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BRAF V600E mutations are frequently found in histiocytic/dendritic cell neoplasms such as Erdheim-Chester disease (ECD) and Langerhans cell histiocytosis (LCH), but few reports have also described BRAF mutations in Rosai-Dorfman disease (RDD), and even these cases may predominantly represent mixed histiocytosis. BRAF mutations have been studied in histiocytic/dendritic cell neoplasms and described to be associated with increased risk of relapse and long-term consequences, but few studies have examined BRAF V600E mutation in RDD, which is recognized as a neoplasm given the high frequency of MAPK pathway alterations. Here, we report a case of BRAF V600E-mutated RDD in a patient who presented with generalized lymphadenopathy. During our evaluation of this patient, we also found expression of PD-L1 in neoplastic histiocytes. During our review period, only few cases of RDD reported to harbor BRAF mutation or were evaluated for the expression of PDL1 by neoplastic cells. Given the potential challenges in distinguishing RDD from other histiocytic/dendritic cell neoplasms, including mixed histiocytosis with similar clinicopathological manifestations, we will discuss the current state of knowledge regarding the frequency and clinical impact of BRAF V600E in RDD, as well as the role of BRAF mutations in RDD pathogenesis. Distinction of BRAF V600E mutated histiocytic/dendritic cell neoplasms requires consideration of distinctive histopathological and immunophenotypic findings in appropriate clinical and radiologic setting. Given the increasing use of BRAF inhibitors as well as checkpoint blockade inhibitors to treat a number of cancers, we will discuss the clinical implications of the presence of BRAF V600E mutation and PD-L1 expression in RDD., Competing Interests: Declarations. Ethical approval: For this type of study, formal consent is not required. Informed consent: For this type of study, informed consent is not required. Consent for publication: Consent for publication was obtained for every individual person’s data included in the study. Conflict of interest: The authors declare no competing interests., (© 2024. The Author(s).)

We present a case of a 64-year-old man with refractory acute myeloid leukemia and trisomy 8 who developed leukemia cutis. Interphase fluorescence in situ hybridization (FISH) was performed on a paraffin-embedded skin section. FISH confirmed a population of cells with trisomy 8 in the blastic infiltrates involving the skin. This case illustrates a novel application of interphase FISH to confirm the diagnosis of leukemia cutis., (Copyright © 2012 John Wiley & Sons A/S.)

The association of idiopathic venous thrombosis with occult cancer is generally recognized. However, it has not been fully appreciated that thrombin generated during thrombosis can augment the malignant phenotype. Thrombin activates tumor cell adhesion to platelets, endothelial cells, and subendothelial matrix proteins; enhances tumor cell growth; increases tumor cell seeding and spontaneous metastasis; and stimulates tumor cell angiogenesis. These mechanisms are reviewed. Evidence is also presented to support the hypothesis that thrombin serves to preserve dormant tumor cells in individuals, preventing host eradication. It is proposed that tumor malignancy may be regulated by a procoagulant/anticoagulant axis.

Because thrombin-treated tumor cell-induced metastasis increases tumor nodule volume(12) greater than nodule number, we studied the effect of thrombin on tumor cell growth in vitro and in vivo (murine B16F10 melanoma, human HCT8 colon carcinoma, DU145 prostate carcinoma). Tumor cell growth was measured after 3 to 7 days in 1% fetal calf serum (FCS) + RPMI 1640. We found that, whereas relatively low concentrations of thrombin, 0.1 to 0.5 U/mL (1-5 nmol/L) enhance tumor cell growth in vitro approximately 2- to 3-fold, higher concentrations, 0.5 to 1 U/mL (5-10 nmol/L) impaired cell growth approximately 2- to 4-fold. Impaired cell growth was associated with cell cycle arrest at G(2)M and increased pre-G(o) DNA, as well as apoptosis, measured by tumor cell binding to Annexin V and propidium iodide. Apoptosis was reversed with the general caspase inhibitor, FK-011. The enhancing and inhibiting effects were specific for thrombin (reversed with inactive diisopropyl-fluorophosphate [DFP]-thrombin) and mediated via the protease-activated receptor 1 (PAR-1). PAR-1 activation was demonstrated by (1) use of a cell line, B16F10, devoid of the 3 other thrombin receptors, PAR-3, PAR-4, and GPIb; and (2) greater sensitivity of PAR-1 transfected B16F10 and HCT8 cells to impaired cell growth/apoptosis, 3- and 14-fold, respectively. Thus, thrombin has a bimodal effect on PAR-1 in tumor cells: enhanced growth at low concentration, impaired growth/apoptosis at higher concentration.

