Ann Janssens

Molecular cytogenetic and clinical findings in ETV6/ABL1-positive leukemia

Rearrangements of 12p, resulting from deletions or translocations, are common findings in hematologic malignancies. In many cases, these rearrangements target the ETV6 gene (previously called TEL) located at 12p13. Various partner genes have been implicated in the formation of fusion genes with ETV6. These include PDGFRB, JAK2, NTRK3, ABL2, and ABL1, each of which encodes for proteins with tyrosine kinase activity. To date, ETV6/ABL1 transcripts have been detected in only four patients with a leukemic disorder. Here, we describe one adult with chronic myeloid leukemia and a child with T‐cell acute lymphocytic leukemia with ETV6/ABL1. Molecular cytogenetic analysis confirmed that formation of an ETV6/ABL1 fusion in these patients required at least three chromosomal breaks and showed that each of these translocations is the result of a complex chromosomal rearrangement. Molecular analysis showed the presence of two fusion transcripts in both patients as the result of alternative splicing, questioning the suggested role of these transcripts in the lineage specificity. Clinical findings of these patients were compared to those of previously reported cases, and the possible clinical and biological similarities between ETV6/ABL1 and other fusion genes leading to increased tyrosine kinase activity are discussed.© 2000 Wiley‐Liss, Inc.

High-risk clonal evolution in chronic B-lymphocytic leukemia: single-center interphase fluorescence in situ hybridization study and review of the literature

We studied the relation of clonal evolution (CE) in Chronic B‐lymphocytic leukemia (CLL) with prognostic factors and the correlation between CE and disease progression and overall survival.

Pyoderma gangrenosum as an Early Revelator of Acute Leukemia

Bullous pyoderma gangrenosum is an atypical, more superficial variety of the classical pyoderma and is often associated with myeloproliferative disorders. We present the case of a patient who presented initially with subcutaneous nodules and who developed bullous lesions afterwards. Histological evaluation showed the presence of neutrophilic infiltrates in both lesions. A few months after the diagnosis of bullous pyoderma gangrenosum, an underlying leukemia was revealed. Our case illustrates the importance of regular blood and bone marrow examinations in patients with atypical bullous pyoderma gangrenosum, resulting in a rapid diagnosis of the underlying disease.

Steroid effects on ZAP-70 and SYK in relation to apoptosis in poor prognosis chronic lymphocytic leukemia

There is resurgent interest in glucocorticoids (GCs) in the treatment of poor prognosis chronic lymphocytic leukemia (CLL). Little is known however on how GCs induce apoptosis in CLL. Methylprednisolone (MP) induces apoptosis in ZAP-70 positive CLL more readily than in ZAP-70 negative CLL, which is in contrast to the effects of radiation and chemotherapy. The increased GC sensitivity of ZAP-70+ CLL was studied in relation to the expression status of ZAP-70 and the related signal transducing tyrosine kinase SYK. Both ZAP-70 and SYK were downregulated by GC treatment. Moreover, SYK was dephosphorylated by the phosphatase PTP1B of which the expression and translation levels were induced by GCs. Inhibition of PTP1B successfully restored ZAP-70 expression and SYK phosphorylation but did not interfere with GC-induced apoptosis. Therefore, the downregulation of ZAP-70 and P-SYK per se during treatment with GCs is not sufficient to induce apoptosis, and different mechanisms must therefore be responsible for the increased steroid sensitivity of ZAP-70+ CLL.

HA14-1 potentiates apoptosis in B-cell cancer cells sensitive to a peptide disrupting IP3 receptor / Bcl-2 complexes

