Case Report: ANXA2 Associated Life-Threatening Coagulopathy With Hyperfibrinolysis in a Patient With Non-APL Acute Myeloid Leukemia

Patients with acute promyelocytic leukemia (APL) often present with potentially lifethreatening hemorrhagic diathesis. The underlying pathomechanisms of APLassociated coagulopathy are complex. However, two pathways considered to be APLspecific had been identified: 1) annexin A2 (ANXA2)-associated hyperfibrinolysis and 2) podoplanin (PDPN)-mediated platelet activation and aggregation. In contrast, since disseminated intravascular coagulation (DIC) is far less frequent in patients with non- APL acute myeloid leukemia (AML), the pathophysiology of AML-associated hemorrhagic disorders is not well understood. Furthermore, the potential threat of coagulopathy in non- APL AML patients may be underestimated. Herein, we report a patient with non-APL AML presenting with severe coagulopathy with hyperfibrinolysis. Since his clinical course resembled a prototypical APL-associated hemorrhagic disorder, we hypothesized pathophysiological similarities. Performing multiparametric flow cytometry (MFC) and immunofluorescence imaging (IF) studies, we found the patient’s bone-marrow mononuclear cells (BM-MNC) to express ANXA2 - a biomarker previously thought to be APL-specific. In addition, whole-exome sequencing (WES) on sorted BM-MNC (leukemiaassociated immunophenotype (LAIP)1: ANXAlo, LAIP2: ANXAhi) demonstrated high intratumor heterogeneity. Since ANXA2 regulation is not well understood, further research to determine the coagulopathy-initiating events in AML and APL is indicated. Moreover, ANXA2 and PDPN MFC assessment as a tool to determine the risk of life-threatening DIC in AML and APL patients should be evaluated

Progenitor Renin Lineage Cells are not involved in the regeneration of glomerular endothelial cells during experimental renal thrombotic microangiopathy.

Endothelial cells (EC) frequently undergo primary or secondary injury during kidney disease such as thrombotic microangiopathy or glomerulonephritis. Renin Lineage Cells (RLCs) serve as a progenitor cell niche after glomerular damage in the adult kidney. However, it is not clear whether RLCs also contribute to endothelial replenishment in the glomerulus following endothelial injury. Therefore, we investigated the role of RLCs as a potential progenitor niche for glomerular endothelial regeneration. We used an inducible tet-on triple-transgenic reporter strain mRen-rtTAm2/LC1/LacZ to pulse-label the renin-producing RLCs in adult mice. Unilateral kidney EC damage (EC model) was induced by renal artery perfusion with concanavalin/anti-concanavalin. In this model glomerular EC injury and depletion developed within 1 day while regeneration occurred after 7 days. LacZ-labelled RLCs were restricted to the juxtaglomerular compartment of the afferent arterioles at baseline conditions. In contrast, during the regenerative phase of the EC model (day 7) a subset of LacZ-tagged RLCs migrated to the glomerular tuft. Intraglomerular RLCs did not express renin anymore and did not stain for glomerular endothelial or podocyte cell markers, but for the mesangial cell markers α8-integrin and PDGFRβ. Accordingly, we found pronounced mesangial cell damage parallel to the endothelial injury induced by the EC model. These results demonstrated that in our EC model RLCs are not involved in endothelial regeneration. Rather, recruitment of RLCs seems to be specific for the repair of the concomitantly damaged mesangium

Case Report: ANXA2 Associated Life-Threatening Coagulopathy With Hyperfibrinolysis in a Patient With Non-APL Acute Myeloid Leukemia

Patients with acute promyelocytic leukemia (APL) often present with potentially lifethreatening hemorrhagic diathesis. The underlying pathomechanisms of APLassociated coagulopathy are complex. However, two pathways considered to be APLspecific had been identified: 1) annexin A2 (ANXA2)-associated hyperfibrinolysis and 2) podoplanin (PDPN)-mediated platelet activation and aggregation. In contrast, since disseminated intravascular coagulation (DIC) is far less frequent in patients with non- APL acute myeloid leukemia (AML), the pathophysiology of AML-associated hemorrhagic disorders is not well understood. Furthermore, the potential threat of coagulopathy in non- APL AML patients may be underestimated. Herein, we report a patient with non-APL AML presenting with severe coagulopathy with hyperfibrinolysis. Since his clinical course resembled a prototypical APL-associated hemorrhagic disorder, we hypothesized pathophysiological similarities. Performing multiparametric flow cytometry (MFC) and immunofluorescence imaging (IF) studies, we found the patient’s bone-marrow mononuclear cells (BM-MNC) to express ANXA2 - a biomarker previously thought to be APL-specific. In addition, whole-exome sequencing (WES) on sorted BM-MNC (leukemiaassociated immunophenotype (LAIP)1: ANXAlo, LAIP2: ANXAhi) demonstrated high intratumor heterogeneity. Since ANXA2 regulation is not well understood, further research to determine the coagulopathy-initiating events in AML and APL is indicated. Moreover, ANXA2 and PDPN MFC assessment as a tool to determine the risk of life-threatening DIC in AML and APL patients should be evaluated