Thrombin-treated tumor cells induce a metastatic phenotype in experimental pulmonary murine metastasis. Thrombin binds to a unique protease-activated receptor (PAR-1) that requires N-terminal proteolytic cleavage for activation by its tethered end. A 14-mer thrombin receptor activation peptide (TRAP) of the tethered end induces the same cellular changes as thrombin. Four murine tumor cells (Lewis lung, CT26 colon CA, B16F10 melanoma, and CCL163 fibroblasts) contain PAR-1, as detected by reverse transcriptase-polymerase chain reaction (RT-PCR). B16F10 cells did not contain the two other thrombin receptors, PAR-3 and glycoprotein Ib. TRAP-treated B16F10 tumor cells enhance pulmonary metastasis 41- to 48-fold (n = 17). Thrombin-treated B16F10 cells transfected with full-length murine PAR-1 sense cDNA (S6, S7, S14, and S22) enhanced their adhesion to fibronectin 1.5- to 2.4-fold (n = 5, P <.04), whereas thrombin-treated wild-type cells do not. S6 (adhesion index, 1.5-fold) and S14 (index, 2.4-fold) when examined by RT-PCR and Northern analysis showed minimal expression of PAR-1 for S6 over wild-type and considerable expression for S14. Immunohistochemistry showed greater expression of PAR-1 for S14 compared with wild-type or empty-plasmid transfected cells. In vivo experiments with the thrombin-treated S14 transfectant showed a fivefold to sixfold increase in metastases compared with empty-plasmid transfected thrombin-treated naive cells or S6 cells (n = 20, P =.0001 to .02). Antisense had no effect on thrombin-stimulated tumor mass. Thus, PAR-1 ligation and expression enhances and regulates tumor metastasis.

Thrombin-treated tumour cells enhance their adhesion to platelets, fibronectin and von Willebrand factor in vitro, and enhanced their pulmonary metastasis in mice in vivo. A unique seven transmembrane spanning thrombin receptor has recently been cloned which is activated following thrombin proteolysis of the N-terminal end of the receptor with exposure of a tethered ligand. An N-terminal 14-mer (SFLLRNPNKYEPF) or 6-mer (SFLLRN) of the tethered ligand can serve as a thrombin receptor activation peptide (TRAP) by mimicking the action of thrombin on platelets, endothelial cells and smooth muscle cells. We have examined six human tumour cell lines for their response to TRAP, for the presence of this thrombin receptor mRNA by RT-PCR, protein by immunoblot and for their in vitro and in vivo response to TRAP. All six cell lines contain the receptor mRNA, and when treated with 100 microM 6-mer TRAP or 1 u/ml thrombin increase their adhesion to platelets 2-3-fold. Four of the six cell lines undergo tyrosine phosphorylation within 30 s to 1 min after exposure to 6-mer TRAP or thrombin. Thus tumour cells respond to thrombin via activation of their seven transmembrane spanning thrombin receptor.

Six monoclonal IgG1-k antibodies (LK2, LK3r, LK4-55, LK5, LK6-55, LK7r) were raised against platelet membrane GPIIIa in order to study the structure-function relationship of this molecule. Antibodies were selected on their ability to react with GPIIIa by ELISA on adherent platelets, by immunoblot on platelet lysates and by fluorescence flow cytometry on intact platelets. Fluorescence reactivity varied from 3- to 202-fold greater than isotype control fluorescence. Two MoAbs reacted on immunoblot under reduced conditions (LK7r and LK3r). Two reacted with a 55 kD chymotrypsin/subtilisin digest of GPIIIa which is likely to exclude amino acids 121-348 (LK4-55 and LK6-55). Four of the MoAbs (LK5, LK3r, LK2 and LK4-55) inhibited tyrosine phosphorylation of one to four distinct bands on immunoblot. LK4-55 reacted with an N-terminal 66 amino acid fusion protein of GPIIIa near the PLA epitope (Leu 33). LK7r reacted with a 212-222 peptide reported to be an RGD fibrinogen binding site. LK2 reacted near a disintegrin-RGD binding site. Except for LK5, all inhibited ADP, collagen and thrombin-induced platelet aggregation in a heterogeneous fashion. Percentage inhibition of 125I-fibrinogen binding to platelets varied from 18% to 98%. No correlation was noted between inhibition of fibrinogen binding, location of MoAb binding on GPIIIa, reactivity of MoAb binding with GPIIIa, inhibition of thrombin-induced tyrosine phosphorylation or inhibition of platelet aggregation induced by ADP, collagen or thrombin. Thus MoAbs, binding to platelet GPIIIa at different sites, inhibit platelet aggregation in a heterogeneous manner.

