Primary splenic histiocytic sarcoma associated with hemophagocytic lymphohistiocytosis: A case report and review of literature of next‐generation sequencing involving FLT3 , NOTCH2 , and KMT2A mutations

Abstract Background Histiocytic sarcoma is a very rare monocyte/macrophage‐derived hematopoietic system tumor with a poor prognosis whose diagnosis is pathologically challenging due to its extreme rarity and histological overlap with various mimicking entities in which histiocytes also predominate. Case We report the case of a 33‐year‐old male patient with hemophagocytic lymphohistiocytosis, purpuric syndrome, and significant splenomegaly. The patient underwent splenectomy; subsequent macroscopic examination revealed a spleen weighing 2065 grams with hyperemic red pulp and multiple infarcts at the periphery. The histological and immunohistochemical study established a diagnosis of primary splenic histiocytic sarcoma with frequent hemophagocytosis. Next‐generation sequencing demonstrated mutations in FLT3, NOTCH2, and KMT2A, microsatellite stability, and a tumor mutational burden of 2 mut/Mb. The patient's condition deteriorated clinically from the appearance of the first symptoms and he died 6 months later from multi‐organ failure. Conclusion Primary splenic histiocytic sarcoma is one of the rarest tumors of the hematopoietic system. We report the first case with mutations in FLT3, NOTCH2, and KMT2A, and associated hemophagocytic lymphohistiocytosis.

histiocytes or dendritic cells. 1 It may be primary (of the skin, lymph nodes, digestive system, central nervous system, or disseminated) or secondary to another hematological neoplasm. There are no global epidemiological data concerning HS; however, the largest known case series, conducted by the U.S. National Cancer Institute, determined its overall incidence to be 0.17 per million people. Out of a total of 159 cases, only 5% were primary HS of the spleen and reticuloendothelial system. 2 The analyzed literature describes the association of PSHS and hemophagocytosis without detailing the fulfillment of the diagnostic criteria to classify HLH secondary to PSHS. Because of its poor prognosis, early HS diagnosis is critical to improving patient survival, but such diagnosis is a clinical and pathological challenge because of the more than 100 recently described histiocytosis subtypes, varying between benign and malignant forms, that can only be differentiated through immunohistochemical, chromosomal, and molecular studies. 3 We present the first case of HLH-associated PSHS with an NGS study identifying FLT3, NOTCH2, and KMT2A gene mutations that suggest new therapeutic strategies.

| Case presentation
A 33-year-old Hispanic male patient without previous medical and family history of illness, came in for consultation regarding weight loss and fever. He was admitted to a hospital 4 months later with a diagnosis of HLH, multiple organ dysfunction, pneumonia, splenic infarction, and cytomegalovirus (CMV) infection. In the fifth month, he presented generalized pallor, purpura over the chest and lower limbs, edema in the legs, and hemiparesis of the left foot. Corticosteroids, intravenous immunoglobulin, and valganciclovir were administered. A simple and contrasted tomography showed a significant left pleural effusion reaching the pulmonary hilum, collapse of the ipsilateral lower lobe, hepatomegaly, and supermassive (2065 g) splenomegaly with peripheral infarcts ( Figure 1(A)). No masses, retroperitoneal, mediastinal, or pulmonary hilar lymph node enlargements were found. Pleural and abdominal cytology tests were negative. Bone marrow flow cytometry showed no monoclonality, blasts, dysplasia, or myelofibrosis; neither acute myeloid leukemia nor Non-Hodgkin lymphoma infiltration was found. The iliac crest bone marrow biopsy was hypocellular without hemophagocytosis. Splenectomy, distal pancreatectomy, and left adrenalectomy were performed in the sixth month. The electrophysiological study of the four extremities found distal symmetric axonal sensorimotor polyneuropathy. A control tomography revealed hepatomegaly (a liver volume of 5500 cc) without tumors, as well as preaortic, para-aortic, and intercavo-aortic adenopathies. The tumor was subjected to NGS (FoundationOne ® Heme) to find possible therapeutic targets. The patient died of multiple organ failure 6 months after the onset of his condition.

| Histopathological findings
We received the postsurgical samples of the spleen, distal pancreas, and left adrenal gland. Macroscopically, the spleen weighed 2065 g and measured 26 Â 19 Â 12 cm, with a grayish lobed capsule interspersed with firm whitish areas (Figure 1(B)). The section revealed no nodular lesions, but multiple wedge-shaped, preferentially subcapsular ischemic lesions from 3 to 6 cm in diameter were observed, and 10 lymph nodes ranging from 0.2 to 0.6 cm in diameter were isolated.
Microscopically, the spleen showed expansion of the red pulp cords and sinuses due to diffuse proliferation of medium to large neoplastic cells with pleomorphic nuclei, vesicular chromatin, prominent nucleolus and clear to eosinophilic cytoplasm, atypical mitotic figures, and apoptotic bodies (Figure 1(C)). In addition, we observed frequent hemophagocytosis produced by reactive and neoplastic histiocytes ( Figure 1(D)). Furthermore, all examined lymph nodes showed lymphatic invasion and tumor involvement; acute pancreatitis was also found. Immunohistochemical stains showed tumor cell positivity for S100, CD68 (Figure 1 We ruled out myeloid differentiation due to CD34, CD117 and myeloperoxidase negativity. The established histopathological diagnosis was primary splenic histiocytic sarcoma (PSHS).

