BackgroundMultiple myeloma is a life-threatening disease and despite the introduction of stem cell transplantation and novel agents such as thalidomide, lenalidomide, and bortezomib most patients will relapse and develop chemoresistant disease. Therefore, alternative therapeutic modes for myeloma are needed and cancer-testis antigens such as MAGE-C1/CT7 and MAGE-A3 have been suggested to represent a class of tumor-specific proteins particularly suited for targeted immunotherapies. Surprisingly, the biological role of cancer-testis genes in myeloma remains poorly understood. Design and MethodsWe performed the first investigation of the function of two cancer-testis antigens most commonly expressed in myeloma, MAGE-C1/CT7 and MAGE-A3, using an RNA interferencebased gene silencing model in myeloma cell lines. Functional assays were used to determine changes in proliferation, cell adhesion, chemosensitivity, colony formation, and apoptosis resulting from gene-specific silencing. ResultsWe show that the investigated genes are not involved in regulating cell proliferation or adhesion; however, they play an important role in promoting the survival of myeloma cells. Accordingly, knock-down of MAGE-C1/CT7 and MAGE-A3 led to the induction of apoptosis in the malignant plasma cells and, importantly, both genes were also essential for the survival of clonogenic myeloma precursors. Finally, silencing of cancer-testis genes further improved the response of myeloma cells to conventional therapies. ConclusionsCancer-testis antigens such as MAGE-C1/CT7 and MAGE-A3 play an important role in promoting the survival of myeloma cells and clonogenic precursors by reducing the rate of spontaneous and chemotherapy-induced apoptosis and might, therefore, represent attractive targets for novel myeloma-specific therapies.Key words: cancer-testis antigens, gene function, RNAi, apoptosis, tumor immunology, multiple myeloma, stem cell transplantation.Citation: Atanackovic D, Hildebrandt Y, Jadczak A, Cao Y, Luetkens T, Meyer S, Kobold S, Bartels K, Pabst C, Lajmi N, Gordic M, Stahl T, Zander AR, Bokemeyer C, promote the survival of multiple myeloma cells. Haematologica 2010;95:785-793. doi:10.3324/haematol.2009 This is an open-access paper. © F e r r a t a S t o r t i F o u n d a t i o n Cancer-testis antigens MAGE-C1/CT7 and MAGE-A3 promote the survival of multiple myeloma cells

The online version of this article has a Supplementary Appendix. BackgroundTo date, multiple myeloma remains an incurable malignancy due to the persistence of minimal residual disease in the bone marrow. In this setting, monoclonal antibodies against myelomaspecific cell surface antigens represent a promising therapeutic approach, which is however hampered by a lack of appropriate target structures expressed across all pathogenic myeloma cell populations. We, therefore, investigated functionally relevant immunoreceptors specifically associated with myeloma cells as well as their clonogenic precursors. Design and MethodsPotential target proteins were identified using antibody arrays against phosphorylated immunoreceptors with lysates from myeloma cell lines. CD229 expression was confirmed in primary myeloma cells by reverse transcriptase polymerase chain reaction, western blot, fluorescence-activated cell sorting, and immunohistochemistry. Apoptosis, clonogenic growth, and sensitivity to chemotherapy were determined following short-interfering RNA-mediated downregulation of CD229. Antibody-dependent cellular and complement-dependent cytotoxicity were analyzed using a monoclonal antibody against CD229 to demonstrate the antigen's immunotherapeutic potential. ResultsOur screening assay identified CD229 as the most strongly over-expressed/phosphorylated immunoreceptor in myeloma cell lines. Over-expression was further demonstrated in the CD138-negative population, which has been suggested to represent myeloma precursors, as well as on primary tumor cells from myeloma patients. Accordingly, CD229 staining of patients' bone marrow samples enabled the identification of myeloma cells by flow cytometry and immunohistochemistry. Down-regulation of CD229 led to a decreased number of viable myeloma cells and clonal myeloma colonies, and enhanced the anti-tumor activity of conventional chemotherapeutics. Targeting CD229 with a monoclonal antibody resulted in complement-and cell-mediated lysis of myeloma cells. ConclusionsOur results demonstrate that the immunoreceptor CD229 is specifically over-expressed on myeloma cells including their clonogenic precursors and contributes to their malignant phenotype. Monoclonal antibodies against this protein may represent a promising diagnostic and immunotherapeutic instrument in this disease. Haematologica 2011;96(10):1512-1520. doi:10.3324/haematol.2010 Surface molecule CD229 as a novel target for the diagnosis and treatment of multiple myeloma