The role of platelets and thrombin was examined in tumor cell adhesion in vitro and metastasis in vivo. Adhesion of tumor cells to platelets was inhibited by agents inhibiting platelet integrin IIb-IIIa receptor occupancy (MoAb 10E5 and tetrapeptide RGDS) as well as IIb-IIIa ligands (polyclonal antibodies against fibronectin and von Willebrand factor (vWF)). In vivo murine experimental pulmonary metastasis (tail vein injection) could be inhibited by antibody-induced induction of thrombocytopenia and reconstituted by simultaneous injection of human platelets. Preincubation of human platelets with MoAb 10E5 (vs IIb-IIIa) inhibited reconstitution of metastasis. Thrombin activates tumor cell adhesion to platelets by activating platelets as well as tumor cells. Thrombin-activated tumor cells also enhance their adhesion to endothelial cells as well as adhesive ligands fibronectin and vWF. Experimental pulmonary metastasis is enhanced 4-400 fold by preinfusion of thrombin into mice or 10-160 fold by prior treatment of tumor cells with thrombin. The in vitro and in vivo effects of thrombin were mimicked by thrombin receptor activation peptides SFLLRNPNDKYEPF and SFLLRN. Nine of nine tumor cell lines have the seven transmembrane spanning thrombin receptor detected by the polymerase chain reaction. Thus, both platelets and thrombin contribute to experimental tumor metastasis by fostering and enhancing IIb-IIIa integrin platelet interaction with tumor cells. Since many tumor cells generate thrombin, it is proposed that tumor cells may autoactivate a metastatic phenotype.

Seven different tumor cell lines (human melanoma SK MEL 28; hamster melanoma HM29; murine melanomas B16F10 and amelanotic melanoma B16a; human colon carcinoma HCT8; murine colon carcinoma CT26; and murine Lewis lung carcinoma) were treated with thrombin at 0.5-1 unit/ml and examined for their ability to bind to adherent platelets; HM29 was studied for its ability to bind to fibronectin and von Willebrand factor; CT26, B16F1, B16F10, and B16a were studied for their ability to form pulmonary metastasis after i.v. injection of thrombin-treated tumor cells; CT26 was studied for its ability to grow s.c. Five of 7 thrombin-treated tumor cell lines increased their adhesion to adherent platelets 2-to 3-fold. HM29 increased its adherence to fibronectin and von Willebrand factor 2- to 3-fold. CT26, B16F1, B16F10, and B16a increased experimental pulmonary metastasis 10- to 156-fold. Thrombin-treated CT26 cells demonstrated 2-fold greater growth in vivo after s.c. injection. The mechanism of enhanced adhesion of thrombin-treated tumor cells to platelets required the platelet integrin GPIIb-GPIIIa since it could be inhibited by agents known to block adhesion of ligands to GPIIb-GPIIIa (monoclonal antibody 10E5, tetrapeptide RGDS, disintegrin Albolabrin); as well as a "GPIIb-GPIIIa-like" structure on tumor cells since it could be inhibited by treatment of thrombin-treated tumor cells with 10E5 and RGDS. The thrombin effect on tumor cells was optimum at 1 h of incubation with thrombin, did not require active thrombin on the tumor cell surface, and did not require protein synthesis (not inhibited by cycloheximide). Thus, thrombin-treated tumor cells markedly enhance pulmonary metastasis. It is suggested that this may be secondary to thrombin-induced enhanced adhesion as well as growth of tumor cells.

Recent studies have revealed a role for platelets and the platelet-adhesive proteins, fibronectin and von Willebrand factor (vWF) in platelet-tumor cell interaction in vitro and metastasis in vivo. The present report documents the effect of thrombin treatment of platelets on this interaction in vitro and in vivo. In vitro, thrombin at 100-1,000 mU/ml maximally stimulated the adhesion of six different tumor cell lines from three different species two- to fivefold. As little as 1-10 mU/ml was effective. The effect of thrombin was specific (inhibitable by hirudin, dansyl-arginine N-(3-ethyl-1,5 pentanediyl) amide and unreactive with the inactive thrombin analogue N-P-tosyl-L-phenylchloromethylketone-thrombin and D-phenylalanyl-L-propyl-L-arginine chloromethylketone-thrombin (PPACK-thrombin), and required high-affinity thrombin receptors (competition with PPACK-thrombin but not with N-P-tosyl-L-lysine-chloromethyl-ketone-thrombin). Functionally active thrombin was required on the platelet surface. Binding of tumor cells to thrombin-activated platelets was inhibitable by agents known to interfere with the platelet GPIIb-GPIIIa integrin: monoclonal antibody 10E5, tetrapeptide RGDS and gamma chain fibrinogen decapeptide LGGAKQAGDV, as well as polyclonal antibodies against the platelet adhesive ligands, fibronectin and vWF. In vivo, thrombin at 250-500 mU per animal increased murine pulmonary metastases fourfold with CT26 colon carcinoma cells and 68-413-fold with B16 amelanotic melanoma cells. Thus, thrombin amplifies tumor-platelet adhesion in vitro two- to fivefold via occupancy of high-affinity platelet thrombin receptors, and modulation of GPIIb-GPIIIa adhesion via an RGD-dependent mechanism. In vivo, thrombin enhances tumor metastases 4-413-fold with two different tumor cell lines.