Case Report: ANXA2 Associated Life-Threatening Coagulopathy With Hyperfibrinolysis in a Patient With Non-APL Acute Myeloid Leukemia

Patients with acute promyelocytic leukemia (APL) often present with potentially lifethreatening hemorrhagic diathesis. The underlying pathomechanisms of APLassociated coagulopathy are complex. However, two pathways considered to be APLspecific had been identified: 1) annexin A2 (ANXA2)-associated hyperfibrinolysis and 2) podoplanin (PDPN)-mediated platelet activation and aggregation. In contrast, since disseminated intravascular coagulation (DIC) is far less frequent in patients with non- APL acute myeloid leukemia (AML), the pathophysiology of AML-associated hemorrhagic disorders is not well understood. Furthermore, the potential threat of coagulopathy in non- APL AML patients may be underestimated. Herein, we report a patient with non-APL AML presenting with severe coagulopathy with hyperfibrinolysis. Since his clinical course resembled a prototypical APL-associated hemorrhagic disorder, we hypothesized pathophysiological similarities. Performing multiparametric flow cytometry (MFC) and immunofluorescence imaging (IF) studies, we found the patient’s bone-marrow mononuclear cells (BM-MNC) to express ANXA2 - a biomarker previously thought to be APL-specific. In addition, whole-exome sequencing (WES) on sorted BM-MNC (leukemiaassociated immunophenotype (LAIP)1: ANXAlo, LAIP2: ANXAhi) demonstrated high intratumor heterogeneity. Since ANXA2 regulation is not well understood, further research to determine the coagulopathy-initiating events in AML and APL is indicated. Moreover, ANXA2 and PDPN MFC assessment as a tool to determine the risk of life-threatening DIC in AML and APL patients should be evaluated

Mesangial cells and not podocytes display considerable transient damage in the course of the EC model.

<p>Mice were analyzed at baseline (day 0), day 1, and day 7 following disease induction. A. Quantification of glomerular mesangial injury by α8-integrin (mesangial cell marker) staining of kidney slices. Data are presented as mean ± SEM, n = 5/5/10 for day 0 /day 1 /day 7, respectively. **p<0.01, ***p<0.001 by one-way ANOVA; B. Representative α8-integrin staining images in the course of the EC model. Scale bars correspond to 25 μm; C. Quantification of podocyte depletion and injury on kidney slices by WT1 and nephrin staining, respectively. Data are presented as mean ± SEM, n = 5/5/10 for day 0 /day 1 /day 7, respectively. *p<0.05, **p<0.01, ns- not significant by one-way ANOVA; D. Representative WT1/nephrin co-staining images in the course of the EC model. 4′,6-diamidino-2-phenylindole (DAPI) was used as a nuclear marker. Scale bars correspond to 25 μm.</p

RLCs selectively differentiate to intraglomerular mesangial cells during EC model.

<p>A. Representative confocal microscopy images for day 0 and day 7 of β-gal and the EC markers ERG (upper panels) or CD31 (lower panels) co-stained kidney slices; B. Representative confocal microscopy images for day 0 and day 7 of β-gal and the mesangial cell markers PDGFβR (upper panels) or α8-integrin (lower panels) co-stained kidney slices; C. Representative confocal microscopy images for day 0 and day 7 of β-gal/WT1 (podocyte marker) co-stained kidney slices. 4′,6-diamidino-2-phenylindole (DAPI) was used as a nuclear marker throughout. The channels for green (β-gal) and red (corresponding cell marker) fluorescent signals in the dashed squares on day 7 are separately shown in the small right panels. Scale bars correspond to 25 μm.</p

RLCs invade the glomerulus during EC model.

