Lucia Zanatta

EWSR1-CREB1 and EWSR1-ATF1 Fusion Genes in Angiomatoid Fibrous Histiocytoma

Purpose: Angiomatoid fibrous histiocytoma (AFH) is a low-grade mesenchymal neoplasm which usually occurs in children and adolescents. Either FUS-ATF1 or EWSR1-ATF1 have been detected in the few cases published, pointing to the interchangeable role of FUS and EWSR1 in this entity. EWSR1-ATF1 also represents the most frequent genetic alteration in clear cell sarcoma, suggesting the existence of a molecular homology between these two histotypes. We investigated the presence of EWSR1-CREB1, recently found in gastrointestinal clear cell sarcoma, and FUS-CREB1, as well as the already reported FUS-ATF1 and EWSR1-ATF1 in a series of AFH. Experimental Design: Fourteen cases were analyzed by fluorescence in situ hybridization (FISH) on paraffin-embedded tissue sections, using a commercial EWSR1 probe and custom-designed probes for FUS, ATF1, and CREB1. In two cases, four-color FISH was also done. Reverse transcription-PCR for the four hypothetical fusion genes was done in one case, for which frozen material was available. Results: Thirteen cases showed rearrangements of both EWSR1 and CREB1, whereas one case showed the rearrangement of both EWSR1 and ATF1. Four-color FISH confirmed the results in two selected cases. Reverse transcription-PCR showed EWSR1-CREB1 transcript in the case analyzed. Conclusion: We identified the presence of either EWSR1-CREB1 or EWSR1-ATF1 in all the cases, strengthening the concept of chromosomal promiscuity between AFH and clear cell sarcoma. Either the occurrence of a second unknown tumor-specific molecular event or, perhaps more likely, divergent differentiation programs of the putatively distinct precursor cells of AFH and clear cell sarcoma might be invoked in order to explain the two different phenotypes.

Concomitant chronic lymphocytic leukemia and acute myeloid leukemia: evidence of simultaneous expansion of two independent clones.

The simultaneous appearance of chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML) has been rarely reported, with AML occurring more frequently as a secondary event in patients receiving cytotoxic drugs for a primary lymphoproliferative disorder. We describe a case of simultaneous CLL and AML documented by morphological and cytometric analysis in a previously untreated patient. In particular, on the basis of morphological and immunological features, the patient was diagnosed as being affected by CD34 + /CD13 + /CD33 + /HLA-DR + /CD7 + FAB-M2 AML, along with a B-CLL characterized by neoplastic cells expressing a VH3-53/D3-22/JH4 Ig, bearing, on average, 3.9% IgVH mutations without evidence of antigen-driven selection. To establish whether the two neoplastic cell populations shared some common molecular signature, we performed IgH gene rearrangement studies on CD34 + /CD19− and CD34−/CD19 + immunomagnetically sorted cell populations: only genomic DNA from the CD19 + /CD34− cell fraction revealed the presence of the IgH gene rearrangement. These results provide evidence that the rare concomitant association of CLL and AML likely arises from simultaneous expansion of two independent clones.

Endothelin-1 receptor blockade as new possible therapeutic approach in multiple myeloma

New effective treatments are needed to improve outcomes for multiple myeloma (MM) patients. Receptors with restricted expression on plasma cells (PCs) represent attractive new therapeutic targets. The endothelin‐1 (EDN1) axis, consisting of EDN1 acting through EDN‐receptor A (EDNRA) and B (EDNRB), was previously shown to be overexpressed in several tumours, including MM. However, there is incomplete understanding of how EDN1 axis regulates MM growth and response to therapy. Besides EDNRA, the majority of MM cell lines and primary malignant PCs express high levels of EDNRB and release EDN1. Similarly, bone‐marrow microenvironment cells also secrete EDN1. Investigating the extent of epigenetic dysregulation of EDNRB gene in MM, we found that hypermethylation of EDNRB promoter and subsequent down‐regulation of EDNRB gene was observed in PCs or B lymphocytes from healthy donors compared to EDNRB‐expressing malignant PCs. Pharmacological blockade with the dual EDN1 receptor antagonist bosentan decreased cell viability and MAPK activation of U266 and RPMI‐8226 cells. Interestingly, the combination of bosentan and the proteasome inhibitor bortezomib, currently approved for MM treatment, resulted in synergistic cytotoxic effects. Overall, our data has uncovered EDN1‐mediated autocrine and paracrine mechanisms that regulate malignant PCs growth and drug response, and support EDN1 receptors as new therapeutic targets in MM.

