Acquired Isolated Factor VII Deficiency in Plasma Cell Dyscrasias: A Brief Presentation of Two Plasma-Cell-Leukemia-Related Cases and Review of Literature.

Acquired isolated factor VII (FVII) deficiency is a rare but important discovery in patients with plasma cell disorders with significant therapeutic and prognostic implications. The present analysis and review of cases reported in the literature is intended to highlight disease-related characteristics associated with this rare clotting defect, clinical manifestations and outcome, and potential underlying mechanisms, and to provide guidance on how to manage these patients in terms of prophylactic and therapeutic measures. The discovery of acquired FVII deficiency in a patient with multiple myeloma (MM) or monoclonal gammopathy of uncertain significance (MGUS) should prompt an evaluation for AL amyloidosis, particularly for amyloid hepatosplenic involvement, whenever not previously documented. Acquired FVII deficiency in patients with MM and AL amyloidosis is frequently associated with severe bleeding diathesis, also related to a number of concomitant predisposing factors, adversely affecting the outcome. The prompt institution of a rapidly acting therapy is crucial to prevent severe bleeding complications and positively impact outcome. Recombinant activated factor VII (rVIIa) may represent a useful supportive care measure, both in treating active bleeding and in the peri-procedural setting. However, further clinical experience is needed to optimize the therapeutic management of this rare disorder.

Analysis of the role of intratecal liposomal cytarabine in the prophylaxis and treatment of central nervous system lymphomatosis: The Balearic Lymphoma Group experience.

Central nervous system (CNS) lymphomatosis is a fatal complication of aggressive non-Hodgkin lymphoma (NHL). In lymphoblastic or Burkitt lymphoma, without specific CNS prophylaxis the risk of CNS relapse is 20-30%. DLBCL has a lower risk of relapse (around 5%) but several factors increase its incidence. There is no consensus or trials to conclude which is the best CNS prophylaxis. Best results seem to be associated with the use of intravenous (iv) high-dose methotrexate (HDMTX) but with a significant toxicity. Other options are the administration of intrathecal (IT) MTX, cytarabine or liposomal cytarabine (ITLC). Our aim is to analyze the experience of the centers of the Balearic Lymphoma Group (BLG) about the toxicity and efficacy of ITLC in the prophylaxis and therapy of CNS lymphomatosis. We retrospectively reviewed cases from 2005 to 2015 (n = 58) treated with ITLC. Our toxicity results were: 33% headache, 20% neurological deficits, 11% nausea, 9% dizziness, 4% vomiting, 4% fever, 2% transient blindness and 2% photophobia. In the prophylactic cohort (n = 26) with a median follow-up of 55 months (17-81) only 3 CNS relapses (11%) were observed (testicular DLBCL, Burkitt and plasmablastic lymphoma, with a cumulative incidence of 8%, 14% and 20% respectively). In the treatment cohort (n = 32), CSF complete clearance was obtained in 77% cases. Median OS was 6 months (0-16). Death causes were lymphoma progression (19 patients, 79%), treatment toxicity (2 patients) and non-related (3 patients, 12%). Toxicity profile was good especially when concomitant dexamethasone was administered. In the prophylactic cohort the incidence of CNS relapse in DLBCL group was similar to previously reported for HDMTX and much better than IT MTX. A high number of ITLC injections was associated with better rates of CSF clearance, clinical responses, PFS and lower relapses. Survival is still poor in CNS lymphomatosis and new therapeutic approaches are still needed.

Acute lymphoblastic leukemia with e1a3 BCR/ABL fusion protein. A report of two cases.

B Acute Lymphoblastic leukemia (B-ALL) with Philadelphia chromosome (Ph') is a neoplasm of lymphoblast committed to the B cell lineage. The clinical presentation of B-ALL Ph'+ is similar to B-ALL, but is more common in adults than in children. The e1a3 rare variant is produced by the fusion of BCR exon 1 to ABL exon 3. The presence of this translocation has been associated with good disease outcome for chronic myeloid leukemia in a very small series of only 5 cases; there is no such evidence for B-ALL. We report two new cases of B-ALL Ph+ with the rare e1a3 fusion transcript. The e1a3 and e1a2 (p190) transcripts have been reported to have a similar molecular weight and probably a similar clinical profile, thus in these cases the presence of e1a3 was associated with extramedullary infiltration and disease acceleration.

Spindle cell tumor with EWS-WT1 transcript and a favorable clinical course: a variant of DSCT, a variant of leiomyosarcoma, or a new entity? Report of 2 pediatric cases.

We report 2 intra-abdominal tumors originally diagnosed as leiomyosarcomas, occurring in adolescents, one as a second malignancy after a Hodgkin lymphoma. Both tumors exhibited unusual morphologic features characterized by spindle cells arranged in sheets or in fascicles, devoid of the typical desmoplastic stroma. Cytokeratins and mesenchymal markers, including smooth muscle actin, desmin, and muscle specific actin, were coexpressed in the tumor cells, whereas EMA was negative. Reverse transcription-polymerase chain reaction analysis showed an EWS-WT1 fusion transcript. Both patients are alive and in complete remission at 3 and 13 years after diagnosis, respectively. These tumors raise a variety of diagnostic possibilities. They could represent intra-abdominal desmoplastic small round cell tumor, with histologic features of epithelioid leiomyosarcoma or an unusual subtype of leiomyosarcoma with an EWS-WT1 transcript. Alternatively, they could represent an unrecognized subgroup of tumors with spindle cell morphology, bearing the same translocation as desmoplastic small round cell tumor, but characterized by a more favorable clinical course.

