indicate rising danger regarding resistant agents. We also highlight that, most of the patients receiving broad-spectrum antifungal prophylaxis are more fragile and more complicated patients, which put them at increased baseline risk for mucormycosis, and deserve more attention.

Potential of Synthetic and Natural Compounds as Novel Histone Deacetylase Inhibitors for the Treatment of Hematological Malignancies.

Histone deacetylases (HDACs) and histone acetyltransferases (HATs) are enzymes that remove or add acetyl groups to lysine residues of histones, respectively. Histone deacetylation causes DNA to more snugly encircle histones and decreases gene expression, whereas acetylation has the opposite effect. Through these small alterations in chemical structure, HATs and HDACs regulate DNA expression. Recent research indicates histone deacetylase inhibitors (HDACis) may be used to treat malignancies, including leukemia, B-cell lymphoma, virus-associated tumors, and multiple myeloma. These data suggest that HDACis may boost the production of immune-related molecules, resulting in the growth of CD8-positive T-cells and the recognition of nonreactive tumor cells by the immune system, thereby diminishing tumor immunity. The argument for employing epigenetic drugs in the treatment of acute myeloid leukemia (AML) patients is supported by evidence that both epigenetic changes and mutations in the epigenetic machinery contribute to AML etiology. Although hypomethylating drugs have been licensed for use in AML, additional epigenetic inhibitors, such as HDACis, are now being tested in humans. Preclinical studies evaluating the efficacy of HDACis against AML have shown the ability of specific agents, such as anobinostat, vorinostat, and tricostatin A, to induce growth arrest, apoptosis, autophagy and cell death. However, these inhibitors do not seem to be successful as monotherapies, but instead achieve results when used in conjunction with other medications. In this article, we discuss the mounting evidence that HDACis promote extensive histone acetylation, as well as substantial increases in reactive oxygen species and DNA damage in hematological malignant cells. We also evaluate the potential of various natural product-based HDACis as therapeutic agents to combat hematological malignancies.

Facing challenges with hope: universal immune cells for hematologic malignancies.

Many patients have achieved a favorable overall survival rate since allogenic hematopoietic stem cell transplantation (allo-HSCT) has been widely implemented to treat hematologic malignancies. However, graft-versus-host disease (GVHD) and complications of immunosuppressive drugs after allo-HSCT are the main causes of non-relapse mortality and a poor quality of life. In addition, GVHD and infusion-induced toxicity still occur with donor lymphocyte infusions (DLIs) and chimeric antigen receptor (CAR) T-cell therapy. Because of the special immune tolerance characteristics and anti-tumor ability of universal immune cells, universal immune cell therapy may strongly reduce GVHD, while simultaneously reducing tumor burden. Nevertheless, widespread application of universal immune cell therapy is mainly restricted by poor expansion and persistence efficacy. Many strategies have been applied to improve universal immune cell proliferation and persistence efficacy, including the use of universal cell lines, signaling regulation and CAR technology. In this review we have summarized current advances in universal immune cell therapy for hematologic malignancies with a discussion of future perspectives.

CAR-modified immune cells as a rapidly evolving approach in the context of cancer immunotherapies

Nowadays, one of the main challenges clinicians face is malignancies. Through the progression of technology in recent years, tumor nature and tumor microenvironment (TME) can be better understood. Because of immune system involvement in tumorigenesis and immune cell dysfunction in the tumor microenvironment, clinicians encounter significant challenges in patient treatment and normal function recovery. The tumor microenvironment can stop the development of tumor antigen-specific helper and cytotoxic T cells in the tumor invasion process. Tumors stimulate the production of proinflammatory and immunosuppressive factors and cells that inhibit immune responses. Despite the more successful outcomes, the current cancer therapeutic approaches, including surgery, chemotherapy, and radiotherapy, have not been effectiveenough for tumor eradication. Hence, developing new treatment strategies such as monoclonal antibodies, adaptive cell therapies, cancer vaccines, checkpoint inhibitors, and cytokines helps improve cancer treatment. Among adoptive cell therapies, theinteraction between the immune system and malignancies and using molecular biology led tothe development of chimeric antigen receptor (CAR) T cell therapy. CAR-modified immune cells are one of the modern cancer therapeutic methods with encouraging outcomes in most hematological and solid cancers. The current study aimed to discuss the structure, formation, subtypes, and application of CAR immune cells in hematologic malignancies and solid tumors.

