Plerixafor with and without chemotherapy in poor mobilizers: results from the German compassionate use program.

The CXCR4-inhibitor plerixafor mobilizes hematopoietic stem cells amplifying the effects of granulocyte-CSF (G-CSF). Before approval plerixafor was used in a compassionate use program (CUP) for patients who failed a previous mobilization. In the German CUP 60 patients from 23 centers (median age 56.5 years (2-75)) were given 240 µg/kg plerixafor SC 9-11 h before apheresis. A total of 78.3% (47/60) received G-CSF for 4 days before plerixafor administration; 76.6% of those (36/47) yielded at least 2.0 × 10(6) CD34(+) cells/µL. The median cell yield was 3.35 × 10(6) CD34+ cells/kg (0-29.53). Nine patients received plerixafor alone or with G-CSF for less than 4 days mobilizing a median of 3.30 × 10(6) CD34+ cells/kg (1.6-5.6). There was no significant difference between G-CSF application for 4 days and for a shorter period of time (P=0.157). A total of 47 patients received plerixafor plus G-CSF combined with chemotherapy yielding a median of 3.28 × 10(6) CD34+ cells/kg (0-24.79). In all, 40 of 60 patients (66.7%) proceeded to transplantation, and achieved a timely and stable engraftment. Side effects were rare and manageable. In conclusion, mobilization with plerixafor in poor mobilizers is safe and results in a sufficient stem cell harvest in the majority of patients.

Epigenetic dysregulation of secreted Frizzled-related proteins in multiple myeloma.

We analysed the clinical impact of epigenetic dysregulation of the Wnt pathway in malignant plasma cell disorders. In multiple myeloma (MM) cell lines, aberrant promoter hypermethylation of the secreted Frizzled-related protein (SFRP) genes was a common event, and hypermethylation of SFRP1,-2 and -5 was associated with transcriptional silencing. Among 76 primary patient samples, the frequency of aberrant methylation was 35.5% for SFRP1, 52.6% for SFRP2, 1.3% for SFRP4 and 6.9% for SFRP5. Hypermethylation of SFRP1 and -2 genes was detected in monoclonal gammopathy of undetermined significance and all MM stages including plasma cell leukaemia (PCL), while SFRP5 methylation was restricted to advanced MM stages and PCL. Our data indicate that epigenetic silencing of Wnt antagonists is an early event in MM pathogenesis and that SFRP5 hypermethylation may play a role in disease progression.

Epigenetic inactivation of secreted Frizzled-related proteins in acute myeloid leukaemia.

The Wnt signalling pathway has a key function in stem cell maintenance and differentiation of haematopoietic progenitors. Secreted Frizzled-related protein genes (SFRPs), functioning as Wnt signalling antagonists, have been found to be downregulated by promoter hypermethylation in many tumours. To analyse epigenetic dysregulation of SFRPs in acute myeloid leukaemia (AML), we examined the promoter methylation status of SFRP1, -2, -4 and -5 in AML cell lines by methylation-specific polymerase chain reaction (MSP). Aberrant CpG island methylation was found for all four SFRP genes. By real-time reverse transcription-PCR, corresponding transcriptional silencing for SFRP1 and -2 was demonstrated and treatment of cell lines with 5-aza-2'-deoxycytidine resulted in re-expression. The methylation status of the SFRP genes was analysed in 100 specimens obtained from AML patients at diagnosis. The frequencies of aberrant methylation among the patient samples were 29% for SFRP1, 19% for SFRP2, 0% for SFRP4 and 9% for SFRP5. For SFRP2, a correlation between promoter hypermethylation and transcriptional downregulation was found in primary AML samples. Among AML cases with a favourable karyotype, hypermethylation of SFRP genes was restricted to patients with core binding factor (CBF) leukaemia, and aberrant methylation of the SFRP2 promoter was an adverse risk factor for survival in CBF leukaemia.

Epigenetic alterations complement mutation of JAK2 tyrosine kinase in patients with BCR/ABL-negative myeloproliferative disorders.

An acquired autoactivating mutation with a V617F amino-acid substitution in the JAK2 tyrosine kinase is frequently found in BCR/ABL-negative myeloproliferative disorders (MPD). Hypermethylation of CpG islands within gene promoter regions is associated with transcriptional inactivation and represents an important mechanism of gene silencing in the pathogenesis of hematopoietic malignancies. In this study, we determined the DNA methylation status of 13 cancer-related genes in the context of JAK2 mutations in 39 patients with MPD. Genes analyzed for hypermethylation were SOCS-1, SHP-1, E-cadherin, MGMT, TIMP-2, TIMP-3, p15, p16, p73, DAPK1, RASSF1A, RARbeta2 and hMLH1. We found at least one hypermethylated gene in 15/39 MPD patient specimens, and in 6/39 samples aberrant methylation of the negative cytokine regulator SOCS-1 was present. The JAK2V617F mutation was found in 21/39 patients as determined by allele-specific polymerase chain reaction. Hypermethylation of SOCS-1 was observed in 3/21 patients with an autoactivating JAK2 mutation and in 3/18 patients with wild-type JAK2. Our results suggest that epigenetic inactivation of SOCS-1 may be a complementary mechanism to the JAK2V617F mutation in the pathogenesis of MPD that leads to dysregulation of JAK-STAT signal transduction and thus contributes to growth factor hypersensitivity.

Aberrant methylation of the Wnt antagonist SFRP1 in breast cancer is associated with unfavourable prognosis.