Primary cardiac onset of disseminated Burkitt’s lymphoma

Dear Editor, We report a case of a 61-year-old woman with a prior history of hypertension, dyslipidemia, and ischemic cardiopathy who was admitted to our hospital complaining of dyspnea and cardiopalmus. Physical examination and laboratory tests including HIV serology and lactate dehydrogenase (LDH) were normal. Transthoracic echocardiography demonstrated a large mass arising from the right ventricle with associated pericardial and pleural effusions. Magnetic resonance imaging scans displayed also an involvement of the tricuspidal valve with secondary stenosis and complete infiltration of the ventricular wall. The patient underwent exploratory thoracotomy that revealed a mass extending from the atrium to the ventricle and impairing diastolic filling. This mass was almost completely excised. Histological section disclosed a Burkitt’s Non-Hodgkin’s Lymphoma: tumor consisted of small round cleaved cell with small nuclei surrounded by a deeply basophilic cytoplasm and admixed starry-sky histiocytes. Immunohistochemical examinations showed positivity of lymphoma cells for CD20, IgM, bcl6, CD10, Ki67 100% (EBV-; see Fig. 1a,c,b,d). A total body computed tomography (CT) scan did not demonstrate any lymph node enlargement or visceral involvement. Bone marrow cytology and histology showed a focal, minimal infiltration by lymphoma cells. After a short rehabilitation program, the patient was treated with a pre-phase of high-dose Dexametasone. During this phase, an epigastric palpable mass appeared, and an EGDS showed a thickening of the gastric wall due to lymphoma localization as showed by biopsy. A PET/CT scan demonstrated moreover an elevated pathological uptake of stomach, duodenum, omentum, retroperitoneal lymph nodes, pelvic bone, femur, and vertebral column. A second examination of bone marrow cytology at that time demonstrated 100% infiltration by blasts consistent with classical Burkitt’s Non-Hodgkin’s Lymphoma (BL) in both morphology and immunophenotype. After G-banding and fluorescence in situ hybridization (FISH) analyses, the karyotype was 49, XX, +X, t(8;14)(q24;q32), −14, +der(14)(3;8;14) (q27;q24;q32), +18, +20 (see Fig. 1d). In spite of Dexametasone administration, LDH level markedly increased. The patient started hyper-CVAD regimen, but she died before completing the first cycle. Incidence of cardiac neoplasia is very low, and lymphoproliferative disorders account for about 1% of the primary tumors of heart [1]. Primary cardiac BL has been reported very rarely [2, 3] both in immunocompetent and immunocompromised patients and is associated with a very unfavorable outcome. However, these reports frequently lack complete cytogenetic data fulfilling WHO criteria. Sporadic variants of BL and Burkitt-like lymphoma (BLL) present secondary genomic aberrations more frequently than endemic BL, which could explain their worse prognosis. To our knowledge, only two further cases of Burkitt’s lymphoma have been reported [4, 5] sharing cytogenetic abnormality Ann Hematol (2008) 87:487–488 DOI 10.1007/s00277-007-0414-3

Reagent and labor cost optimization through automation of fluorescence in situ hybridization (FISH) with the VP 2000: an Italian case study.

In the modern molecular diagnostic laboratory, cost considerations are of paramount importance. Automation of complex molecular assays not only allows a laboratory to accommodate higher test volumes and throughput but also has a considerable impact on the cost of testing from the perspective of reagent costs, as well as hands-on time for skilled laboratory personnel. The following study tracked the cost of labor (hands-on time) and reagents for fluorescence in situ hybridization (FISH) testing in a routine, high-volume pathology and cytogenetics laboratory in Treviso, Italy, over a 2-y period (2011–2013). The laboratory automated FISH testing with the VP 2000 Processor, a deparaffinization, pretreatment, and special staining instrument produced by Abbott Molecular, and compared hands-on time and reagent costs to manual FISH testing. The results indicated significant cost and time saving when automating FISH with VP 2000 when more than six FISH tests were run per week. At 12 FISH assays per week, an approximate total cost reduction of 55% was observed. When running 46 FISH specimens per week, the cost saving increased to 89% versus manual testing. The results demonstrate that the VP 2000 processor can significantly reduce the cost of FISH testing in diagnostic laboratories.

Cytogenetic evidence of metastatic myxoid liposarcoma and therapy-related myelodysplastic syndrome in a bone marrow biopsy

Myxoid liposarcoma exhibits a peculiar clinical behavior, with a tendency to spread to serosal membranes, distant soft tissues, and bones, even in the absence of lung metastases. Therapy-related hematological neoplasms are well-known side effects of cytotoxic chemotherapy. We describe an exceptional case of metastatic myxoid liposarcoma of the spine associated with therapy-related refractory anemia with excess of blasts in a 37-year-old woman who underwent multi-agent chemotherapy for a myxoid liposarcoma of the left thigh. Microscopic examination of the bone marrow biopsy revealed dysplastic features, with abnormal localization of immature precursors and micromegakaryocytes, and islands of undifferentiated oval small/medium-size cells, suggestive of acute myeloid leukemia arising in the setting of a myelodysplastic syndrome. Immunohistochemistry was not discriminant. Cytogenetic analyses of bone marrow aspirate disclosed the presence of 2 different rearrangements, subsequently confirmed by fluorescent in situ hybridization and was crucial in making the correct diagnosis.