Modulation of T-cell function by myeloid-derived suppressor cells in hematological malignancies.

Myeloid-derived suppressor cells (MDSCs) are pathologically activated neutrophils and monocytes that negatively regulate the immune response to cancer and chronic infections. Abnormal myelopoiesis and pathological activation of myeloid cells generate this heterogeneous population of myeloid-derived suppressor cells. They are characterized by their distinct transcription, phenotypic, biochemical, and functional features. In the tumor microenvironment (TME), myeloid-derived suppressor cells represent an important class of immunosuppressive cells that correlate with tumor burden, stage, and a poor prognosis. Myeloid-derived suppressor cells exert a strong immunosuppressive effect on T-cells (and a broad range of other immune cells), by blocking lymphocyte homing, increasing production of reactive oxygen and nitrogen species, promoting secretion of various cytokines, chemokines, and immune regulatory molecules, stimulation of other immunosuppressive cells, depletion of various metabolites, and upregulation of immune checkpoint molecules. Additionally, the heterogeneity of myeloid-derived suppressor cells in cancer makes their identification challenging. Overall, they serve as a major obstacle for many cancer immunotherapies and targeting them could be a favorable strategy to improve the effectiveness of immunotherapeutic interventions. However, in hematological malignancies, particularly B-cell malignancies, the clinical outcomes of targeting these myeloid-derived suppressor cells is a field that is still to be explored. This review summarizes the complex biology of myeloid-derived suppressor cells with an emphasis on the immunosuppressive pathways used by myeloid-derived suppressor cells to modulate T-cell function in hematological malignancies. In addition, we describe the challenges, therapeutic strategies, and clinical relevance of targeting myeloid-derived suppressor cells in these diseases.

Abstract 2919: A novel method for clinical scale production of natural killer cells from clonal master induced pluripotent stem cells with CISH knockout for next generation, off-the-shelf cancer immunotherapy

Abstract Natural killer (NK) cells are innate immune cells that play a key role in in tumor immune surveillance. NK cells are self-tolerant to healthy cells and can kill tumor cells in both hematological and solid malignancies. Recent advances allow for the derivation of immune cells such as NK cells, from human induced pluripotent stem cells, that can be utilized for cancer immunotherapy. Recent clinical trials suggest that high doses of NK cells (approx. 0.5-5 x 109Jaya cells per dose) and multiple doses are both safe and likely necessary for clinical efficacy. Manufacturing large number of NK cells from a clonal master iPSC line provides a promising strategy to enable next generation, off-the-shelf, affordable, cancer immunotherapies. We have developed a novel method to produce high purity, clinical scale iNK cells and improved the process for the expansion of NK cells efficiently and consistently. This method does not require cell sorting step and the usage of murine derived stromal cells. Briefly, hematopoietic progenitor cells were induced using an improved spin embryoid body (EB) method and the hematopoietic progenitor cells were subsequently differentiated into mature iNK cells in the absence of cell sorting and independent of stroma cells support. Using this novel method, differentiated NK cells could be expanded more than 8,000-fold to enable us to potentially produce 1 x 1012 iNK cells starting from 1 x 106 undifferentiated iPSC. This cell production scale can supply hundreds of doses in clinic from one cGMP manufacturing campaign. iNK cells produced using this method display the typical phenotype of NK cell activating receptor including NKG2D, NKp44, NKp46, DNAM-1 etc. Importantly, these iNK cells demonstrate better anti-tumor activities and higher levels of IFNγ and TNFα, compared to primary peripheral blood-derived NK cells isolated from healthy donors. Moreover, this protocol has been adapted and optimized for clinical scale manufacturing of iNK cells from genetically engineered, clonal master iPSC cell line with knock out of CISH (CISH KO iNK), a key intracellular checkpoint of NK cell anti-tumor immunity. CISH KO iNK cells produced using this optimized method show significantly better anti-tumor activities against multiple liquid and solid tumor cell lines including K562, Raji, Daudi, SUP-B15, MOLT-4, SKOV-3, OVCAR-4, HCC-827, ZR-75, and BT-474 etc. in vitro and in vivo in an AML xenograft mouse model, compared with unmodified iNK cells. Overall, this novel cell production strategy paves the way for clinical trials using higher doses of iPSC-derived NK cells with increased potency, creating next-generation NK cell-based immunotherapies. Citation Format: Davide Bernareggi, Caryn Gonsalves, Max Schabla, Alejandra Gárate-Carrillo, Duygu Ozmadenci, Jaya Thangaraj, Qin Li, Leah Mitchell, Dan S. Kaufman, Robert Hollingsworth, Huang Zhu. A novel method for clinical scale production of natural killer cells from clonal master induced pluripotent stem cells with CISH knockout for next generation, off-the-shelf cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2919.

