Acute promyelocytic leukemia (APL) is a distinct subtype of acute myeloid leukemia (AML) characterized by the accumulation of promyelocytes in the bone marrow and a high risk of severe hemorrhagic complications. Despite significant therapeutic advancements with all-trans retinoic acid (ATRA) and arsenic trioxide (Arsenic Trioxide) [1–3], early mortality remains a major concern, potentially reaching 5–20 %, depending on clinical and geographical factors. The primary cause of early death is coagulopathy, which often leads to fatal intracranial hemorrhages [4]. Therefore, early diagnosis and prompt initiation of treatment are crucial for reducing mortality and improving patient outcomes. In addition, the identification of novel biomarkers and therapeutic targets of APL coagulopathy could bring benefits to these patients.

Podoplanin is a small transmembrane glycoprotein expressed in various cell types, including lymphatic endothelial cells, kidney podocytes, and certain tumor cells [5]. Functionally, podoplanin interacts with the C-type lectin-like receptor 2 (CLEC-2) on platelets, triggering their activation and contributing to thrombotic processes [6]. This mechanism is particularly relevant in cancer-associated thrombosis and inflammatory diseases, where podoplanin overexpression has been linked to prothrombotic states [5–7].

In the context of APL, podoplanin expression was first identified in leukemic promyelocytes in 2018, suggesting its potential role as a distinguishing marker for this leukemia subtype [8]. More recently, its expression was validated in a prospective cohort using flow cytometry, reinforcing its diagnostic and pathophysiological relevance in APL [9]. However, the extent to which circulating podoplanin correlates with disease characteristics and coagulation abnormalities remains unexplored.

Soluble forms of membrane proteins can serve as biomarkers, reflecting their biological activity and disease status. For instance, soluble fms-like tyrosine kinase-1 (sFLT1) is well established as a biomarker in preeclampsia and soluble urokinase-type plasminogen activator receptor (suPAR) as a biomarker in different types of cancers [10–13]. Similarly, soluble podoplanin (sPDPN) has been identified as a biomarker in malignancies and inflammatory diseases such as cancer and COVID-19, highlighting its potential and prognostic significance [14–18].

Based on this evidence, this study investigated whether quantification of sPDPN in plasma could be associated with the diagnosis of APL, as well as its relationship with biomarkers involved in APL coagulopathy.

In total, 35 patients with APL were included in this study, as well as 35 patients with non-APL AML, matched by age, sex and year of diagnosis, used as a comparator group. The clinical and laboratory characteristics of these groups are presented in Table 1. As expected, APL patients presented more frequently with alterations in hemostasis biomarkers, and a lower rate of early mortality.

APL coagulopathy still represents a major challenge to the early management of APL, and the availability of biomarkers capable of differentiating APL from other forms of AML, and of providing information about APL coagulopathy could potentially facilitate the management of these patients [20]. The main contribution of the present study was the demonstration of sPDPN as a potential biomarker. Its expression is markedly higher in APL compared to other forms of AML, and it correlates with an important thrombo-inflammatory mediator. This suggests that podoplanin expression may be a unique biological feature contributing to the well-known predisposition of these patients to hemorrhagic and thrombotic complications.

The first demonstration that podoplanin expression as a hallmark of APL was published in 2018 and included data obtained by flow cytometry and RNA sequencing [8]. That study also presented evidence linking podoplanin expression to platelet activation in vitro. While the diagnosis of APL using flow cytometry and molecular techniques is well established, reliance on methods that are not usually available outside specialized cancer centers may delay diagnosis and jeopardize patient outcomes [20]. Immune-based methods such as ELISA are widely available and have the advantage of being potentially performed with diagnostic kits in almost every setting. Based on the demonstration that circulating levels of podoplanin was capable of identifying discrete subgroups of patients with other conditions [14–18], and that soluble levels of membrane proteins can serve as useful disease biomarkers [10–13], we hypothesized that sPDPN levels might contribute to the differential diagnosis of APL from other forms of AML. In fact, this study demonstrates a significant difference in sPDPN levels between these two patient subgroups, although a subgroup of patients with non-APL AML also presents higher sPDPN values.