Aleukemic granulocytic sarcoma with associated T-cell lymphoblastic lymphoma in the same lymph node: Morphologic features and molecular signatures

The concept of bilineal acute leukemia is defined in the WHO classification, where, along with the undifferentiated acute leukemia and the biphenotypic acute leukemia, it is included in the group of the acute leukemias of ambiguous lineage. The diagnosis is made on the basis of the detection of a dual population of blasts expressing specific markers of the two different myeloid and lymphoid lineages. Cases of Tcell lymphoblastic leukemia/lymphoma with eosinophilia and coexistent acute myeloid leukemia (AML) have been described in the literature [1]. In some of these cases an association of this disorder with the t(8;13) translocation has been observed, but it represents an inconstant report [2]. Much less is known about its solid counterpart. Only very few cases of association of T-cell lymphoblastic lymphoma (TLL) with granulocytic sarcoma (GS) have been reported so far [3–6], one of which was typically associated with eosinophilia [3]. It is not known whether the bilineal solid acute leukemia results from a single cellular clone differentiating toward two different lineages, is the expression of a lineage switch or alternatively represents a collision neoplasm ab initio. The detection of the same molecular signatures, such as the same Ig heavy chain or T-cell receptor (TcR) rearrangements, would support the first hypothesis, despite the different morphology and immunophenotype of the two components. A 31-year-old man was admitted to our hospital with chest pain and a 13-cm mass in the anterior mediastinum with pleural infiltration. The excision of a right supraclavicular lymph node revealed a neoplasm with morphological and immunohistochemical features most consistent with GS. The blood cell count was normal and there was neither morphologic nor cytogenetic evidence of bone marrow involvement. Immunocytoflowmetric analysis from bone marrow revealed the presence of a cell population positive for CD45 and CD34, which also expressed myeloid markers and represented 4% of the bone marrow cells, whereas T and B cell markers and TdT were negative. The patient was started on chemotherapy (FLAIE). After 3 months, cytogenetic analysis of the bone marrow revealed the presence of a cell population with a complex karyotype (10354n4XXXY, þ3, del(4)(?), þ7, 79, þ12, 714, þ15, 717, þ18, þ19, þ21, þ7mar). The minimal response to the chemotherapy and the complex karyotype, more in keeping with a lymphoblastic lymphoma rather than a GS, led us to send the lymph node biopsy material in consultation. A diagnosis of GS associated with TLL was made. A different chemotherapy scheme (ICE-EORTC/GIMEMA) was instituted. However, the patient died of gastrointestinal bleeding 4 months later. For immunohistological studies, sections from formalin-fixed, paraffin-embedded lymph node tissue were stained with hematoxylin–eosin, Giemsa stain and with the following antibodies: CD30 (clone ber-H2, prediluted, MW pH7, Dako,

A Pediatric Intra-Axial Malignant SMARCB1-Deficient Desmoplastic Tumor Arising in Meningioangiomatosis

SMARCB1 inactivation is a well-established trigger event in atypical teratoid/rhabdoid tumor. Recently, a role for SMARCB1 inactivation has emerged as a mechanism of clonal evolution in other tumor types, including rare brain tumors. We describe an unusual malignant intra-axial SMARCB1-deficient spindle cell desmoplastic neoplasm, occurring in a 6-year-old child with meningioangiomatosis and a long history of seizures. Striking features of the tumor were a storiform pattern and strong CD34 expression. Undifferentiated round cell areas with isolated rhabdoid cells showing high mitotic index and focal necrosis with INI1 expression loss were present. The meningioangiomatosis component showed few chromosomal imbalances, including chromosomal 22 monosomy (where SMARCB1 maps) and gain at 6q14.3. In addition to these abnormalities, the spindle cell desmoplastic neoplasm and its dedifferentiated SMARCB1-deficient component shared several other aberrations, including homozygous deletion at 9p21.3, losses at 1p, 3p, 3q, 10p, and 13q, gains and losses at 5p and 11p. In line with INI1 loss, the dedifferentiated component showed remarkably decreased levels of SMARCB1 transcript. The residual SMARCB1 allele was wildtype. Our findings suggest progression from the meningioangiomatosis to the malignant desmoplastic neoplasm through the occurrence of complex chromosomal abnormalities, and point to functional silencing of SMARCB1 in the dedifferentiation component.

In reply to Schäfer et al: new evidence on the role of endothelin-1 axis as a potential therapeutic target in multiple myeloma

frequently found among those with positive correlation whereas upregulated genes were more frequent among those with negative expression correlation. Table SI. Biological features of the 85 CLL cases from CRO (Italy). Table SII. Baseline characteristics of the 296 CLL cases (ICGC series). Table SIII. Summary of probes for transcripts overlapping UCR loci found significantly differentially expressed by CpGODN in the discovery CLL series. Table SIV. Univariate Cox regression analyses of TTT in the 67 CLL series. Table SV. Multivariate Cox regression analyses of TTT in the 67 CLL series. Table SVI. Statistical analyses of uc.70-related transcripts impact on TTT in the 296 CLL ICGC series. Table SVII. Pearson correlation of ICGC CLL gene expression in reference to the transcript AC092652.2-202 (5th percentile). Data S1. Supplementary methods.