The peculiar challenge of bringing CAR-T cells into the brain: Perspectives in the clinical application to the treatment of pediatric central nervous system tumors.

Childhood malignant brain tumors remain a significant cause of death in the pediatric population, despite the use of aggressive multimodal treatments. New therapeutic approaches are urgently needed for these patients in order to improve prognosis, while reducing side effects and long-term sequelae of the treatment. Immunotherapy is an attractive option and, in particular, the use of gene-modified T cells expressing a chimeric antigen receptor (CAR-T cells) represents a promising approach. Major hurdles in the clinical application of this approach in neuro-oncology, however, exist. The peculiar location of brain tumors leads to both a difficulty of access to the tumor mass, shielded by the blood-brain barrier (BBB), and to an increased risk of potentially life-threatening neurotoxicity, due to the primary location of the disease in the CNS and the low intracranial volume reserve. There are no unequivocal data on the best way of CAR-T cell administration. Multiple trials exploring the use of CD19 CAR-T cells for hematologic malignancies proved that genetically engineered T cells can cross the BBB, suggesting that systemically administered CAR-T cell can be used in the neuro-oncology setting. Intrathecal and intra-tumoral delivery can be easily managed with local implantable devices, suitable also for a more precise neuro-monitoring. The identification of specific approaches of neuro-monitoring is of utmost importance in these patients. In the present review, we highlight the most relevant potential challenges associated with the application of CAR-T cell therapy in pediatric brain cancers, focusing on the evaluation of the best route of delivery, the peculiar risk of neurotoxicity and the related neuro-monitoring.

Mucosal-associated invariant T cells in hematological malignancies: Current knowledge, pending questions.

Non-classical HLA restricted T cell subsets such as γδ T and NK-T cells are showing promises for immune-based therapy of hematological malignancies. Mucosal-Associated Invariant T cells (MAIT) belong to this family of innate-like T cell subsets and are the focus of many studies on infectious diseases, owing to their unusual recognition of bacterial/fungal metabolites. Their ability to produce type 1 cytokines (IFNγ, TNFα) as well as cytotoxic effector molecules endows them with potential anti-tumor functions. However, their contribution to tumor surveillance in solid cancers is unclear, and only few studies have specifically focused on MAIT cells in blood cancers. In this review, we wish to recapitulate our current knowledge on MAIT cells biology in hematological neoplasms, at diagnosis and/or during treatment, as well as tentative approaches to target them as therapeutic tools. We also wish to take this opportunity to briefly elaborate on what we think are important question to address in this field, as well as potential limitations to overcome in order to make MAIT cells the basis of future, novel therapies for hematological cancers.

Early administration of SARS‐CoV‐2 monoclonal antibody reduces the risk of mortality in hematologic malignancy and hematopoietic cell transplant patients with COVID‐19

Data on severe acute respiratory distress syndrome coronavirus 2 monoclonal antibody (SARS-CoV-2-specific mAb) use in hematologic malignancy and hematopoietic cell transplantation (HM/HCT) patients are limited. Here, we describe our experience with the use of casirivimab-imdevimab or bamlanivimab for the treatment of coronavirus disease 2019 (COVID-19) in HM/HCT patients. This was a retrospective chart review at the University of Miami Hospital and Sylvester Comprehensive Cancer Center for HM/HCT patients with COVID-19 who received casirivimab-imdevimab or bamlanivimab from November 21, 2020, to September 30, 2021. Outcomes measured were mortality, hospital admission, and infusion reaction to SARS-CoV-2-specific mAbs. We identified 59 HM/HCT patients with mild to moderate COVID-19 who received casirivimab-imdevimab or bamlanivimab. Median age was 57 years (interquartile range [IQR]: 45-65). Among the 59 patients, 25 (42%) received cellular therapy: 14 (24%) had undergone allogeneic HCT, nine (15%) autologous HCT, and two (3%) received chimeric antigen receptor T-cell therapy. The median time from COVID-19 symptom onset to SARS-CoV-2-specific mAb administration was 4 (IQR: 3-6) days. Forty-six (78%) patients received SARS-CoV-2-specific mAbs as outpatients and 13 (22%) patients received SARS-CoV-2-specific mAbs during hospitalization. Among patients who received SARS-CoV-2-specific mAbs as outpatients, only four (9%) visited the emergency department at days 10, 11, 15, and 35 after SARS-CoV-2-specific mAb administration. None of these four patients required hospital admission. Among the hospitalized patients, five (38%) were admitted to the hospital with neutropenic fever, four (31%) were already hospitalized for transplantation and cellular therapy, three (23%) were admitted for monitoring of COVID-19 symptoms, and one (8%) was admitted with acute kidney injury. Three hospitalized patients (23%) died at 14, 35, and 59 days after SARS-CoV-2-specific mAb administration; two of these three deaths were attributed to COVID-19 infection. One patient developed an immediate infusion reaction to bamlanivimab, and no infusion reactions were reported to casirivimab-imdevimab use. During the alpha and delta variant surges, early administration of bamlanivimab or casirivimab-imdevimab prevented hospitalization and death when given in the outpatient setting. Among patients who received mAbs at or after hospital admission, the risk of COVID-19 disease progression and death remains significant. Larger studies of the use of mAb therapy to treat COVID-19 in this population are needed.