| MATERIALS AND METHODS
We performed immunohistochemical staining on 4 μm formalin-fixed and paraffin-embedded (FFPE) tissue sections using the VENTANA Benchmark system (Roche, Tucson, AZ) according to standardized laboratory procedures. The following antibodies were used during the diagnostic study: CD68 (KP1), CD4, CD56, CD45, CD1a, CD163, CD8, CD3, CD5, CD20, CD30, CD35, CD21, S100, lysozyme, CD34, CD117, myeloperoxidase, ALK, pan-melanoma panel, and Ki-67. which included a case of HS of the nasal cavity. 6 It is important to note that the BRAF mutation was not found in our case, despite it has been described in other cases of HS. 5 The fms-related receptor tyrosine kinase-3 (FLT3) is a type III tyrosine kinase that functions as a hematopoietic progenitor of cell proliferation, survival, and differentiation. 7 Studies have shown that FLT3 activation is involved in leukemogenesis through serine/threonine kinase AKT phosphorylation or activation, which occurs in onethird of acute myeloid leukemia cases and confers a worse prognosis. 7 Presumably, FLT3 ligands can activate the RAS/MAPK pathway, and MAP kinase activation is needed for mitogenic signaling of FLT3. 8 Despite the clear association between FLT3 and acute myeloid leukemia, we were unable to find any mention of a possible association between FLT3 and HS in our search of medical literature. Experiments in mice demonstrated that FLT3 is one of the regulators of dendritic cell progenitors in the bone marrow and peripheral dendritic cells; it is also essential in regulating homeostasis in splenic dendritic cell development. 9 Despite the important role of FLT3 in vivo, no overexpression of this gene was found in Langerhans cell histiocytosis (LCH). 10  Mixed leukemia lineage (MLL) is a family of proteins whose function is histone-H3K4 methylation to regulate active gene transcription. 15 It is known that the KMT2A gene is necessary to generate an adequate number of hematopoietic progenitors. 16 Mutation of this gene is found in 5-10% of adult acute myeloid and lymphocytic leukemias; such cases are characterized by poor prognosis and refractory to treatment. 14 Bao et al. highlight the importance of KMT2A mutations and rearrangements found in MS with HS-like morphology, leading us to consider the relationship that these two neoplasms may have in addition to their common oncogenic pathway. We have so far found no literature describing the possibility of transdifferentiation from HS to MS. 11 We ruled out myeloid sarcoma among other entities due to our histological and immunohistochemical findings. Other authors have reported molecular similarities with variants like those of our patient (NTRK1 in LCH), pointing to the possible importance of these mutations in histiocytosis and other hematological neoplasms. 17 Our case is exceptional in that it is, to our knowledge, only the second reported case of PSHS with no macroscopic splenic nodules 18 and constitutes an unusual presentation of HLH associated with a primary splenic tumor, which has been scantly described in the literature. 19 4,20 Despite the paucity of reported PSHS cases, neoplastic cells and reactive histiocytes have been shown to present phagocytosis in one or all hematological cell lines, including proteins such as immunoglobulins and albumin, 22 and PSHS may thus be a secondary cause of HLH. 19 Despite the possibility for neoplastic HS cells to produce hemophagocytosis, there is no information regarding the cause nor the consequence for reactive histiocytes to lose their physiological activity and start acting like neoplastic cells; we hypothesize that PSHS can be the responsible for the aberrant activity of histiocytic cells, which can range from normal to reactive, and finally neoplastic. 21,22 Furthermore, fulfillment of the Histiocyte Soci-  We present the first report of PSHS with NGS-identified mutations in FLT3, NOTCH2, and KMT2A. We suggest that these mutations may be key in the genetic origin of HS and in particular PSHS. We recognize the importance of epigenetic modifiers, such as KMT2A, as one of the possible oncogenic pathways for these neoplasms. A PSHS diagnosis should be considered as a possibility in patients presenting with HLH.

| Molecular profiling
Molecular studies have become an essential diagnostic tool that differentiates similar pathologies and identifies common molecular pathways to establish targeted therapies. Neither our patient's mutations nor the accompanying VUS have been described to date, and further NGS studies are therefore needed to demonstrate their genomic and clinical significance in the future.

ACKNOWLEDGMENTS
The authors wish to thank Roche ® for sponsoring the FoundationOne ® Heme next-generation sequencing.

CONFLICT OF INTEREST
The authors have stated explicitly that there are no conflicts of interest in connection with this article. Conceptualization; investigation; writing-original draft.

ETHICAL STATEMENT
The article complied with the local ethics committee (Ethics Committee, Hospital Metropolitano, Quito, Ecuador) and adhered to the principles outlined in the Declaration of Helsinki. Written informed consent for publication of their clinical details and/or clinical images were obtained from the patient's next of kin.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.