Fatal herpesvirus-6 encephalitis in a recipient of a T-cell-depleted peripheral blood stem cell transplant from a 3-loci mismatched related donor

Human herpesvirus-6 (HHV-6), like all the other herpes viruses, remains latent in host cells after primary infection but can be reactivated In immuno-compromised patients causing fever, skin rash, bone marrow (BM) suppression, pneumonitis, Sinusitis and meningoencephalitis. We describe the case of a man with chronic myelogenous leukemia who developed encephalitis associated with acute graft-versus-host disease two months after a T-cell-depleted mismatched peripheral blood stem cell transplant. Magnetic resonance images of the brain revealed multiple bilateral foci of signal abnormality. HHV-6 was the only pathogen detected in cerebrospinal fluid by PCR. treatment with both ganciclovir and foscarnet was unsuccessful and the patient gradually deteriorated and died. Other cases of HHV-6 encephalitis after bone marrow transplantation are reviewed

Improved achiral and chiral hplc-uv analysis of ruxolitinib in two different drug formulations

In this paper, two new reversed-phase (RP) HPLC-UV methods enabling the quantitative achiral and chiral analysis of ruxolitinib in commercial tablets (containing 20 mg of active pharmaceutical ingredient, API) and not commercially available galenic capsules (with 5 mg of API) are described. For the achiral method based on the use of a water/acetonitrile [70:30, v/v; with 0.1% (v) formic acid] eluent, a “research validation” study was performed mostly following the “International Council for Harmonization” guidelines. All the system suitability parameters were within the acceptance criteria: tailing factor, between 1.7 and 2.0; retention factor, 2.2; number of theoretical plates, >9000. The linearity curve showed R2 = 0.99 (Rxv2 = 0.99), while trueness (expressed as recovery) was between 96.3 and 106.3%. Coefficient of variations (CVs) (repeatability: CVw and intermediate precision: CVIP ) did not exceed 1.3% and 2.9%, respectively. Moreover, the use of the enantiomeric Whelk-O1 chiral stationary phases operated under similar RP eluent conditions as for the achiral analyses, and the “inverted chirality columns approach (ICCA)” allowed us to establish that the enantiomeric purity of ruxolitinib is retained upon reformulation from tablets to capsules. The two developed methods can allow accurate determinations of ruxolitinib in drug formulations for medical use

Frequent traces of EBV infection in Hodgkin and non-Hodgkin lymphomas classified as EBV-negative by routine methods: expanding the landscape of EBV-related lymphoma

peer-reviewedThe Epstein–Barr virus (EBV) is linked to various B-cell lymphomas, including Burkitt lymphoma (BL), classical Hodgkin lymphoma (cHL) and diffuse large B-cell lymphoma (DLBCL) at frequencies ranging, by routine techniques, from 5 to 10% of cases in DLBCL to >95% in endemic BL. Using higher-sensitivity methods, we recently detected EBV traces in a few EBV-negative BL cases, possibly suggesting a “hit-and-run” mechanism. Here, we used routine and higher-sensitivity methods (qPCR and ddPCR for conserved EBV genomic regions and miRNAs on microdissected tumor cells; EBNA1 mRNA In situ detection by RNAscope) to assess EBV infection in a larger lymphoma cohort [19 BL, 34 DLBCL, 44 cHL, 50 follicular lymphomas (FL), 10 T-lymphoblastic lymphomas (T-LL), 20 hairy cell leukemias (HCL), 10 mantle cell lymphomas (MCL)], as well as in several lymphoma cell lines (9 cHL and 6 BL). qPCR, ddPCR, and RNAscope consistently documented the presence of multiple EBV nucleic acids in rare tumor cells of several cases EBV-negative by conventional methods that all belonged to lymphoma entities clearly related to EBV (BL, 6/9 cases; cHL, 16/32 cases; DLBCL, 11/30 cases), in contrast to fewer cases (3/47 cases) of FL (where the role of EBV is more elusive) and no cases (0/40) of control lymphomas unrelated to EBV (HCL, T-LL, MCL). Similarly, we revealed traces of EBV infection in 4/5 BL and 6/7 HL cell lines otherwise conventionally classified as EBV negative. Interestingly, additional EBV-positive cases (1 DLBCL, 2 cHL) relapsed as EBV-negative by routine methods while showing EBNA1 expression in rare tumor cells by RNAscope. The relapse specimens were clonally identical to their onset biopsies, indicating that the lymphoma clone can largely loose the EBV genome over time but traces of EBV infection are still detectable by high-sensitivity methods. We suggest EBV may contribute to lymphoma pathogenesis more widely than currently acknowledged