Anti-HLA antibodies in recipients of CD19 versus BCMA-targeted CAR T-cell therapy

Antibodies against foreign human leukocyte antigen (HLA) molecules are barriers to successful organ transplantation. B cell–depleting treatments are used to reduce anti-HLA antibodies but have limited efficacy. We hypothesized that the primary source for anti-HLA antibodies is long-lived plasma cells, which are ineffectively targeted by B cell depletion. To study this, we screened for anti-HLA antibodies in a prospectively enrolled cohort of 49 patients who received chimeric antigen receptor T-cell therapy (CARTx), targeting naïve and memory B cells (CD19-targeted, n = 21) or plasma cells (BCMA-targeted, n = 28) for hematologic malignancies. Longitudinal samples were collected before and up to 1 year after CARTx. All individuals were in sustained remission. We identified 4 participants with anti-HLA antibodies before CD19-CARTx. Despite B cell depletion, anti-HLA antibodies and calculated panel reactive antibody scores were stable for 1 year after CD19-CARTx. Only 1 BCMA-CARTx recipient had pre-CARTx low-level anti-HLA antibodies, with no follow-up samples available. These data implicate CD19neg long-lived plasma cells as an important source for anti-HLA antibodies, a model supported by infrequent HLA sensitization in BCMA-CARTx subjects receiving previous plasma cell–targeted therapies. Thus, plasma cell–targeted therapies may be more effective against HLA antibodies, thereby enabling improved access to organ transplantation and rejection management.

First-in-class small molecule inhibitors of ICOS/ICOSL interaction as a novel class of immunomodulators.

The interaction of the inducible co-stimulator (ICOS) with its ligand (ICOSL) plays key roles in T-cell differentiation and activation of T-cell to B-cell functions. The ICOS/ICOSL pathway is a validated target for T-cell lymphomas induced by the proliferation of T-follicular helper (Tfh) cells. Moreover, the inhibition of ICOS/ICOSL interaction can decrease the enhancement of immunosuppressive regulatory T cells (Tregs) in both hematologic malignancies and solid tumors. However, targeting ICOS/ICOSL interaction is currently restricted to monoclonal antibodies (mAbs) and there are no small molecules in existence that can block ICOS/ICOSL. To fill this gap, we report herein the first time-resolved fluorescence resonance energy transfer (TR-FRET) assay to evaluate the ability of small molecules to inhibit ICOS/ICOSL interaction. Implementation of the developed TR-FRET assay in high-throughput screening (HTS) of a focused chemical library resulted in the identification of AG-120 as a first-in-class inhibitor of ICOS/ICOSL interaction. We further employed docking studies and molecular dynamics (MD) simulations to identify the plausible mechanism of blocking ICOS/ICOSL complex formation by AG-120. Using the structure-activity relationship (SAR) by catalog approach, we identified AG-120-X with an IC50 value of 4.68 ± 0.47 μM in the ICOS/ICOSL TR-FRET assay. Remarkably, AG-120-X revealed a dose-dependent ability to block ICOS/ICOSL interaction in a bioluminescent cellular assay based on co-culturing Jurkat T cells expressing ICOS and CHO-K1 cells expressing ICOSL. This work will pave the way for future drug discovery efforts aiming at the development of small molecule inhibitors of ICOS/ICOSL interaction as potential therapeutics for cancer as well as other diseases.

