Histopathological Features of Cancer-Associated Venous Thromboembolism: Presence of Intrathrombus Cancer Cells and Prothrombotic Factors

Abstract

Background: Cancer-associated venous thromboembolism (VTE) is a critical complication in patients with cancer. However, the pathological findings of VTE are limited. Here, we investigated the histopathological features of cancer-associated VTE in human autopsy cases.

Methods: We clinically examined the autopsy cases of VTE with (n=114) and without cancer (n=66) and immunohistochemically analyzed the expression of prothrombotic factors in intrathrombus cancer cells, the thrombus contents of erythrocytes, fibrin, platelets, citrullinated histone H3, and degree of organization.

Results: Vascular wall invasion or small cell clusters of cancer cells was observed in thrombi in 27.5% of deep vein thrombosis and 25.9% of pulmonary embolism cases. The majority of the cancer cells in deep vein thrombi appeared to be invading the vessel wall, whereas the majority of pulmonary thrombi had cancer cell clusters, consistent with embolization via blood flow. These cancer cells were immunohistochemically positive for TF (tissue factors) or podoplanin in up to 88% of VTE cases. The frequency of TF-positive monocyte/macrophages in thrombi was higher in cancer-associated VTE than that in VTE without cancer. Citrullinated histone H3 was predominantly observed in the early stages of organizing thrombi. There was no significant difference in thrombus components between VTE with cancer and without cancer groups.

Conclusions: Vascular wall invasion or cancer cell clusters in thrombi might influence thrombogenesis of cancer-associated VTE. TF and podoplanin in cancer cells and in monocyte/macrophages may induce coagulation reactions and platelet aggregation. Neutrophil extracellular traps may play a role in the early stages of VTE, regardless of cancer status.

Keywords: carcinoma; extracellular traps; fibrin; pathology; platelet aggregation; tissue factor; venous thromboembolism.

Figure 1.

Histopathology of cancer cells in venous thromboembolism (VTE) tissue of autopsy cases. A, Venous thrombus of inferior vena cava with pancreatic adenocarcinoma. Dashed line indicates the interface between the thrombus (Th) and the venous wall (V). The thrombus occupies the venous lumen. Cancer cells, CK (cytokeratin)-positive cells, show direct invasion into the thrombus from the venous wall, associated with thrombus formation. B, Pulmonary thrombi with gastric adenocarcinoma. Cancer clusters are observed in a variety of sizes. There is no direct cancer invasion from the pulmonary artery (PA). Squares of the upper figures are corresponding to the lower figures in high-power fields (A and B). C, Frequency of cancer pattern in cancer-associated VTE tissue with cancer cells (specimen n=29). There was a significant difference in the frequency between deep vein thrombosis (DVT) and pulmonary embolism (PE; P=0.040, χ² test). D, CK-positive area per total thrombus area with cancer cells (specimen n=25). There was no significant difference between DVT and PE (Mann-Whitney U test). E, Necrosis of cancer cells in pulmonary thrombus with gastric adenocarcinoma. Cytoplasmic lysis and nuclear fragmentation indicating necrosis (arrow) in the CK immunopositive area adjacent to cancer cells (*). The cancer and necrotic cells are surrounded by fibrin and platelets (GPIIb/IIIa [glycoprotein IIb/IIIa]). Fibrin formation is also observed in the necrotic area (arrow). F, Frequency of necrotic changes in VTE tissue with cancer cells (specimen n=29). There was no significant difference in the frequency between DVT and PE (P=0.88, χ² test). HE indicates hematoxylin and eosin.

Figure 2.

Expression of prothrombotic factors of cancer cells in venous thromboembolism tissue. Representative microphotographs of cancer cells in a venous thrombus, accompanied by TF (tissue factor) expression in a case of pancreatic adenocarcinoma (A) and in a case of gastric adenocarcinoma (B). Immunohistochemically, cancer cells in thrombi correspond to CK (cytokeratin)-positive cells (A). Additionally, the cancer cells express TF (A). No podoplanin is expressed in the cancer cells. Immunofluorescence shows heterogeneous TF expression in CK-positive cells (B). Representative microphotographs of cancer cells in a pulmonary thrombus, accompanied by podoplanin expression (C and D) in a case of cutaneous squamous cell carcinoma. The cancer cells are highlighted with CK, and diffusely expressed podoplanin (C). In contrast, the cancer cells weakly and focally expressed TF (C). Immunofluorescence shows that CK-positive cells are also positive for podoplanin (D). In these cases, fibrin formation and platelet (glycoprotein [GP] IIb/IIIa) aggregation are observed in the surrounding cancer cells (A and C). DAPI indicates 4’,6-diamidino-2-phenylindole; and HE indicates hematoxylin and eosin.

Figure 3.

Analysis of NETs (neutrophil extracellular traps) in venous thromboembolism (VTE) tissue. A, Representative micrographs of Cit-H3 (citrullinated histone H3; as a NET marker) in a venous thrombus. Hematoxylin and eosin (HE) staining shows lysis and fragmentation of neutrophil chromatin, corresponding to Cit-H3 expression. The surrounding thrombus is composed of fibrin and platelets. Nonlytic neutrophils are immuno-negative for Cit-H3 (arrows). B, Representative immunofluorescence images of NETs in VTE tissue. Lytic cells show delobulation and fragmentation of nuclei (4’,6-diamidino-2-phenylindole [DAPI], blue) and dispersed CD66b (neutrophil marker, red) immunoreaction. Triple immunofluorescence images reveal the presence of Cit-H3 positive (green) DNA (cyan) with CD66b in the lytic area. C, Representative immunofluorescent images of neutrophils without NET formation in VTE tissue. This image is obtained from different fields within the same thrombus from B. Nonlytic segmented cells express CD66b (neutrophil marker, red). Triple immunofluorescence images reveal the absence of Cit-H3–positive DNA (blue) in the nonlytic area. D, Immuno-positive area ratio of Cit-H3 between VTE without cancer (non-CAT) and cancer-associated VTE (CAT). The Mann-Whitney U test was used for statistical analysis. E, Immuno-positive area ratio of Cit-H3 between organizing grades (G0–G3) of total VTE, non-CAT, and CAT. The Kruskal-Wallis test with Dunn multiple comparison test was used for statistical analysis. DVT indicates deep vein thrombosis; and PE, pulmonary embolism.