Novel NPM1 exon 5 mutations and gene fusions leading to aberrant cytoplasmic nucleophosmin in AML

Nucleophosmin (NPM1) mutations in acute myeloid leukemia (AML) affect exon 12, but also sporadically affect exons 9 and 11, causing changes at the protein C-terminal end (tryptophan loss, nuclear export signal [NES] motif creation) that lead to aberrant cytoplasmic NPM1 (NPM1c+), detectable by immunohistochemistry. Combining immunohistochemistry and molecular analyses in 929 patients with AML, we found non–exon 12 NPM1 mutations in 5 (1.3%) of 387 NPM1c+ cases. Besides mutations in exons 9 (n = 1) and 11 (n = 1), novel exon 5 mutations were discovered (n = 3). Another exon 5 mutation was identified in an additional 141 patients with AML selected for wild-type NPM1 exon 12. Three NPM1 rearrangements (NPM1/RPP30, NPM1/SETBP1, NPM1/CCDC28A) were detected and characterized among 13 979 AML samples screened by cytogenetic/fluorescence in situ hybridization and RNA sequencing. Functional studies demonstrated that in AML cases, new NPM1 proteins harbored an efficient extra NES, either newly created or already present in the fusion partner, ensuring its cytoplasmic accumulation. Our findings support NPM1 cytoplasmic relocation as critical for leukemogenesis and reinforce the role of immunohistochemistry in predicting AML-associated NPM1 genetic lesions. This study highlights the need to develop new assays for molecular diagnosis and monitoring of NPM1-mutated AML

A novel patient-derived tumorgraft model with TRAF1-ALK anaplastic large-cell lymphoma translocation.

Although anaplastic large-cell lymphomas (ALCL) carrying anaplastic lymphoma kinase (ALK) have a relatively good prognosis, aggressive forms exist. We have identified a novel translocation, causing the fusion of the TRAF1 and ALK genes, in one patient who presented with a leukemic ALK+ ALCL (ALCL-11). To uncover the mechanisms leading to high-grade ALCL, we developed a human patient-derived tumorgraft (hPDT) line. Molecular characterization of primary and PDT cells demonstrated the activation of ALK and nuclear factor kB (NFkB) pathways. Genomic studies of ALCL-11 showed the TP53 loss and the in vivo subclonal expansion of lymphoma cells, lacking PRDM1/Blimp1 and carrying c-MYC gene amplification. The treatment with proteasome inhibitors of TRAF1-ALK cells led to the downregulation of p50/p52 and lymphoma growth inhibition. Moreover, a NFkB gene set classifier stratified ALCL in distinct subsets with different clinical outcome. Although a selective ALK inhibitor (CEP28122) resulted in a significant clinical response of hPDT mice, nevertheless the disease could not be eradicated. These data indicate that the activation of NFkB signaling contributes to the neoplastic phenotype of TRAF1-ALK ALCL. ALCL hPDTs are invaluable tools to validate the role of druggable molecules, predict therapeutic responses and implement patient specific therapies

Development of a distributed international patient data registry for hairy cell leukemia

Hairy cell leukemia (HCL) is a rare lymphoproliferative disorder, comprising only 2% of all leukemias. The Hairy Cell Leukemia Foundation (HCLF) has developed a patient data registry to enable investigators to better study the clinical features, treatment outcomes, and complications of patients with HCL. This system utilizes a centralized registry architecture. Patients are enrolled at HCL Centers of Excellence (COE) or via a web-based portal. All data are de-identified, which reduces regulatory burden and increases opportunities for data access and re-use. To date, 579 patients have been enrolled in the registry. Efforts are underway to engage additional COE’s to expand access to patients across the globe. This international PDR will enable researchers to study outcomes in HCL in ways not previously possible due to the rarity of the disease and will serve as a platform for future prospective research

Tumor protein D52 (TPD52): A novel B-cell/plasma-cell molecule with unique expression pattern and Ca2+-dependent association with annexin VI