Design, synthesis and biological evaluation of novel quinazolinone derivatives as CRBN E3 ligase modulators

CRBN E3 ligase modulators, also anteriorly called immunomodulatory drugs (IMiDs), exhibit excellent pharmacological activity by degrading cereblon (CRBN) associated multiple substrates and have become an important field for drug development. These modulators such as Thalidomide, Lenalidomide and CC-122 abduct CRBN to adhere to IKZF1/3 and other neosubstrates, and then induce the degradation of these substrates, thus retarding the further development of related diseases. Herein, we reported a series of CC-122 derivatives that inhibit the proliferation of hematological malignant tumor cell lines. Studies further confirmed that several derivatives which exhibit strong anti-proliferation effect induce the significant degradation of IKZF1/3. In addition, we found that the best compound 14 (SIAIS355035) exhibits better degradation activity and better anti-proliferation activities than CC-122, especially in diffuse large B lymphoma cell lines. Moreover, the PK properties of compound 14 are pretty promising with excellent oral bioavailability. These results clarified the SAR of CC-122 derivatives preliminarily and suggested that compound 14 has great value for further studies as an ideal novel CRBN E3 ligase modulation drug.

2118. Patient Characteristics and Clinical Outcomes of Leptotrichia spp. Bacteremia

Abstract Background Leptotrichia spp. are anaerobic, gram-negative bacilli that are part of the normal oral and intestinal microbiota. Although traditionally considered non-pathogenic, these bacteria can result in invasive infections including bacteremia in immunosuppressed patients, particularly those with neutropenia. There are limited published data to inform best management strategies in those with Leptotrichia bacteremia. Methods All cases of Leptotrichia spp. bacteremia between January 2012 and 2022 at our tertiary academic medical center were retrospectively reviewed to determine patient risk factors, clinical outcomes, and antimicrobial susceptibilities. Descriptive statistical methods were used. Antimicrobial susceptibilities were performed by agar dilution. Results 26 cases of Leptotrichia spp. bacteremia were identified (Figure 1). The mean patient age was 55 years (SD 17), with 9 female patients (35%). All 26 patients were immunocompromised, predominantly due to hematologic malignancy (69%) or hematopoietic stem cell transplant (23%) (HSCT). 25 of 26 patients were actively neutropenic, with a median duration of neutropenia of 21 days (13-26) (Table 1). The most frequent sources of Leptotrichia bacteremia were gastrointestinal translocation (60%), followed by catheter-related infection (35%). 10 patients had polymicrobial bacteremia (38.5%). The primary antibiotics utilized to treat Leptotrichia bacteremia included metronidazole (42%), piperacillin-tazobactam (27%), and carbapenems (19%). Overall, the mean duration of treatment was 11 days, with a 60-day all-cause mortality of 19% (Table 1) with no cases of microbiologic relapse. In the 22 clinical isolates evaluated for susceptibility, Leptotrichia spp. were largely susceptible to metronidazole, penicillin, ertapenem, and piperacillin-tazobactam, but uniformly resistant to moxifloxacin (Table 2). Figure 1:Leptotrichia spp. identified as cause of bacteremia in 26 patientsTable 1:Baseline characteristics of patients with Leptotrichia bacteremia (n=26) between January 2012 to January 2022Table 2:Antimicrobial susceptibility profile of the Leptotrichia spp. (n=22) Conclusion Leptotrichia spp. may be a rare cause of bacteremia in neutropenic hosts, particularly those with underlying hematologic malignancies and HSCT. The pathogen has a favorable susceptibility profile to penicillins and carbapenems, but has high degree of resistance to fluoroquinolones. Disclosures All Authors: No reported disclosures.

Granulocyte Transfusion Therapy: Institutional Experience of Benefit in Cancer Patients with Prolonged Neutropenic Sepsis—A Retrospective Study

