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Blood Podcast
Hypercalcemia in monoclonal gammopathy of undetermined significance, neutrophil gelatinase-associated lipocalin in hemostasis, and CD19-targeted NK- or T-cell therapy combined with anti-CD19 monoclonal antibodies
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Welcome to the February 27, 2025 episode of Blood Podcast, your source for innovative ideas and cutting edge information. Our topics are based on articles published in Blood, A Journal of the American Society of Hematology. Today we'll learn more about the significance of hypercalcemia in monoclonal gammopathy of undetermined significance, the role of neutrophil gelatinase associated lipokalin in hemostasis, and the feasibility of combining CD19 targeted NK or T cell therapy with anti CD19 monoclonal antibodies. We first examine data in the Blood article entitled Approaching hypercalcemia in gammopathy of Undetermined Significance. Insights from the ISTOPMM study by Astrun Johnstotter of the University of Iceland in Reykjavik, Iceland, and colleagues. Hypercalcemia is a hallmark symptom of multiple myeloma, together with renal impairment, anemia and osteolytic bone lesions. Hypercalcemia can also occur in patients with monoclonal gammopathy of undetermined significance or MGUs, a precursor condition that sometimes progresses to myeloma. Development of hypercalcemia in patients with MGUs has long been considered a warning sign that myeloma progression has begun. However, there are few data on which to base this assumption. The authors of the first paper we'll be discussing set out to fill this data gap by examining the prevalence and causes of hypercalcemia in patients with MGUs enrolled in the Iceland Screens, Treats or Prevents Multiple Myeloma study, also known as ISTOPMM. MGUS is a premalignant condition that carries a 1 to 1.5% risk of progression to multiple myeloma or a related lymphoproliferative disease. MGUS occurs in about 4% of people in the general population over the age of 50. Hypercalcemia is similarly prevalent, occurring in 1 to 4% of the general population. Common causes of hypercalcemia include hyperparathyroidism or cancer. Patients diagnosed with mgus are typically followed for blood M protein levels and signs of end organ damage, including blood calcium, in order to detect progression to multiple myeloma as early as possible. The ISTOPMM study is a population screening study in which about 75,000 people over the age of 40 living in Iceland have been screened for MGUs. About 2/3 of those who tested positive for MGUs have been invited for regular screenings, which includes serum calcium measurements. The retrospective study we're discussing here looked at the prevalence and clinical correlates of hypercalcemia in in a little over 2,500 of those patients who attended follow up visits between April 2017 and February 2022, 191, or about 7.5% of the study cohort, had at least one instance of hypercalcemia. 98 of these, or about half, had hypercalcemia that either disappeared on its own or was found to be an artifact by subsequent testing. Based on this finding, the authors recommended that hypercalcemia be confirmed by repeat testing, preferably with a test that measures serum ionized calcium rather than total serum calcium. The remaining 93 patients had confirmed persistent hypercalcemia. The cause of hypercalcemia could not be determined in 15 patients in some cases because of inadequate documentation. Another three had died from causes unrelated to MGUs before they could be evaluated. In three of the remaining 85 patients, hypercalcemia was caused by progression to multiple myeloma. These patients all had other typical myeloma symptoms at diagnosis. Most of these patients had hypercalcemia that was caused by conditions other than multiple myeloma. This included 56% who had primary hyperparathyroidism and 16% who had other malignancies. Five patients had hypercalcemia as a result of taking vitamin D supplements. These causes of persistent hypercalcemia are very similar to those found in the general population. The study authors concluded that patients with mgus who have hypercalcemia should be evaluated similarly to members of the general population because it occurs for similar reasons. These results also suggest that hypercalcemia is not a reliable marker for progression to multiple myeloma in patients with MGUs when no other myeloma symptoms are present. This work represents the first and largest longitudinal study of hypercalcemia in patients with mgus. The authors noted, however, that Iceland is a genetically and ethnically homogeneous population, which may limit the generalizability of their findings. In an accompanying commentary, Hirami Ann of McMaster University in Hamilton, Canada, and Alyssa Visram of Ottawa Hospital Research Institute in Ontario, Canada, described this work as a hallmark study with important implications for clinical practice. They note that this study will enable providers to better counsel patients, reducing anxiety. It will also decrease unnecessary investigations and premature treatment interventions. This study also adds to our understanding of the underlying biology of premalignant conditions like MGUs. Next up, we'll discuss the findings from the Blood article