<p>A. Intraglomerular RLCs tagged by β-gal, but negative for renin appear in the regenerative phase of the EC model (day 7). Representative confocal microscopy images for day 0 and day 7 of β-gal/renin co-stained kidney slices. 4′,6-diamidino-2-phenylindole (DAPI) was used as a nuclear marker. The channels for green (β-gal) and red (renin) fluorescent signals in the dashed square on day 7 are separately shown in the small right panels. Scale bars correspond to 25 μm; B. Representative 3D reconstruction of glomeruli and β-gal labelled RLCs (blue) on day 0 and day 7 of the EC model. The mesangial cell marker α8-integrin (red) was used to visualize the glomeruli. Scale bars correspond to 20 μm; C. Quantification of glomeruli with tufts containing β-gal expressing RLCs in the regenerative phase of the EC model (day 7). Data are presented as mean ± SEM, n = 5/10 for day 0 (baseline) /day 7, respectively. n.d.—not detectable; D. The intraglomerular RLCs observed during the EC model are not of hematopoietic origin. Representative confocal microscopy images for day 0 and day 7 of β-gal/CD45 (hematopoietic marker) co-stained kidney slices. 4′,6-diamidino-2-phenylindole (DAPI) was used as a nuclear marker. The channels for green (β-gal) and red (CD45) fluorescent signals in the dashed square on day 7 are separately shown in the small right panels. Scale bars correspond to 25 μm.</p

Reversible glomerular EC damage in mRen-rtTAm2/LC1/LacZ mice during thrombotic microangiopathy-like injury model.

<p>Mice were analyzed at baseline (day 0), day 1, and day 7 following disease induction. A. Quantification of intraglomerular fibrin thrombi (pinkish) as revealed by Acid Fuchsin Orange G (AFOG) staining of kidney slices. Data are presented as mean ± SEM, n = 5/5/10 for day 0 /day 1 /day 7, respectively. ***p<0.001, ns- not significant by one-way ANOVA; B. Representative AFOG staining images in the course of the EC model. Scale bars correspond to 25 μm; C. Quantification of glomerular EC depletion and injury on kidney slices by erythroblast transformation-specific related gene (ERG) and platelet endothelial cell adhesion molecule-1 (CD31) staining, respectively. Data are presented as mean ± SEM, n = 5/5/10 for day 0 /day 1 /day 7, respectively. *p<0.05, **p<0.01, ns- not significant by one-way ANOVA; D. Representative ERG/CD31 co-staining images in the course of the EC model. 4′,6-diamidino-2-phenylindole (DAPI) was used as a nuclear marker. Scale bars correspond to 25 μm.</p

Using stroma-anchoring cytokines to augment ADCC: a phase 1 trial of F16IL2 and BI 836858 for posttransplant AML relapse

Natural killer (NK) cells are key effectors in cancer immunosurveillance and posttransplant immunity, but deficiency of environmental signals and insufficient tumor recognition may limit their activity. We hypothesized that the antibody-mediated anchoring of interleukin-2 (IL-2) to a spliced isoform of the extracellular matrix (ECM) glycoprotein tenascin-C would potentiate NK-cell–mediated antibody-dependent cellular cytotoxicity against leukemic blasts. In this novel-novel combination, dose-escalation, phase 1 trial, we enrolled patients with posttransplant acute myeloid leukemia (AML) relapse to evaluate the safety, pharmacokinetics, pharmacodynamics, and preliminary activity of the antibody-cytokine fusion F16IL2 (10 3 106 to 20 3 106 IU IV; days 1, 8, 15, and 22 of each 28-day cycle) in combination with the anti-CD33 antibody BI 836858 (10-40 mg IV, 2 days after each F16IL2 infusion). Among the 15 patients (median [range] age, 50 [20-68] years) treated across 4 dose levels (DLs), 6 (40%) had received 2 or 3 prior transplantations. The most frequent adverse events were pyrexia, chills, and infusion-related reactions, which were manageable, transient and of grade #2. One dose-limiting toxicity occurred at each of DLs 3 (pulmonary edema) and 4 (graft-versus-host disease). Three objective responses were observed among 7 patients treated at the 2 higher DLs, whereas no responses occurred at the 2 starting DLs. Combination therapy stimulated the expansion and activation of NK cells, including those expressing the FcgRIIIA/CD16 receptor. ECM-targeted IL-2 combined with anti-CD33 immunotherapy represents an innovative approach associated with acceptable safety and encouraging biologic and clinical activity in posttransplant AML relapse. This trial was registered at EudraCT as 2015-004763-37