Abstract Introduction Patients undergoing intensive chemotherapy for hematological malignancy and stem cell transplantation are at increased risk of neutropenia.Neutropenia is among the frequent side effects of intensive treatments, and when absolute neutrophil count (ANC) falls < 500/µL, the risk of microbial and fungal infection increases significantly.As neutropenia is the main cause of these infections, transfusion of granulocyte immediately as a replacement is a life-saving therapeutic option to support these patients by restoring neutrophil counts and aiding in the resolution of infection. Objective The present study is a retrospective single institutional analysis of granulocyte transfusion therapy in children and young adults with cancer who received treatment with GT during prolonged and profound life threatening neutropenia. Materials and Methods This study was a retrospective analysis of 66 granulocyte transfusions in 36 patients of hematological and solid malignancy with severe and prolonged neutropenia in the department of Medical Oncology, Sri Aurobindo Institute of Medical Sciences Indore, between September 2019 and March 2022.Donors were either patients' relatives or voluntary donors without comorbidities.All granulocyte concentrates were collected by centrifugation leukapheresis and irradiated with 2500 centigray and immediately transfused in full, to the patient over 60 ot 120 minutes with appropriate premedication. Results A total of 36 patients (M:F, 19:17) with a median age of 16 years (2–43) received 66 granulocyte transfusions. The diagnosis of patients included acute myelogenous leukemia (n = 17), B cell acute lymphoblastic leukemia (n = 10), non-Hodgkin lymphoma (n = 3), Ewing's sarcoma (n = 2), neuroblastoma (n = 1), malignant melanoma (n = 1), aplastic anemia (n = 1), osteosarcoma (n = 1). All had severe neutropenia with absolute neutrophil count < 0.5 × 109/L. The median duration of severe neutropenia was 16 days. Patients received a median cell dose of granulocytes 2.9 × 1010/L. A favorable response was seen in 28 (78%) patients, whereas an unfavorable response was seen in 8 patients (23%). Conclusion A granulocyte therapy was effective in many critically sick patients with prolonged and profound neutropenia. Granulocyte transfusions may be more beneficial in selected patients where it provides more time to overcome refractory infections with broad-spectrum antibiotics. Granulocyte transfusion are at best a “bridge” that gives time to marrow recovery. The challenges to using GT are clinical, finding patients who may get benefitted, and logistical, selection of donors and harvest technique. Randomized trials with large numbers of patients are required to prepare guidelines for granulocyte use.

Comparing Invasive Pulmonary Aspergillosis Mortality Between Liposomal Amphotericin B and Voriconazole in Patients With Hematological Malignancy or Hematopoietic Stem Cell Transplantation.

Objectives We evaluated liposomal amphotericin B versus voriconazole for the treatment of invasive pulmonary aspergillosis (IPA) in patients with hematological malignancy or hematopoietic stem cell transplantation (HSCT). Methods This retrospective cohort, single-center study included patients with compatible radiological diagnosis of IPA between 2016 and 2021. Results Forty-six patients with hematological malignancy or HSCT were diagnosed with IPA. Thirty-nine of them fulfilled the criteria for comparing liposomal amphotericin B (n=15) with voriconazole (n=24). Their median age was 48.5 years. Stem cell transplant recipients were 45.65%, and nearly half of the patients (47.83%) had acute myeloid leukemia. Twenty-six (56.52%) of the patients did not require oxygen therapy. The 12-week mortality was 13.33% (two out of 15) in patients who received liposomal amphotericin B compared to 25% (six out of 24) in patients who received voriconazole. There was no mortality judged to be related to IPA. Success or global clinical response was not different between the two drugs: 80% for liposomal amphotericin B versus 83.33% for voriconazole. However, the safety profile favored liposomal amphotericin B. Conclusion In this small cohort, there was an equipoise in the mortalityand clinical and radiological outcomes obtained using liposomal amphotericin B or voriconazole for the treatment of IPA in hematological malignancy or HSCT.

Newly Synthesized Melphalan Analogs Induce DNA Damage and Mitotic Catastrophe in Hematological Malignant Cancer Cells.