entitled Deficiency of neutrophil Gelatinase Associated Lipokalen is elicits hemophilia like bleeding and Clotting disorder by Mix Xiu of Qingdao University in Qingdao, China and colleagues. Neutrophil gelatinase associated lipocalen, also known as N gal, is a small protein that promotes inflammation and helps defend against bacterial infections. As part of the innate immune system, NGAL is secreted into blood, urine and other body fluids by neutrophils, macrophages and other immune cells in response to the presence of inflammatory cytokines. In recent years, NGAL has been under investigation as a biomarker for disease conditions that involve inflammation, including acute kidney injury and chronic kidney disease, diabetic complications and cardiovascular disease. Both neutrophils and NGAL have been implicated in processes that lead to thrombosis formation and vascular injury, suggesting that they form part of the link between cardiovascular disease and inflammation. Neutrophils, for example, are recruited to sites of thrombosis and may contribute to clot formation by forming web like structures known as neutrophil extracellular traps. Increased NGAL production correlates with atherosclerotic plaque instability and myocardial damage and has been implicated in the pathological mechanisms of stroke. The authors of the study we will discuss next set out to investigate the link between inflammation and cardiovascular disease more closely by studying the effects of NGAL on hemostasis in vitro in human plasma and in vivo in a number of mouse models. The author's investigations into human plasma lends support to the idea that that NGAL is involved in hypercoagulability and promotes thrombosis through multiple mechanisms. NGAL levels were significantly increased in plasma from patients who had experienced deep vein thrombosis compared with healthy individuals or patients who had an ST segment elevation. Myocardial infarction NGAL enhanced the activities of clotting factors in plasma, including thrombin, kallikrine factor 11a and factor VIIa, while inhibiting antithrombin activity. ENGAL also increased thrombin induced platelet aggregation, reduced clot reaction time and increased clot firmness as measured by thromboelastography. Conversely, addition of an anti NGAL monoclonal antibody to human plasma prolonged clotting time and reduced thrombin activity. The authors also showed by several methods that NGAL directly interacts with clotting factors, including the detection of protein complexes composed of NGAL and clotting factors in thrombi collected from patients with deep vein thrombosis. The authors used a number of mouse models to investigate the effects of manipulating NGAL levels in vivo. NGAL levels were increased by by infecting mice with an NGAL expressing adenovirus or by administering NGAL intravenously, NGAL levels were reduced using the anti ngal monoclonal antibody as well as by creating an NGAL knockout mouse. Results were similar to those of the in vitro studies. Increasing NGAL promoted coagulation and thrombosis while decreasing NGAL had the opposite effect. Increased NGAL reduced bleeding time in mouse tail bleeding and saphenous vein tests. Tests in the ngal knockout mice, on the other hand, revealed a prolonged bleeding phenotype that resembled that of mouse hemophilia models. The authors also tested the effects of NGAL in a mouse model in which deep vein thrombosis was induced with the use of ferric chloride. Higher NGAL led to increased thrombosis formation while administration of the anti NGAL antibody had the opposite effect. They noted that the ENGAL knockout mice had quite prolonged clotting times compared with wild type mice in this model as well. Mice injected with pro inflammatory agents such as bacterial lipopolysaccharide or carrageenan can also be induced to form thrombi. This effect was amplified when ngal levels were higher and reduced when they were lower. Introduction of carrageenan into the ENGAL knockout mice also led to substantially less thrombosis formation compared with wild type mice. The study authors concluded that, taken together, these results indicate that ENGAL plays an important role in the regulation of coagulation, thrombosis and overall hemostasis. They suggested that ENGAL may act as a mediator between the inflammatory effects of the innate immune system and thrombosis. This work has important implications for hypercoagulability related to infectious disease, cardiovascular disease and cancer. They further suggested that engal's interactions with proteins involved in the coagulation cascade could could serve as targets for novel antithrombotic therapies. In an accompanying commentary, Bader Keilani and Constantin Stark of Ludwig Maximilian University Munich in Munich, Germany, said that this paper provides strong evidence for NGAL as a mediator of hypercoagulability and thrombosis. They noted, however, that several questions remain unanswered, in particular whether and how NGAL might interact with the complement system, which also plays a key role in linking inflammation and thrombosis. They agreed with the authors that NGAL might serve as a productive target for treatments aimed at managing clotting disorders, adding autoimmune disorders to the list of conditions in which ENGAL might contribute to the