The canonical Wnt signalling pathway plays a key role during embryogenesis and defects in this pathway have been implicated in the pathogenesis of various types of tumours, including breast cancer. The gene for secreted frizzled-related protein 1 (SFRP1) encodes a soluble Wnt antagonist and is located in a chromosomal region (8p22-p12) that is often deleted in breast cancer. In colon, lung, bladder and ovarian cancer SFRP1 expression is frequently inactivated by promoter methylation. We have previously shown that loss of SFRP1 protein expression is a common event in breast tumours that is associated with poor overall survival in patients with early breast cancer. To investigate the cause of SFRP1 loss in breast cancer, we performed mutation, methylation and expression analysis in human primary breast tumours and breast cell lines. No SFRP1 gene mutations were detected. However, promoter methylation of SFRP1 was frequently observed in both primary breast cancer (61%, n=130) and cell lines analysed by methylation-specific polymerase chain reaction (MSP). We found a tight correlation (P<0.001) between methylation and loss of SFRP1 expression in primary breast cancer tissue. SFRP1 expression was restored after treatment of tumour cell lines with the demethylating agent 5-aza-2'-deoxycytidine. Most interestingly, SFRP1 promoter methylation was an independent factor for adverse patient survival in Kaplan-Meier analysis. Our results indicate that promoter hypermethylation is the predominant mechanism of SFRP1 gene silencing in human breast cancer and that SFRP1 gene inactivation in breast cancer is associated with unfavourable prognosis.

Heart and muscle involvement by extra-medullary myeloid leukemia: a case report and review of the literature.

Extra-medullary myeloid tumours (EMT) have been described after curative treatment for acute myeloid leukaemia (AML) in increasing numbers after allogeneic stem cell transplantation. The sites of manifestations are ubiquitous and the discovery is most frequently guided by symptoms reported by the patient or by findings on clinical examination. This study reports a case of EMT in muscles and the heart 1.5 years after allogeneic transplantation for an AML with t(8;21)(q22;23) who achieved a complete remission by use of an idarubicine-based combination chemotherapy. Pathological and imaging findings are presented and treatment options are discussed.

DNA methylation changes in multiple myeloma.

Using a candidate gene approach, we analyzed the methylation status of the promoter-associated CpG islands of 11 well-characterized tumor suppressor genes by methylation-specific polymerase chain reaction in five multiple myeloma (MM) cell lines and 56 patients with malignant plasma cell disorders. The frequency of aberrant methylation among the patient samples was 46.4% for SOCS-1, 35.7% for p16, 21.4% for E-cadherin, 12.5% for DAP kinase and p73, 1.8% for p15, MGMT as well as RARbeta, and 0% for TIMP-3, RASSF1A and hMLH1. We found at least one hypermethylated gene in 80.4% of the primary patient samples, while 33.9% harbored two or more hypermethylated genes. For the first time, we show that p73 may be hypermethylated in MM and thus be involved in the pathogenesis of plasma cell disorders. Hypermethylation of p16 at diagnosis was associated with a poorer prognosis. In patients with plasma cell leukemia, we found frequent simultaneous hypermethylation of p16, E-cadherin and DAP kinase. We conclude that aberrant methylation of tumor suppressor genes is a common event in malignant plasma cell disorders and that there is a correlation between methylation patterns and clinical characteristics in MM patients.

Pneumatosis intestinalis following cytotoxic or immunosuppressive treatment.

Pneumatosis intestinalis (PI) is an uncommon condition characterized by the presence of gas within the bowel wall. We describe 5 cases of PI that occurred after cytotoxic or immunosuppressive treatment for hematological disorders. All patients were neutropenic shortly before or at the time of diagnosis of PI, but did not show specific symptoms. The diagnosis was made by conventional X-ray and confirmed by abdominal computed tomography. Since there were no signs of secondary complications such as peritonitis, ischemia, or perforation, conservative treatment with broad-spectrum antibiotics and parenteral nutrition was initiated. All patients but 1 achieved complete resolution of PI after recovery from myelosuppression. Benign pneumoperitoneum due to PI should be considered in the differential diagnosis of free intra-abdominal air after chemotherapeutic or immunosuppressive therapy. It can be managed successfully by conservative treatment in the absence of secondary complications, if there is recovery of myelopoiesis.

[An unusual etiology of erythrocytosis in a 23-year-old primiparous woman in the 22nd week of pregnancy]. / Ungewöhnliche Ursache einer Erythrozytose bei einer 23-jährigen Erstgebärenden in der 22. Schwangerschaftswoche.

CASE REPORT: A 23-year-old pregnant woman presented with erythrocytosis and a spuriously elevated HbA1c. Family history revealed that her father has been treated with phlebotomies for the last 2 years because of erythrocytosis of unknown cause. An examination of the family members demonstrated that the patient and her father were carriers of the hemoglobin (Hb) variant Hb Andrew-Minneapolis. DISCUSSION: Hb Andrew-Minneapolis belongs to a group of hemoglobin variants with a high oxygen affinity resulting in compensatory erythrocytosis. The carriers of such hemoglobin variants are usually clinically asymptomatic, exercise tolerance appears unimpaired and there is no higher incidence of cardiovascular diseases. There is no clear-cut evidence that a maternal hemoglobinopathy with high oxygen affinity is accompanied by negative consequences for the fetus or a higher abortion rate. CONCLUSION: Hemoglobinopathies with a high oxygen affinity are a rare but important differential diagnosis of polycythemia. Under these circumstances erythrocytosis has to be accepted as the primary mode of compensation and does not require treatment, as long as blood viscosity is kept within tolerable limits. An excessively elevated or lowered HbA1c without a history or symptoms of diabetes should lead to further investigations concerning the possibility of hemoglobinopathy.