Myeloablative therapy with highdoses of the cytostatic drug melphalan (MEL) in preparation for hematopoietic cell transplantation is the standard of care for multiple myeloma (MM) patients. Melphalan is a bifunctional alkylating agent that covalently binds to nucleophilic sites in the DNA and effective in the treatment, but unfortunately has limited therapeutic benefit. Therefore, new approaches are urgently needed for patients who are resistant to existing standard treatment with MEL. Regulating the pharmacological activity of drug molecules by modifying their structure is one method for improving their effectiveness. The purpose of this work was to analyze the physicochemical and biological properties of newly synthesized melphalan derivatives (EE-MEL, EM-MEL, EM-MOR-MEL, EM-I-MEL, EM-T-MEL) obtained through the esterification of the carboxyl group and the replacement of the the amino group with an amidine group. Compounds were selected based on our previous studies for their improved anticancer properties in comparison with the original drug. For this, we first evaluated the physicochemical properties using the circular dichroism technique, then analyzed the zeta potential and the hydrodynamic diameters of the particles. Then, the in vitro biological properties of the analogs were tested on multiple myeloma (RPMI8226), acute monocytic leukemia (THP1), and promyelocytic leukemia (HL60) cells as model systems for hematological malignant cells. DNA damage was assessed by immunostaining γH2AX, cell cycle distribution changes by propidium iodide (PI) staining, and cell death by the activation of caspase 2. We proved that the newly synthesized derivatives, in particular EM-MOR-MEL and EM-T-MEL, affected the B-DNA conformation, thus increasing the DNA damage. As a result of the DNA changes, the cell cycle was arrested in the S and G2/M phases. The cell death occurred by activating a mitotic catastrophe. Our investigations suggest that the analogs EM-MOR-MEL and EM-T-MEL have better anti-cancer activity in multiple myeloma cells than the currently used melphalan.

KLRG1 Depletion Is a Novel Therapeutic Strategy for Patients with Mature T and NK/T-Cell Lymphomas

Outcomes for patients with aggressive T-cell lymphomas (TCLs) remain dismal. Killer cell lectin-like receptor G1 (KLRG1) is a co-inhibitory immune checkpoint receptor expressed on late differentiated CD8+ T effector memory (TEM) and TEMRA cells. Monotherapy with KLRG1 neutralizing antibodiesextended survival in solid tumor models. Here, we report the therapeutic potential of a novel and distinct strategy characterized by depletion of KLRG1+ malignant cells in hematologic malignancies and specifically in TCLs. We defined the molecular epidemiology of KLRG1 by determining tumor cell-specific RNA and protein expression on T cell subsets from 8 healthy donors, 26 TCL lines, 15 patient-derived xenografts (PDXs), and 165 tumor and blood specimens representing various subtypes of T and NK/TCLs. Microarray andRNA-seqdata demonstrated highertranscript levels of KLRG1 in patients with T-LGLL and PDXs of T-PLL and HSTCL relative to other subtypes, which were corroborated by protein expression. We quantified tumor cell-specific protein expression of KLRG1 by IHC by generating H scores (% of KLRG1+ tumor cells × measure of staining intensity) ranging from 0-300 across 141 primary TCL biopsies. PTCL-NOS and AITL had heterogeneous KLRG1 expression, whereas ALCL, CTCL, and EATL demonstrated none. The majority of patients with T-PLL (median 180), δ/γ TCLs (median 140), and NK/TCLs (median 100) exhibited higher H scores relative to other subtypes. Up to 99% of the CD3+/CD8+/CD57+ and up to 97% of the CD3-/CD56+/CD57+/CD16+/CD94+ leukemic cells in the blood of patients with T-LGLL (n=12) and CLPD-NK (n=12) respectively expressed KLRG1 protein (Fig.1). None of the TCL cell lines expressed surface KLRG1. Therefore, SMZ1 cells (TP63 rearranged PTCL-NOS line) were stably transduced to express KLRG1. We utilized two human KLRG1 binding antibodies, 13F12F2 (a commercial mIgG2a Ab) and mAb208 (a humanized afucosylated mIgG2a Ab) for in vitro and in vivo studies. Antibody binding capacity assays confirmed a higher number of KLRG1 receptors per cell on SMZ1 KLRG1+ (175,800), HSTCL (23,586), and CD8+ T cells (16,326) compared with B-cell lymphoma cells (242). Competition binding assays demonstrated that concentrations of 0.1 ug/ml of mAb208 saturated receptors on SMZ1 KLRG1+ cells. mAb208 induced a 2-5-fold dose-dependent increase in antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC), but not apoptosis of SMZ1 KLRG1+ cells relative to SMZ1 KLRG1- WT and isotype control (CTL). This was exhibited across a variety of human and murine effectors such as human PBMCs, human and murine macrophages, and human and murine complements. Reporter-based murine FcγRIV ADCC bioassay demonstrated a dose-dependent 2-6 fold induction of bioluminescence by mAb208 in SMZ1 KLRG1+ cells relative to WT (p=<0.0001). In 4/4 tested T-LGLL patient samples, mAb208 induced ADCC of CD8+/CD57+ LGLs with autologous PBMCs (p=0.03) relative to CTL. Finally, mAb208 induced selective ADCC of KLRG1+ CD8+ TEM and TEMRA cells while sparing KLRG1- naive and central memory T cells in 3 healthy donors (p=0.06). Duvelisib (Duv), a dual PI3K-δ/γ inhibitor (PI3Ki), has demonstrated a high ORR of 50% in phase II trials in TCLs and its ability to reprogram macrophages to an M1 phenotype in TCL PDXs. Thus, we hypothesized that the combination of PI3Ki with anti-KLRG1 mAbs would enhance the clearance of apoptotic and opsonized KLRG1+ TCL cells by proinflammatory macrophages in several PTCL subtypes. We tested this combination in vivo using SMZ1 KLRG1+ subcutaneous xenografts in NSG mice. After 17 days of treatment, combination (mAb208 + Duv)- treated mice had a marked reduction in their mean tumor volumes (159 mm3) relative to mAb208 (1003 mm3 ; p=0.03), Duv (465.6 mm3 ; p=0.03) and CTL (2104 mm3 ; p=0.03) mice resulting in prolonged survival (Fig. 2). Studies in PDX models of T-PLL and HSTCL with varying levels of KLRG1 expression (30% and 80% respectively) have been initiated and will be reported at the meeting. Selective depletion of tumor cells with an anti-KLRG1 depleting mAb in specific subtypes of TCLs, T-LGLL, and CLPD-NK is effective in vitro and in vivo, and its combination with Duv has marked activity in an aggressive PTCL-NOS xenograft model. A multicenter phase I/II trial of the KLRG1 depleting antibody ABC008 (Abcuro) in patients with R/R T-LGLL has been initiated. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Novel Method for Efficient cGMP Production of Natural Killer Cells from Clonal Master Induced Pluripotent Stem Cells for Next Generation, Off-the-Shelf Cancer Immunotherapy