link between inflammation and thrombosis. And in the final part of today's podcast, we'll discuss findings in the Blood article entitled Anti CD19 Antibody Co Treatment Enhances Serial Killing Activity of Anti CD19 CAR T&NK cells and reduces trogocytosis by Seung Kwan Ko of Sung Kyung Kwan University in Seoul, South Korea, and colleagues. CD19 targeted therapies have become increasingly important in treating B cell malignancies. FDA approved CD19 targeted therapies take the form of chimeric antigen receptor or CAR T cell therapies, cytolytic monoclonal antibodies, and antibody drug conjugates. As use of these therapies has become more widespread, researchers are investigating whether these agents might be combined by to provide greater antitumor efficacy. The authors of the study we'll be discussing next investigated this question in vitro and in vivo using preclinical models. Their studies shed light on ways to potentially overcome some important CAR T cell limitations, including CD19 antigen loss and the common CAR T cell toxicity known as cytokine release syndrome. The study authors combined a series of anti CD19 antibodies with car therapies engineered from a natural killer or NK immortalized cell line as well as from primary human NK cells and T cells. These combination therapies were tested against a B cell precursor leukemia cell line derived from a patient with acute lymphoblastic leukemia. The immortalized tumor cells were used for in vitro experiments and in vivo as xenografts in a strain of immunodeficient mice. Initial studies showed that cotreatment with an anti CD19 monoclonal antibody enhanced the tumor cell killing activities of all three types of CD19 targeted car, NK and car T cells. This was true even under conditions where the CAR cells were repeatedly challenged by the addition of more tumor cells. This result showed that the two types of therapies could be used together despite competition for the CD19 binding site. The authors corroborated these results in the mouse xenograft model, showing that CD19 targeted combination therapy both improved antitumor efficacy and and prolonged mouse survival compared with car, NK or T cell therapy alone in vitro. These results also held true for Multiple different anti CD19 antibody clones. The extent to which tumor cell killing was enhanced depended on the different clones. Affinities for CD19 clones with less affinity for CD19 interfered less with the car, NK or T cell's ability to to bind to and kill tumor cells and led to more pronounced improvements in CAR cell efficacy. The authors investigated the underlying mechanisms that led to this effect. CAR cells killing abilities depend on their ability as effector cells to form transient interactions with target tumor cells. CO treatment with an anti CD19 monoclonal antibody modulated these interactions, causing the NK or T cells to let go of target cells more quickly. This increased off rate had two effects that led to enhanced tumor cell killing. First, the car, NK or T cells that released quickly were free to go on and kill additional tumor cells, improving their overall kill rates. Second, the increased off rate led to reduced trogocytosis. Trogocytosis is a process by which interacting cells take up bits of membrane and proteins from each other. In car, NK or T cell therapy, this can result in the effector cells taking up the targeted antigen, causing antigen loss on the target cells and thus loss of efficacy. This process is recognized as a leading mechanism of acquired resistance to CAR, NK&T cell therapies in B cell malignancies. In the current study, the authors showed that CO treatment with anti CD19 antibodies reduced trogocytosis, thus reducing antigen loss on the target tumor cells. Because reducing trogocytosis also reduces the amount of CD19 antigen displayed on the car, NK or T cells, these cells were also less likely to be attacked and killed by their CAR cell siblings. Further investigation of the effects of anti CD19 antibodies on car, NK and T cell activities suggested that by delaying and otherwise modulating the course of CAR cell activation, these antibodies might also help to prevent overactivation and thus reduce the probability of cytokine release syndrome. The authors concluded that using anti CD19 monoclonal antibodies to modulate the interactions of CD19 targeted car, NK and T cells with their targets might be a fruitful avenue to improving efficacy and reducing resistance in patients with B cell malignancies. In an accompanying commentary, Alexandre V. Hirayama and Marie Bleakley of the Fred Hutchinson Cancer center in Seattle, Washington, said that this study adds to growing evidence that competition for CD19 binding can modulate CD19 targeted car T cell activation and increase efficacy. They expressed reservations related to the heterogeneity of CD19 expression in B cell malignancies, which may limit this approach if pretreatment CD 19 levels are low. They noted that controlled clinical trials will be needed to optimize dosing strategies for anti CD19 combination therapy. For a list of additional authors, as well as more detailed articles and commentaries on which this podcast is based, please go to bloodjournal.org be sure to join us next week for another episode of Blood Podcast. Thank you for listening.