Anti-apoptotic B-cell lymphoma 2 (Bcl-2) is commonly upregulated in hematological cancers, including B-cell chronic lymphocytic leukemia (B-CLL) and diffuse large B-cell lymphoma (DLBCL), thereby protecting neoplastic cells from oncogenic-stress-induced apoptosis. Bcl-2 executes its anti-apoptotic function at two different sites in the cell. At the mitochondria, Bcl-2 via its hydrophobic cleft interacts with pro-apoptotic Bcl-2 family members to inhibit apoptosis. At the endoplasmic reticulum (ER), Bcl-2 via its Bcl-2 homology (BH)4 domain, prevents excessive Ca(2+) signals by interacting with the inositol 1,4,5-trisphosphate receptor (IP3R), an intracellular Ca(2+)-release channel. A peptide tool (BIRD-2) that targets the BH4 domain of Bcl-2 reverses Bcl-2s inhibitory action on IP3Rs and can trigger pro-apoptotic Ca(2+)signals in B-cell cancer cells. Here, we explored whether HA14-1, a Bcl-2 inhibitor that also inhibits sarco/endoplasmic reticulum Ca(2+)-ATPases (SERCA), could potentiate BIRD-2-induced cell death. We measured apoptosis in Annexin V/7-AAD stained cells using flow cytometry and intracellular Ca(2+) signals in Fura2-AM-loaded cells using an automated fluorescent plate reader. HA14-1 potentiated BIRD-2-induced Ca(2+) release from the ER and apoptosis in both BIRD-2-sensitive DLBCL cell lines (SU-DHL-4) and in primary B-CLL cells. BIRD-2-resistant DLBCL cells (OCI-LY-1) were already very sensitive to HA14-1. Yet, although BIRD-2 moderately increased Ca(2+) levels in HA14-1-treated cells, apoptosis was not potentiated by BIRD-2 in these cells. These results further underpin the relevance of IP3R-mediated Ca(2+) signaling as a therapeutic target in the treatment of Bcl-2-dependent B-cell malignancies and the advantage of combination regimens with HA14-1 to enhance BIRD-2-induced cell death.

Non-Hodgkin's lymphoma presenting with spinal involvement

The case concerns a 38 year old woman with a one year history of a low grade non-Hodgkins lymphoma stage 4 (disseminated involvement of one or more extralymphatic organs with or without lymph node involvement). At the time of diagnosis, nodal involvement was found in the supraclavicular and axillar regions and in the neck; the abdomen presented marked splenomegaly and sternal puncture revealed bone marrow involvement. Restaging after treatment with cyclofosfamide-vincristine-prednisone (CVP) and cyclofosfamide-epirubicine-vincristine-prednisone (CEOP) chemotherapy during 10 months showed the absence of pathological node with computed tomography (CT). One year after diagnosis, the patient consulted the department of haematology with sudden onset of low back pain and sciatica in the right leg. The complaints resolved after treatment with non-steroidal anti-inflammatory drugs (NSAIDs). Three weeks later, she presented a relapse of the sciatica with dysaesthesia and sensory deficit of the right leg and progressive paresis of the right leg, however no systemic features at that time. According to the clinical stigmata, the diagnosis of acute sciatica was made and treatment with oral prednisolone 20 mg daily was started. Despite treatment, she was admitted urgently with acute paresis of the right leg. Clinical examination revealed a paresis of the right quadriceps muscle and tibialis anterior muscle. This is compatible with involvement of the L4, L5 and S1-level. The sciatic nerve stretch test was positive at 30° (right side) and 50° (left side), and the patellar reflex at the right side was absent, with indifferent Babinsky sign, and sensory deficit at the right calf and heel. Percussion of the spine revealed pain from the fifth lumbar vertebra to the presacral region; anteflexion of the spine was limited by pain. Radiographic examination showed a normal lumbar spine; no herniation was seen on CT of the lumbar intervertebral discs and 99mTc total body …

Importance of receptor density- in alpha radioimmunotherapy in B cell malignancies: an in-vitro study

BackgroundExternal beam radiotherapy and beta radioimmunotherapy (RIT) are effective treatments for lymphoid malignancies. The development of RIT with alpha emitters is attractive because of the high linear energy transfer (LET) and short path length, allowing higher tumour cell kill and lower toxicity to healthy tissues. AimTo assess the binding of rituximab to samples of B cell chronic lymphocytic leukaemia (B-CLL) and splenic lymphoma with villous lymphocytes (SLVL), and to evaluate the induction of apoptosis by conventional therapies as well as with 213Bi conjugated to rituximab. Method213Bi was eluted from a 225Ac generator and conjugated to CD20 antibody (rituximab) with CHX-A″-DTPA as chelator. Binding assays with 213Bi-rituximab were correlated to antibody binding capacity obtained by flow cytometry. Apoptosis was scored by flow cytometric analyses of the cells stained with annexin V–FITC and 7-amino-actinomycin D. ResultsBinding of 213Bi-rituximab was significantly lower for B-CLL compared to SLVL samples (12±3 and 42±10 213Bi atoms per cell, respectively, at 370 kBq·ml−1). The induction of apoptosis did not differ significantly between the two groups (B-CLL and SLVL) after external gamma irradiation or treatment with methylprednisolone and fludarabine (17±12% and 18±11%; 23±14% and 21±12%; 9±9% and 11±8%, respectively; all results expressed as percentages of all cells). Rituximab conjugated or not to 213Bi induced significantly more apoptosis in SLVL (42±19% and 42±17%) compared to B-CLL samples (27±12% and 6±8%). ConclusionBinding assays confirm that SLVL samples present more CD20 antigens compared to B-CLL samples. Conventional therapies such as fludarabine, methylprednisolone or external gamma irradiation induce similar responses in the two populations but SLVL samples present higher sensitivity towards 213Bi-rituximab. These data are in favour of alpha-RIT in SLVL patients.

Erythromelalgia: a clue to the diagnosis of polycythemia vera.

We report the case of a 74-year-old woman with recurrent episodes of symmetrical congestion and erythema in the distal lower legs causing a burning distress. Laboratory and clinical investigations revealed an underlying myeloproliferative disorder. The cutaneous symptoms were atypical of erythromelalgia. Salicylates and treatment of the underlying polycythemia were able to eliminate the skin lesions but not entirely suppress the subjective discomfort.

Recipient-derived chronic lymphocytic leukaemia diagnosed shortly after kidney transplantation on protocol biopsy

Here we present the case of a patient with diagnosis of chronic lymphocytic leukaemia (CLL) on routine protocol biopsy 3 months following kidney transplantation. Genetic analysis confirmed the origin of the malignancy, being the recipient. Differential diagnosis with post-transplant lymphoproliferative disorder (PTLD) is extremely important in order to avoid unnecessary devastating treatment. This case is challenging, both in terms of making the correct diagnosis and in terms of optimal treatment. The case underscores that it is extremely important to distinguish between a pre-existing lymphoma diagnosis after transplantation and a true PTLD as the treatment options of both are very divergent.