We generated a murine monoclonal antibody (B28p) detecting an antigenic determinant shared by the immunoglobulin superfamily receptor translocation-associated 1 (IRTA1) receptor (the immunogen used to raise B28p) and an unrelated 28-kDa protein that was subsequently subjected to extensive characterization. The expression of the 28-kDa protein in normal lymphohematopoietic tissues was restricted to B cells and plasma cells and clearly differed from that expected for IRTA1 (selectively expressed by mucosa-associated lymphoid tissue [MALT] marginal zone B cells). Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE)/mass-spectrometry analysis identified the 28-kDa protein as human tumor protein D52 (TPD52), whose expression had been previously described only in normal and neoplastic epithelia. Specific B28p reactivity with TPD52 was confirmed by immunostaining/immunoblotting of TPD52-transfected cells. TPD52 expression pattern in normal and neoplastic B cells was unique. In fact, unlike other B-cell molecules (paired box 5 [PAX5], CD19, CD79a, CD20, CD22), which are down-regulated during differentiation from B cells to plasma cells, TPD52 expression reached its maximum levels at the plasma cell stage. In the Thiel myeloma cell line, TPD52 bound to annexin VI in a Ca2+-dependent manner, suggesting that these molecules may act in concert to regulate secretory processes in plasma cells, similarly to what was observed in pancreatic acinar cells. Finally, the anti-TPD52 monoclonal antibody served as a valuable tool for the diagnosis of B-cell malignancies

Both carboxy-terminus NES motif and mutated tryptophan(s) are crucial for aberrant nuclear export of nucleophosmin leukemic mutants in NPMc(+) AML

We recently identified aberrant cytoplasmic expression of nucleophosmin (NPM) as the immunohistochemical marker of a large subgroup of acute myeloid leukemia (AML) (about one-third of adult AML) that is characterized by normal karyotype and mutations occurring at the exon-12 of the NPM gene. In this paper,we have elucidated the molecular mechanism underlying the abnormal cytoplasmic localization of NPM. All 29 AML-associated mutated NPM alleles so far identified encode abnormal proteins which have acquired at the C-terminus a nuclear export signal (NES) motif and lost both tryptophan residues 288 and 290 (or only the residue 290) which determine nucleolar localization. We show for the first time that both alterations are crucial for NPM mutant export from nucleus to cytoplasm. In fact, the cytoplasmic accumulation of NPM is blocked by leptomycin-Bandratjadones, specific exportin-1/Crm1-inhibitors, and by reinsertion of tryptophan residues 288 and 290, which respectively relocate NPM mutants in the nucleoplasm and nucleoli. NPM leukemic mutants in turn recruit the wild-type NPM from nucleoli to nucleoplasm and cytoplasm. These findings indicate that potential therapeutic strategies aimed to retarget NPM to its physiological sites will have to overcome 2 obstacles, the new NES motif and the mutated tryptophan(s) at the NPM mutant C-terminus

Secretion of Novel SEL1L Endogenous Variants Is Promoted by ER Stress/UPR via Endosomes and Shed Vesicles in Human Cancer Cells

We describe here two novel endogenous variants of the human endoplasmic reticulum (ER) cargo receptor SEL1LA, designated p38 and p28. Biochemical and RNA interference studies in tumorigenic and non-tumorigenic cells indicate that p38 and p28 are N-terminal, ER-anchorless and more stable relative to the canonical transmembrane SEL1LA. P38 is expressed and constitutively secreted, with increase after ER stress, in the KMS11 myeloma line and in the breast cancer lines MCF7 and SKBr3, but not in the non-tumorigenic breast epithelial MCF10A line. P28 is detected only in the poorly differentiated SKBr3 cell line, where it is secreted after ER stress. Consistently with the presence of p38 and p28 in culture media, morphological studies of SKBr3 and KMS11 cells detect N-terminal SEL1L immunolabeling in secretory/degradative compartments and extracellularly-released membrane vesicles. Our findings suggest that the two new SEL1L variants are engaged in endosomal trafficking and secretion via vesicles, which could contribute to relieve ER stress in tumorigenic cells. P38 and p28 could therefore be relevant as diagnostic markers and/or therapeutic targets in cancer