In addition, a positive correlation between sPDPN and CD40L levels was found in APL patients, suggesting a biological interaction between these two markers. CD40L is a transmembrane protein expressed on activated platelets that has been implicated in platelet-leukocyte crosstalk and inflammatory signaling [21]. CD40L is also expressed by other hematopoietic cells such as lymphocytes, neutrophils, dendritic cells, monocytes and macrophages, in response to cytokines [22], which are key elements of thrombo-inflammatory pathways [23]. So, the correlation described in the current study could represent either evidence of the participation of podoplanin in platelet activation, in alignment with Lavallé et al. [8], who demonstrated that podoplanin-expressing APL blasts can activate platelets, or a yet unknown association between podoplanin and CD40L in another hematopoietic compartment. In the present dataset, sPDPN levels were not associated with clinical outcomes, although this study was not powered for this type of analysis.

The results also demonstrate that a significant proportion of AML patients present detectable sPDPN levels, while some APL patients present low sPDPN levels. This is in contrast with data from flow cytometry and reverse transcription polymerase chain reaction (RT-PCR) results from our lab that show less overlap between APL and other forms of AML (data not shown). Additional studies are warranted to explore and explain this lower accuracy of sPDPN ELISA for the segregation of APL from other forms of AML compared to flow cytometry.

This study has limitations that should be acknowledged. Although APL is a relatively rare condition and the present sample size was sufficient to demonstrate relevant biological differences in sPDPN levels, this study was not powered to analyze clinical outcomes, which were rare. Another limitation lies in the absence of additional hemostatic biomarkers, which might have enhanced the interpretation of coagulation-related mechanisms. However, these markers are not routinely assessed in the standard clinical workflow of the hospital, and citrate plasma samples were no longer available. Future studies involving larger cohorts and broader biomarker panels are important to further investigate the potential diagnostic and clinical relevance of sPDPN in APL.

In conclusion, the results of this study represent proof-of-concept that the immunodetection (in this case, using ELISA) of circulating levels of sPDPN can serve as a complementary tool in the differential diagnosis of APL. The data also adds support to the concept that podoplanin expression is a distinct hallmark of APL and contributes to the pathogenesis of the unique thrombo-inflammatory complications seen in these patients.

Figure 1, Figure 2

Figure 1.

Plasma levels of podoplanin of acute promyelocytic leukemia (APL) and non-APL acute myeloid leukemia (AML) patients (p-value: Mann-Whitney test) To investigate the potential relationship between sPDPN and platelet activation, plasma levels of P-selectin and CD40L, two established markers of platelet degranulation, were measured. Both P-selectin (7194 ng/mL versus 11,220 ng/mL; p-value = 0.003) and CD40L levels (100.4 ng/mL versus 196.6 ng/mL; p-value = 0.002) were both significantly lower in APL patients compared to those with other AML subtypes (Figure 2A, B). Interestingly, a significant positive correlation was observed between podoplanin and CD40L levels in APL patients (Rs = 0.60; p-value = 0.0004), but not for other AML subtypes (Figure 2C,D). Podoplanin levels were not associated with predefined clinical outcomes (Table 2).

Figure 2.

(A) Plasma levels of P-selectin were significantly lower in acute promyelocytic leukemia (APL) patients compared to non-APL acute myeloid leukemia (AML) patients (Mann-Whitney test; p-value = 0.003). (B) Plasma levels of CD40L were significantly lower in APL patients compared to non-APL AML patients (Mann-Whitney test; p-value = 0.002). (C) A significant positive correlation between podoplanin and CD40L was observed in APL patients (Spearman’s correlation; p-value = 0.0004). (D) No significant correlation between podoplanin and CD40L was found in non-APL AML patients.

All reported data are available for sharing upon a reasonable request to the corresponding author.

The study was performed in accordance with the Declaration of Helsinki and approved by the local institutional research board (protocol CAAE: 39,948,520.8.1001.5404).

CRPM: performed all assays; revised medical records; contributed to data analysis and drafted the manuscript; revised and approved the manuscript. CMAS: revised medical records; contributed to data analysis; revised and approved the manuscript. ITBJ: contributed to data analysis; revised and approved the manuscript. BKLD: contribute to study design; revised and approved the manuscript. PMC: provided laboratory support and infra-structure; revised and approved the manuscript. STOS: provided laboratory support and infra-structure; revised and approved the manuscript. EVDP: designed the study, oversaw and provided resources and infra-structure for ELISA analysis, contributed to data analysis; drafted the manuscript; revised and approved the manuscript.