The aim of this study is to describe the prenatal diagnosis and perinatal outcomes in fetuses with agenesis of the ductus venosus (ADV) and to perform a systematic review of literature. Methods: Retrospective descriptive study of all cases with prenatal diagnosis of ADV, occurring between 2011 and 2018 in three tertiary centres. We reviewed patient data of all ADV cases. We also conducted literature search of ADV in MEDLINE, PUBMED, and SCIELO, data bases. Series with 10 or more cases were included. Results: Ten articles were reviewed and our cases were included in the analysis. A total of 283 cases of ADV were included, 36 ours and 247 previously published. In our series mean maternal age was 34 years (16-43 y) and mean gestational age at diagnosis was 17 weeks (11-37 w). ADV was diagnosed at 11-14 screening in 17 patients (47%), 11 with increased nuchal translucency. 19 (53%) were diagnosed at the second-trimester ultrasound, 17 had a major abnormality associated. Shunt was intrahepatic in 22 patients, extrahepatic in 14. In the extra hepatic group, 11 communicated directly to inferior vena cava, 2 to right atrium, and 1 to the iliac vein. Chromosome analysis was performed in 19 of 36 cases; 12 of the fetuses were aneuploid (9 Turner syndrome and 3 Down's syndrome). As for perinatal results, of 36 patients 9 died; 30 had some anomaly (aneuploidy or major abnormality associated), and 6 were healthy. Conclusions: ADV is a rare condition and is associated with aneuploidies, especially Turner Syndrome. It is highly associated with major abnormalities; especially cardiac malformations and it is associated with poor perinatal outcomes (especially extrahepatic). Systematic review, including our series, shows similar perinatal outcomes. P20.07Review of ultrasound markers in diagnosis of total and partial anomalous venous connection in mid and late gestation

The cancer-testis (CT) class of tumor antigens is a group of proteins, the expression of which is characteristically restricted to cancer and the human germline. Based on their immunogenicity and restricted tissue expression, CT antigens seem ideal targets for immunotherapeutic approaches. This is particularly the case in multiple myeloma (MM) where these tumor antigens are frequently and strongly expressed. Unfortunately, very little is known about the biological function of CT antigens which seems surprising given the strong impact of CT antigen expression on the prognosis of a variety of malignancies including MM. We examined for the first time the impact of the expression of two CT antigens frequently found in MM, MAGE-C1/CT7 and MAGE-A3, on the biology of the disease conducting knock-down experiments using RNA interference (RNAi) technology. As read-out assays, Western Blots as well as a large variety of tests measuring proliferation, cytotoxicity, cell adhesion, and clonogenic growth were performed. Transfecting myeloma cell line MOLP-8 with RNAi specific for MAGE-C1/CT7 and MAGE-A3 we observed a down-regulation of CT antigen protein expression by 80% and 70%, respectively. Importantly, transfection with MAGE-A3 RNA stealth led to a specific decrease in MAGE-A3 expression while the protein expression of other MAGE genes (MAGE-A4, MAGE-C1/CT7, MAGE-C2/CT10) and non-MAGE CT antigens such as Ropporin-1 was not affected. The same was true for knock-down experiments targeting MAGE-C1/CT7 with the exception of a slight decrease in expression of the highly homologous gene MAGE-C2/CT10. Comparing myeloma cell lines treated with gene-specific versus nonsense RNA stealth, we found that knocking down MAGE-C1/CT7 and MAGE-A3 led to the induction of apoptosis in in about 70% of cells at 72h post transfection as indicated by MTT assays, LDH release assays and Annexin V expression analyzed by flow cytometry. No such effect was observed when transfection was performed with nonsense RNAi (p<0.01). Importantly, CD138-negative myeloma precursors, which also expressed the CT antigens investigated, were also affected by downregulation of CT antigen expression as indicated by an 48% decrease in clonogenic growth (p<0.01). Applying chemotherapy with melphalan to the cell cultures we observed that knock-down of MAGE-C1/CT7 or MAGE-A3 increased the chemosensitivity of MM cell lines by 67% (p<0.01). Importantly, knock-down of MAGE-C1/CT7 or MAGE-A3 specifically affected cell survival and did not alter the proliferative capacity of myeloma cells or cell adhesion. In conclusion, we show here for the first time that CT antigens MAGE-C1/CT7 and MAGE-A3, which are also expressed in the majority of patient myeloma samples, are important for the survival of myeloma cell lines and clonogenic myeloma precursors. In addition, the expression of these CT antigens most likely contributes to promoting resistance to chemotherapy in multiple myeloma. Targeting CT antigens expressed by myeloma cells, either by applying CT antigen-specifric immunotherapy or other targeted therapies, might significantly improve myeloma therapy, i.e. by eliminating chemotherapy-resistant clones.

BACKGROUND AND OBJECTIVES: The majority of patients with multiple myeloma (MM) will eventually relapse and succumb to their disease even after high dose chemotherapy and autologous stem cell transplantation, possibly due to the persistence of BM-residing myeloma stem cells. Therefore, new treatment strategies incorporating new therapeutic targets - ideally to be expressed by the bulk of end-stage myeloma cells and their dormant progenitors - are needed to improve the outcome of myeloma patients. DESIGN AND METHODS: We screened myeloma cell lines for the presence of a large number of immunoreceptors and verified expression of potential target molecules on cell lines and patient samples. The function of the respective proteins was evaluated using gene knockdown and their potential as targets for antibody therapies was investigated using in vitro cytotoxicity assays. RESULTS: Of all immunoreceptors analyzed, SLAM family member CD229 showed the strongest expression and was also found on myeloma precursors. Primary myeloma cells of myeloma patients uniformly evidenced CD229 surface expression while the molecule was absent from most healthy human tissues. Flow cytometric analysis of CD229 expression facilitated the detection of myeloma cells in the bone marrow. CD229 seemed to promote the survival of myeloma cells while CD229 silencing increased their susceptibility towards chemotherapy. Importantly, targeting CD229 with a monoclonal antibody resulted in specific lysis of the tumor cells. CONCLUSIONS: These results suggest that CD229 might represent an attractive diagnostic and therapeutic target for the treatment of MM. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2450.