Defining the gene expression signature of rhabdomyosarcoma by meta-analysis.

BACKGROUND: Rhabdomyosarcoma is a highly malignant soft tissue sarcoma in childhood and arises as a consequence of regulatory disruption of the growth and differentiation pathways of myogenic precursor cells. The pathogenic pathways involved in this tumor are mostly unknown and therefore a better characterization of RMS gene expression profile would represent a considerable advance. The availability of publicly available gene expression datasets have opened up new challenges especially for the integration of data generated by different research groups and different array platforms with the purpose of obtaining new insights on the biological process investigated. RESULTS: In this work we performed a meta-analysis on four microarray and two SAGE datasets of gene expression data on RMS in order to evaluate the degree of agreement of the biological results obtained by these different studies and to identify common regulatory pathways that could be responsible of tumor growth. Regulatory pathways and biological processes significantly enriched has been investigated and a list of differentially meta-profiles have been identified as possible candidate of aggressiveness of RMS. CONCLUSION: Our results point to a general down regulation of the energy production pathways, suggesting a hypoxic physiology for RMS cells. This result agrees with the high malignancy of RMS and with its resistance to most of the therapeutic treatments. In this context, different isoforms of the ANT gene have been consistently identified for the first time as differentially expressed in RMS. This gene is involved in anti-apoptotic processes when cells grow in low oxygen conditions. These new insights in the biological processes responsible of RMS growth and development demonstrate the effective advantage of the use of integrated analysis of gene expression studies.

Plakoglobin is differentially expressed in alveolar and embryonal rhabdomyosarcoma and is regulated by DNA methylation and histone acetylation.

Plakoglobin (gamma-catenin) and beta-catenin are pivotal components of cell-cell adherent junctions that link cadherin receptors to the actin cytoskeleton. Whereas beta-catenin overexpression induces cell proliferation and tumor formation, plakoglobin induces tumor suppressor activity. We investigated the expression of plakoglobin in alveolar (ARMS) and embryonal (ERMS) rhabdomyosarcoma (RMS) cell lines and tumors, and found that plakoglobin is present both in the cytoplasm and in the nucleus of ERMS cells, whereas it is absent or detectable at extremely low levels in ARMS. As gene silencing can be mediated by methylation and/or deacetylation of promoter regions, we assessed the effects of the DNA demethylating agent 5-Aza-2'-deoxycytidine (5AzadC) and of the histone deacetylase inhibitor Trichostatin A (TSA), and obtained restoration of plakoglobin expression in ARMS cells cultivated in the presence of 5AzadC and TSA. By methylation-specific PCR, ARMS cells were shown to contain methylated CpG dinucleotides in CpG islands located around the transcriptional start site of one or both alleles, whereas ERMS cells did not. Furthermore, we demonstrated that promoter regions (P1-P3) of plakoglobin gene were associated with hypoacetylated H4 histone in ARMS cells RH4, suggesting that aberrant DNA methylation of the 5' CpG island and histone deacetylation play key roles in silencing the plakoglobin gene. These results demonstrate that plakoglobin is differentially expressed in ARMS and ERMS and that its expression depends on the methylation and acetylation status of the gene.

Results of a prospective minimal disseminated disease study in human rhabdomyosarcoma using three different molecular markers.

BACKGROUND: Rhabdomyosarcoma (RMS) has 2 major histologic subtypes: alveolar (ARMS) and embryonal (ERMS). ARMS is more aggressive and prone to distant tumor dissemination, whereas ERMS tends to expand and recur locally. Little information is available on bone marrow involvement by RMS. METHODS: We determined the sensitivity and specificity of MyoD1, myogenin, and PAX-FKHR transcripts as RMS markers and used them to study prospectively by reverse-transcriptase polymerase chain reaction (RT-PCR) a series of consecutive unselected RMS patients enrolled in the Italian Association of Pediatric Hematology and Oncology national trial. Prevalence of minimal disseminated disease (MDD) and its response kinetics to chemotherapy were assessed. RESULTS: MyoD1 and myogenin were specifically associated with RMS, independently of histologic subtype, whereas PAX3/7-FKHR transcripts were expressed only in ARMS. Sensitivity was higher for MyoD1 compared with myogenin and PAX-FKHR. Out of a cohort of 40 patients, MDD positivity was limited to ARMS, with the sole exception of 1 ERMS. Prevalence of MDD positivity increased when a real-time polymerase chain reaction approach was used on a subgroup of patients. RT-PCR was more sensitive than microscopic examination of bone marrow biopsies. The study of the response kinetics of MDD showed that in approximately half of the cases, bone marrow was cleared of disease after 1 cycle of chemotherapy. CONCLUSIONS: MyoD1 and myogenin transcripts can be used as tumor markers for MDD assessment in virtually all RMS cases, whereas PAX-FKHR is specific for ARMS. Sensitivity of RT-PCR methods was superior compared with standard morphologic assays. Our study suggests that bone marrow involvement is more common in ARMS compared with ERMS, and that MDD can be often cleared by the initial chemotherapy cycles.

Endothelin-3 production by human rhabdomyosarcoma: a possible new marker with a paracrine role.

Several autocrine and paracrine growth factor circuits have been found in human rhabdomyosarcoma cells. In this study we show that endothelin-3 (ET-3), a vasoactive peptide, is produced by human rhabdomyosarcoma cell lines, whereas it is not expressed by human sarcoma cell lines of non-muscle origin. We did not find evidence of a significant autocrine loop; nevertheless ET-3 produced by rhabdomyosarcoma cells can act as a paracrine factor, since it promotes migration of endothelial cells. Moreover ET-3 is present in plasma of mice bearing xenografts of human rhabdomyosarcoma cells, and may be potential new marker of the human rhabdomyosarcoma to be studied further.

Gene expression profiling identifies potential relevant genes in alveolar rhabdomyosarcoma pathogenesis and discriminates PAX3-FKHR positive and negative tumors.

We analyzed the expression signatures of 14 tumor biopsies from children affected by alveolar rhabdomyosarcoma (ARMS) to identify genes correlating to biological features of this tumor. Seven of these patients were positive for the PAX3-FKHR fusion gene and 7 were negative. We used a cDNA platform containing a large majority of probes derived from muscle tissues. The comparison of transcription profiles of tumor samples with fetal skeletal muscle identified 171 differentially expressed genes common to all ARMS patients. The functional classification analysis of altered genes led to the identification of a group of transcripts (LGALS1, BIN1) that may be relevant for the tumorigenic processes. The muscle-specific microarray platform was able to distinguish PAX3-FKHR positive and negative ARMS through the expression pattern of a limited number of genes (RAC1, CFL1, CCND1, IGFBP2) that might be biologically relevant for the different clinical behavior and aggressiveness of the 2 ARMS subtypes. Expression levels for selected candidate genes were validated by quantitative real-time reverse-transcription PCR.

The influence of iontophoresis on acyclovir transport and accumulation in rabbit ear skin.

PURPOSE: The aim of this work was to explore the effect of iontophoresis on acyclovir (ACV) accumulation and permeation. In particular, the objectives were to check the efficacy of the transport mechanisms, electromigration and electrosmosis, on drug accumulation. METHODS: Permeation experiments were performed in vitro, using rabbit ear skin as barrier, from donor solutions at pH 3.0, 5.8, and 7.4. At the end of the experiments, drug accumulation in epidermis and dermis was measured. Anodal and cathodal iontophoresis were applied at pH 3.0, whereas only anodal iontophoresis was used at pH 5.8 (current densities 0.06--0.50 mA/cm(2)) and 7.4. RESULTS: Cathodal iontophoresis was more efficient than anodal iontophoresis on ACV permeation across the skin at pH 3.0. At pH 5.8, ACV flux and accumulation increased with current density during anodal iontophoresis. At pH 7.4, anodal iontophoresis produced a remarkable increase of flux and a modest increase of accumulation. Overall, anodal flux increased as the pH of the donor solution was increased as a result of the increase of the skin net negative charge. CONCLUSIONS: From the results obtained in the present work, it can be concluded that iontophoresis application increases ACV flux and, to a limited extent, accumulation in the skin.

Inhibition of connective tissue growth factor (CTGF/CCN2) expression decreases the survival and myogenic differentiation of human rhabdomyosarcoma cells.

Connective tissue growth factor (CTGF/CCN2), a cysteine-rich protein of the CCN (Cyr61, CTGF, Nov) family of genes, emerged from a microarray screen of genes expressed by human rhabdomyosarcoma cells. Rhabdomyosarcoma is a soft tissue sarcoma of childhood deriving from skeletal muscle cells. In this study, we investigated the role of CTGF in rhabdomyosarcoma. Human rhabdomyosarcoma cells of the embryonal (RD/12, RD/18, CCA) and the alveolar histotype (RMZ-RC2, SJ-RH4, SJ-RH30), rhabdomyosarcoma tumor specimens, and normal skeletal muscle cells expressed CTGF. To determine the function of CTGF, we treated rhabdomyosarcoma cells with a CTGF antisense oligonucleotide or with a CTGF small interfering RNA (siRNA). Both treatments inhibited rhabdomyosarcoma cell growth, suggesting the existence of a new autocrine loop based on CTGF. CTGF antisense oligonucleotide-mediated growth inhibition was specifically due to a significant increase in apoptosis, whereas cell proliferation was unchanged. CTGF antisense oligonucleotide induced a strong decrease in the level of myogenic differentiation of rhabdomyosarcoma cells, whereas the addition of recombinant CTGF significantly increased the proportion of myosin-positive cells. CTGF emerges as a survival and differentiation factor and could be a new therapeutic target in human rhabdomyosarcoma.