Natural killer (NK) cells are innate immune cells that play a key role in in tumor immune surveillance. NK cells are self-tolerant to healthy cells and can kill tumor cells in both hematological and solid malignancies. Recent advances allow for the derivation of immune cells, including NK cells, from human induced pluripotent stem cells that can be utilized for cancer immunotherapy. High doses of NK cells (approx. 0.5-5 x 109 cells per dose) and multiple doses are both safe and likely necessary for clinical efficacy. Manufacturing large number of NK cells from a clonal master iPSC line provides a promising strategy to enable next generation, off-the-shelf, affordable, cancer immunotherapies. We have developed a novel process to produce high purity, clinical scale NK cells efficiently and consistently. This method does not require cell sorting or the usage of murine-derived stromal cells. Briefly, hematopoietic progenitor cells are derived using an improved spin embryoid body (EB) method using defined cytokines including bone morphogenic protein-4 (BMP-4), stem cell factor (SCF), vascular endothelial growth factor (VEGF), fms-like tyrosine kinase 3 (FLT-3) etc. The hematopoietic progenitor cells are subsequently differentiated into mature NK cells using a second set of defined cytokines such as IL-3, IL-7, IL-15, SCF etc. Using this novel method, fully functioning NK cells could be expanded more than 8,000-fold to enable us to produce 1 x 1012 NK cells starting from 1 x 106 undifferentiated iPSCs. This cell production scale can supply >200 clinical doses from one cGMP manufacturing campaign. NK cells produced using this method are CD56-positive and display the typical phenotype of NK cell activating receptors including NKG2D, NKp44, NKp46, DNAM-1. Importantly, these NK cells demonstrate better anti-tumor activities and produce higher levels of IFNγ and TNFα compared to peripheral blood-derived NK cells isolated from healthy donors. Moreover, this protocol has been adapted and optimized for clinical scale manufacturing of NK cells from a genetically engineered iPSC cell line bearing a knock-out of CISH, a key intracellular checkpoint of NK cell activation. CISH KO NK cells produced using this optimized method show significantly better anti-tumor activities against multiple liquid and solid tumor cell lines (Figure 1), including K562, Raji (B cell lymphoma), Daudi (B cell lymphoma), SUP-B15 (B cell lymphoma), MOLT-4 (T cell lymphoma), SKOV-3 (ovarian cancer), OVCAR-4 (ovarian cancer), HCC-827 (lung cancer), ZR-75 (breast cancer), and BT-474 (breast cancer), in standard cytotoxicity assays compared with unmodified NK cells. Overall, this novel cell production strategy paves the way for clinical trials using higher doses of iPSC-derived NK cells with increased potency, thus enabling next-generation CAR-NK cell-based immunotherapies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal