Concepción Prats-Martín

NUP98-HOXA9 bearing therapy-related myeloid neoplasm involves myeloid-committed cell and induces HOXA5, EVI1, FLT3, and MEIS1 expression.

Chromosomal rearrangements involving NUP98 gene have been associated with human leukemias such as de novo AML, therapy‐related AML (t‐AML), myelodysplastic syndrome (MDS), and chronic myeloid leukemia (CML). Genetic fusion NUP98–HOXA9, caused by t(7;11)(p15;p15), is a recurrent cytogenetic alteration in de novo acute myeloid leukemia (AML) usually found in young Asian patients and its description in therapy‐related myeloid neoplasms (t‐MN) is rare. Only one Asian case with molecular demonstration of the NUP98–HOXA9 fusion has been reported in therapy‐related leukemia. NUP98–HOXA9 leukemogenic mechanism is derived from the transcription factor activity of the chimeric protein, which enhances the expression of genes related to cellular differentiation arrest and proliferation.

Intravascular lymphoma: look through the small vessels

Dear Editor, A 56-year-old Caucasian woman was admitted to the hospital after a stepwise presentation of multiple neurological symptoms—unusual headache, dizziness, nightmares, recurrent transient visual loss, and disorientation—during the last month. Finally, she also developed fever and abdominal pain. At examination, she was febrile (38.6 °C) and had pale skin. Neither adenopathy nor organomegaly was found. The neurological examination showed confusion, but it was non-focal. Routine laboratory test showed the following results: hemoglobin, 79 g/L; leucocytes, 4.43 × 10/L (neutrophils, 2.9 × 10/L), and platelets, 215 × 10/L. Further workup revealed abnormally high levels of ferritin (3225 μg/L), triglycerides (294 mg/dL), LDH (1501 IU/L), soluble CD25 (39.09 ng/mL, normal <7.5), and β2-microglobulin (5.1 mg/L). Serology tests were negative, but markers of previous hepatitis B were noted, with an undetectable viral load. Cerebrospinal fluid examination was unremarkable. Brain magnetic resonance images (MRI) showed two lesions in the right parietal lobe (Fig. 1a). The subcortical one exhibited gadolinium enhancement and restriction on diffusion. Microscopic examination of a blood film revealed a leukoerythroblastic reaction with 1–2% of blast-like cells. The bone marrow aspirate was hypercellular showing 40.5% of large undifferentiated cells (Fig. 1b). Nuclei were rounded or lobulated with a finely condensed chromatin pattern and, in some cases, with prominent nucleoli (Fig. 1c–e). Cytoplasms were basophilic and agranular often with a pseudopodium formation (Fig. 1f). The presence of clusters of cells similar to metastasis was repeatedly observed (Fig. 1g). A high number of mitotic figures was also noted. Cytochemical assays showed acid phosphatase positivity with granular pattern. Other techniques, including myeloperoxidase, Sudan Black B, alpha-naphthyl acetate esterase, chloroacetate esterase, and periodic acid-Schiff reaction, were negative. In addition, an abnormal number of histiocytes (24%) with activation signs were observed. One third of which showed phagocytosis, mainly of neutrophils (Fig. 1h) but also of malignant cells. Electronic supplementary material The online version of this article (doi:10.1007/s00277-016-2874-9) contains supplementary material, which is available to authorized users.

Strong periodic acid-schiff reaction in hypervacuolated blastic plasmacytoid dendritic cell neoplasm

The periodic acid-Schiff (PAS) reaction puts on evidence the presence of large polysaccharides. Its positivity is mainly recognized in erythroid and lymphoblastic acute leukemias, although it is not specific for the diagnosis. In blastic plasmacytoid dendritic cells neoplasm (BPDCN), it is well-established that the blasts are negative with alpha-naphthyl-acetate-esterase (ANAE), which is a monocytic/histiocytic esterase, and myeloperoxidase cytochemical stains. PAS positivity has occasionally been described with fine granular pattern in <50% of the blasts. The diagnosis of BPDCN is confirmed by flow cytometry and immunohistochemistry. A 72-year-old man was diagnosed with BPDCN with cutaneous nodular lesions on the thoracic region and nodal involvement (mediastinal and hiliar lymph node). Histopathological examination of the skin demonstrated dermal infiltration by medium-sized cells with irregular nuclei and fine chromatin. Immunohistochemistry showed positivity for CD45/CD43/CD4/CD56, negativity for TdT/ CD34/CD117/cMPO/CD20/CD3/CD30 and epithelial, neuroendocrine, and melanoma markers. Bone marrow (BM) biopsy and aspirate did not show any infiltration. The patient was treated with chemotherapy (four cycles of CHOP and radiotherapy) and reached complete remission. Seventeen months later he relapsed with skin involvement, 0.7 3 10/L blasts in peripheral blood (PB) and BM infiltration. BM aspirate demonstrated 50% of big-sized blasts with monocytic appearance, most of them with hyaline cytoplasm, multiple vacuoles, and a pseudopodia formation. These vacuoles were mainly found peripherally and often had large size (3–4 microns) (Figs. 1A and B). PAS reaction was strongly positive with thick granular pattern located in cytoplasmic vacuoles in 96% of blasts (Fig. 1C). Other cytochemical techniques, including myeloperoxidase (MPO)/Sudan Black-B/ ANAE/cloroacetate-esterase/acid phosphatase were negative. The presence of clusters of blasts was repeatedly observed (Fig. 2A). ANAE intense positive macrophages inside the cluster were shown (Fig. 2B) in addition to strong PAS reaction in blasts (Fig. 2C). Flow cytometry analysis showed a lineage negative blast population expressing the following markers: CD451, CD41, CD12311, CD561, and human leukocyte antigen (HLA)-DR11 . The BM karyotype was normal. The patient was treated with chemotherapy [two cycles of hyper-cyclophosphamide, vincristina, doxorubicin, dexamethasone (CVAD)] being refractory. Currently, he is on palliative care. Department of Hematology, Biomedicine Institute of Sevilla (IBIS), Virgen Del Roc ıo/Virgen Macarena University Hospital/CSIC/Sevilla University, Sevilla, Spain Department of Pathology, Virgen Del Roc ıo University Hospital, Sevilla, Spain *Correspondence to: Dr. Teresa Caballero-Vel azquez., UGC de Hematolog ıa. Hospital Universitario Virgen del Roc ıo, Instituto de Biomedicina de Sevilla (IBIS)/CSIC/Universidad de Sevilla, Sevilla, Spain. E-mail: t.c.velazquez@gmail.com Received 14 June 2015; Revised 11 September 2015; Accepted 24 September 2015 DOI: 10.1002/dc.23372 Published online 15 October 2015 in Wiley Online Library (wileyonlinelibrary.com).

Erythrophagocytosis by peripheral monocytes and histiocytes in diffuse large B cell lymphoma

Dear Editor, An 80-year-old male presented with a 2-month history of weakness, bradypsychia, and fever (38 °C). On physical examination, he was found to be febrile, hypotensive, and tachycardic. No lymphadenopathy was palpable. His full blood count showed hemoglobin concentration 89 g/l, leucocytes 4.63 × 10/l, neutrophils 3.1 × 10/l, monocytes 1.0×10/l, and platelets 65×10/l. Further workup revealed hyperferritinemia (1928 μg/dl), hypertriglyceridemia (352 mg/dl), and elevated soluble CD25 (31.54 ng/ml, normal <7.5). A comprehensive autoimmune and infectious evaluation was negative, including Epstein-Barr virus viral load, serology for hepatitis B, hepatitis C and human immunodeficiency virus. An abdominal and thorax CT scan demonstrated hepatosplenomegaly, with numerous mediastinal and abdominal lymph nodes and pulmonary involvement. Microscopic examination of a blood film revealed monocytes with activation signs such as erythrophagocytosis (Fig. 1a, b, c), pseudopod formation (Fig. 1e, f) and cytoplasm containing a large phagosomal vacuole (Fig. 1g, h, i); even circulating histiocytes showing erythrophagocytosis (Fig. 1d) and prominent vacuolation were identified (Fig. 1j, k). The bonemarrow aspirate was normocellular with adequate global myelopoiesis showing 1 % histiocytes, of which 30 % engulfed erythrocytes, erythroblasts and platelets. The diagnostic criteria for hemophagocytic lymphohistiocytosis (HLH) were met. In addition, a diagnosis for diffuse large B cell lymphoma (DLBCL) was established by fine needle aspiration cytology of a duodenal lymph node (Fig. 2a). Immunohistochemistry revealed a non-germinal centre B cell-like subtype, expressing CD45, CD20, Bcl-6, and MUM-1 with no expression of CD10, or CD3 (Fig. 2b, c, d). The patient died due to respiratory infection 2 days after the bone marrow aspirate. Erythrophagocytosis by monocytes and histiocytes is unusually revealed in a peripheral blood film. This finding has infrequently been described in HLH [1]. It is even rarer if HLH is associated with DLBCL [2]. Hemophagocytosis in peripheral blood may have a worse outcome as has been suggested by the rather few cases reported. Electronic supplementary material The online version of this article (doi:10.1007/s00277-016-2685-z) contains supplementary material, which is available to authorized users.

MLL-rearranged acute myeloid leukemia: Influence of the genetic partner in allo-HSCT response and prognostic factor of MLL 3′ region mRNA expression

MLL gene is involved in more than 80 known genetic fusions in acute leukemia. To study the relevance of MLL partner gene and selected genes expression, in this work, we have studied a cohort of 20 MLL‐rearranged acute myeloid leukemia (AML).

Eosinophil phagocytosis in advanced systemic mastocytosis with eosinophilia

A 69-year-old female was diagnosed with advanced systemic mastocytosis with associated chronic myelomonocytic leukaemia. Her past medical history included renal mucosa-associated lymphoid tissue (MALT) lymphoma, with complete remission having been achieved after chemo-immunotherapy and local radiation 9 years earlier. Peripheral (16%, 2 7 9 10/l) and marrow (17 5%) eosinophilia were detected. Serum tryptase was 169 ng/ml (normal range 1–15). The bone marrow (BM) aspirate was hypercellular with 11% loosely scattered atypical mast cells as well as clusters. Up to 2% of the atypical mast cells were observed to have engulfed mainly eosinophils and some neutrophils (images, see also supplementary material). Cytochemical studies of mast cells showed granular chloroacetate esterase positivity and metachromasia on toluidine blue staining. Flow cytometric immunophenotyping identified a CD25CD2 pathological population of BM mast cells. The karyotype was 45,XX,der (7;18)(q10;q10)[20], confirmed by fluorescence in situ hybridization (FISH). BCR-ABL1, FIP1L1-PDGFRA, and FGFR1 and PDGFRB rearrangements were not detected by FISH. The KIT D816V mutation was negative by real time polymerase chain reaction. CBL (c.1250C>G, p.Pro417Arg), ASXL1 (c.2385delC, p.Trp796fs), RUNX1 (c.367_368insGG, p.Asp123fs) and DNMT3A (c.1643T>C, p.Met548Thr) mutations were identified by next generation sequencing. BM biopsy revealed 100% cellular marrow with spindled mast cell clusters (positive for CD25, CD117 and tryptase) and eosinophilia. Although normal mast cells are known to be involved in immune response, they have no phagocytic capacity. We report an interesting case of advanced systemic mastocytosis in which neoplastic mast cells exhibit a striking phagocytosis of eosinophils and neutrophils.

Bone marrow erythrocyte and neutrophil phagocytosis and cannibalism by neuroendocrine carcinoma

A 61-year-old non-smoking woman, who previously worked at a tobacco company for 36 years, had been diagnosed with stage IV lung adenocarcinoma 4 years earlier. After several lines of treatment, she had achieved a partial response. On this presentation, a full blood count showed pancytopenia (Hb 70 g/l, neutrophil count 0 7 9 10/l, platelet count 4 9 10/l), with a leucoerythroblastic picture. The bone marrow (BM) aspirate showed an infiltration by 86% of non-haematological neoplastic cells, of which 12% showed cell internalisation phenomena: approximately 2/3 were neutrophil (top) or erythrocyte (bottom left) haemophagocytosis and 1/3 were cellular cannibalism. Cellular cannibalism and haemophagocytosis by the same cells, and even phagocytosis of cells that had already phagocytosed other cells (bottom centre), were occasionally seen. BM histological examination showed diffuse neoplastic infiltration by medium-sized to large cells with sporadic haemophagocytosis (bottom right). Immunohistochemistry showed strong diffuse positivity for synaptophysin, and weaker staining for chromogranin A and CD56. Further images are shown in the Supplementary information. BM involvement by neuroendocrine carcinoma with a high proliferation index (Ki67: 80%) was diagnosed. Palliative care was chosen and, 3 weeks later, the patient died of septic shock and multiple organ failure. Neuroendocrine carcinomas rarely involve the BM, while haemophagocytosis by tumour cells, especially of neutrophils, is unusual. Cellular cannibalism has been related to tumour aggression while haemophagocytosis by tumour cells is of uncertain significance.

KIT D816V− chronic myelomonocytic leukemia progressing to KIT D816V+ associated to mast cell leukemia responding to allogeneic hematopoietic cell transplantation

Dear Editor, A 63-year-old male was diagnosed with a chronic myelomonocytic leukemia (CMML) type 1, with normal karyotype and an IgG-kappa monoclonal gammopathy of undetermined significance (MGUS). Blood cell counts showed the following: hemoglobin, 86 g/L; leucocytes, 8.2 × 10/L (neutrophils, 3.7 × 10/L; monocytes 1.4 × 10/L); and platelets, 199 × 10/L. A splenomegaly of 14 cm by ultrasound was observed. Qualitative and quantitative assessment of KIT D816V by realtime polymerase chain reaction (RT-PCR) was negative. Nextgeneration sequencing (NGS) by using PGM sequencer (Ion TorrentTM) was performed including 19 candidate genes (The Ion AmpliSeqTM AML Cancer Research Panel): CEPBA, DNMT3A, GATA2, TET2, TP53, ASXL1, BRAF, CBL, FLT3, IDH1, IDH2, JAK2, KIT, KRAS, NPM1, NRAS, PTPN11, RUNX1,WT1. No additional mutations were identified. After 8 months, the patient’s condition rapidly deteriorated. An abdominal ultrasound demonstrated splenomegaly 30 cm. Laboratory test showed the following: hemoglobin, 78 g/L; leukocytes, 12.3 × 10/L (neutrophils, 9.5 × 10/L; monocytes, 1.4 × 10/L); platelets, 34 × 10/L; serum IgGmonoclonal protein, 1.3 g/dl; serum tryptase, 84 mcg/L (N, 1–15 mcg/ L). Progression of CMML was suspected. The bone marrow (BM) aspirate was normocellular. Additionally, the presence of 25% of atypical mast cells was noted, with scattered clusters on the edges of the films (Fig. 1a). These cells showed a wide morphological variability, sometimes spindle-shaped and occasionally with a pseudopodium formation (Fig. 1b). Nuclei were oval-shaped, lobed, or multinucleated (Fig. 1c). Cells contained azurophilic granules ranging from agranular to hypergranular and vacuoles were occasionally observed (Fig. 1c). Isolated images of hemophagocytosis by mast cells were seen (Fig. 1d). Five percent of leukemic cells showed morphological differences, similar to atypical basophils with hypogranularity. Cytochemical studies showed periodic acid–Schiff positivity and intense metachromasia on toluidine blue staining (Fig. 1e). No evidence of CMML progression was noted. Flow cytometry identified a high forward scatter and intermediate side scatter population CD117/CD203c/ CD123/HLA-DR/CD38/CD25/CD2. In addition, 2.5% pathological plasma cells were observed. Cytogenetic testing revealed a normal karyotype. The presence of KIT D816V mutation was demonstrated in selected cell populations separated by FACSAria: mast cells, CD34+ cells, monocytes, granulocytes, and lymphocytes. BCR/ABL was negative by interphase fluorescence in situ hybridization. KIT D816V mutation was again confirmed in both, mast cells and granulocytes by NGS, without other additional mutations. Aleukemic variant of mast cell leukemia (MCL) with associated CMML and MGUS was diagnosed. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00277-017-3187-3) contains supplementary material, which is available to authorized users.

Neutrophil faggot cells and inv(16): not such a fortuitous association?

Dear Editor, A 36-year-old previously healthy man presented with fever, malaise, and bruising. A full blood count test showed a hemoglobin concentration of 98 g/L, platelet count 42 × 10/L, and leucocyte count 22 × 10/L with 40% blast cells and 6% neutrophils [14% with a few or multiple cytoplasmic Auer rods (Fig. 1a–c)]. No coagulation abnormalities were present. A bone marrow aspirate revealed 48% blasts, 41.5% granulocytes, and 4% eosinophils. Blasts had promonocytoid (28%) or undifferentiated (18%)morphology and showed granular positivity with myeloperoxidase (97%) but negativity with alpha-naphthyl acetate esterase. Eosinophils occasionally contained pre-eosynophilic granulation and showed weak positivity with periodic acid-Schiff or chloroacetate esterase stains. Dysplasia was present in 96% of the cells in the granulocytic lineage: pseudo-Pelger-Hüet anomaly, agranular cytoplasm or presence of Auer rods, from metamyelocytes to mature neutrophils, predominated in the latter. Interestingly, bundles of Auer rods (faggot cells) were seen up to 10% in mature neutrophils (Fig. 1d–f), while only 1 or 2 Auer rods were observed in less than 0.5% of the atypical promonocytoid cells (Fig. 2a–d). Flow cytometry showed that blasts were positive for CD34, HLA-DR, CD123, cyMPO, CD13, CD33, and CD117, with 12.5% positive for CD64 and CD4. Cytogenetic analysis found an inv(16) and, in 4/40 metaphases, a hyperdiploidy of about 52 chromosomes was observed. Fluorescence in situ hybridization (Fig. 3) and molecular studies confirmed inv(16) and CBFB-MYH11 rearrangement, respectively and discarded PML-RARA rear rangement . Nextgeneration sequencing showed a KIT p.Asp816Val mutation. The final diagnosis was acute myeloid leukemia (AML) with inv(16); CBFB-MYH11. The patient received chemotherapy according to the Pethema protocol, achieving a complete remission after induction, but with persistently positive CBFB-MYH11. The presence of 1–2 Auer rods is usually seen in myeloid blasts yet rarely in mature neutrophils, being exceptional in the monocytic lineage [1]. It is evenmore unusual that they are numerous, in bundles, such as those described in the faggot cells. * Concepción Prats-Martín concha_prats@yahoo.es

RUNX1 amplification in AML with myelodysplasia-related changes and ring 21 chromosomes.

Ring 21 is an unstable structural abnormality of chromosome 21 that can lead to RUNX1 gene amplification. We present a unique case with a carrier patient of a constitutional ring chromosome 21 (partial monosomy and trisomy 21) with dysmorphic features and congenital malformations phenotype, who developed acute myeloid leukaemia with myelodysplasia‐related changes and two ring 21 chromosomes with RUNX1 amplification. The patients constitutional ring 21 chromosome showed alterations in tumour suppressor genes, and oncogenes, but not in RUNX1. RUNX1 gene expression at acute myeloid leukaemia diagnosis, showed no upregulation, so other genes may also be the genetic amplification targets in this patient. Copyright