Coxsackievirus A16 Encephalitis during Obinutuzumab Therapy, Belgium, 2013

To the Editor: Enterovirus infections are associated with many clinical manifestations, and specific virus groups or serotypes are associated with specific manifestations. Coxsackievirus A16, a common cause of hand, foot and mouth disease, rarely causes encephalitis. Although most enterovirus infections are cleared by cellular immune responses, invasive enterovirus disease is prevented or controlled by neutralizing antibodies (1). Thus, patients with humoral immunodeficiencies are susceptible to serious enterovirus infections. Nine cases of enteroviral encephalitis (1 caused by echovirus 13, 1 caused by coxsackievirus A16, 2 caused by enterovirus 71, and 5 caused by unknown enteroviruses) have been reported after therapy with rituximab, a monoclonal antibody (MAb) that causes secondary hypogammaglobulinemia (2). We describe coxsackievirus A16 encephalitis in a patient who was receiving treatment with the MAb obinutuzumab. A 67-year-old woman with non-Hodgkin lymphoma showed complete remission after 6 cycles of treatment with bendamustine and obinutuzumab. Induction immunochemotherapy was followed by obinutuzumab maintenance therapy. At admission, she had received 7 of 12 scheduled treatments. The patient was hospitalized because of a history of high-grade fever that did not respond to antimicrobial drugs, confusion, general weakness, and urinary incontinence. She had a neutrophil count of 3.1 × 109 cells/L but had severe lymphocytopenia (0.3 × 109 cells/L and an absolute CD4 cell count of 0.082 × 109 cells/L) and low serum immunoglobulin levels (IgG 3.86 g/L [reference range 7.51–15.6 g/L], IgA 0.07 g/L [reference range 0.82–4.53 g/L], and IgM 0.13 g/L [reference range 0.46–3.04 g/L]). Cerebrospinal fluid (CSF) samples were collected on days 1, 4, and 6. CSF leukocyte counts increased from 14 cells/mm3 (day 1) to 60 cells/mm3 (day 4) (35% and 27% lymphocytes, respectively). Cytologic and immunophenotypic analyses showed no cerebromeningeal lymphoma infiltration. Total protein levels in CSF increased from 561 mg/L on day 1 to 771 mg/L on days 4 and 6. Bacterial and fungal cultures were negative. Cryptococcal antigen was not detected in CSF. Serologic test results were negative for Borrelia spp., Listeria spp., parvovirus B19, measles virus, and galactomannan. PCR results for CSF were repeatedly negative for herpes simplex virus, varicella zoster virus, cytomegalovirus, Toxoplasma gondii, JC polyomavirus, human herpesvirus 6, Epstein-Barr virus, and mumps virus. PCR results and culture were negative for Mycobacterium spp. Serum samples were negative for antibodies against neuronal nuclear (Hu, Ri, and Yo) antigens. However, enterovirus RNA was detected by reverse transcription PCR in all CSF samples. Sequencing of the virion protein 1 gene obtained directly from RNA extracted from CSF identified the virus as coxsackievirus A16 (3). Computed tomography scan of the brain on day 2 showed no abnormalities. However, brain magnetic resonance imaging scans on the third and fourth days showed bilateral, multiple, hyperintense white matter lesions in the periventricular region and cerebral hemispheres. Treatment was started empirically with broad-spectrum antimicrobial drugs and acyclovir; the acyclovir was stopped after infection with herpes simplex virus was excluded. On day 4, imaging indicated development of aphasia and right hemiparesis without new lesions. The patient was transferred for mechanical ventilation after a grand mal seizure. Treatment with intravenous immune globulin (IVIG, 400 mg/kg) was started on day 4 and given for 5 consecutive days, which resulted in marked and continued neurologic improvement. Monthly doses of IVIG (500 mg/kg) resulted in normal serum IgG levels. Four months after initial examination, the virus has been cleared but the patient still has intermittent confusion and language defects. Treatment with IVIG will be continued for an additional 6 months. Use of MAbs against CD20 B-cell antigen has become standard treatment for B-cell lymphomas and an increasing number of autoimmune disorders (4,5). However, resulting hypogammaglobulinemia predisposes patients to opportunistic infections, including progressive multifocal leukoencephalopathy and enterovirus infections (2,6). MAbs with enhanced activity against CD20 (e.g., obinutuzumab) have been developed. Obinutuzumab has been approved by the US Food and Drug Administration for treatment of chronic lymphocytic leukemia. Studies regarding the use of obinutuzumab for other B-cell malignancies are ongoing (7). We anticipate that more cases of enteroviral encephalitis might develop, given the increasingly frequent use of MAbs against CD20 and widespread occurrence of enteroviruses. However, in view of the few reported cases (2), we also suspect that many cases remain undiagnosed despite availability of several pan-enterovirus diagnostic kits, which can detect low viral loads. Thus, clinicians should be suspicious of severe enterovirus infections in patients receiving MAbs. Any patient receiving MAbs against CD20 who has neurologic symptoms should be screened for infection with enterovirus RNA. In contrast to JC polyomavirus–associated progressive multifocal leukoencephalopathy, enteroviral encephalitis can be successfully treated by early administration of IVIG, which might contain neutralizing antibodies, albeit in variable amounts (8). In the absence of double-blinded, placebo-controlled clinical studies of treatment for severe enterovirus infections, no specific antiviral therapy has been approved. However, 3 capsid inhibitors (pleconaril, pocapavir [V-073], and the pirodavir analog BTA-798) that show activity against enteroviruses are being developed (9). Pocapavir has potent activity against poliovirus and appears to be safe and well tolerated (10). In the United States, this drug is available by special request from the Food and Drug Administration.