Judith Dierlamm

Accelerated and safe expansion of human mesenchymal stromal cells in animal serum‐free medium for transplantation and regenerative medicine

Human bone marrow mesenchymal stromal cells (hMSC) are currently investigated for a variety of therapeutic applications. However, most expansion protocols still use fetal calf serum (FCS) as growth factor supplement which is a potential source of undesired xenogeneic pathogens. We established an expansion protocol for hMSC based on the use of GMP‐produced basic medium LP02 supplemented with 5% of platelet lysate (PL) obtained from human thrombocyte concentrates. Compared to FCS‐supplemented culture conditions, we found a significant increase in both colony forming unit‐fibroblast (CFU‐F) as well as cumulative cell numbers after expansion. This accelerated growth is optimized by pooling of at least 10 thrombocyte concentrates. A minimal requirement is the use of 5% of PL with an optimal platelet concentration of 1.5 × 109/ml, and centrifugation of thawed lysate at high speed. Cells expanded by this protocol meet all criteria for mesenchymal stromal cells (MSCs), e.g. plastic adherence, spindle‐shaped morphology, surface marker expression, lack of hematopoietic markers, and differentiation capability into three mesenchymal lineages. MSC at passage 6 were cytogenetically normal and retained their immune‐privileged potential by suppressing allogeneic reaction of T‐cells. Additionally, gene expression profiles show increased mRNA levels of genes involved in cell cycle and DNA replication and downregulation of developmental and differentiation genes, supporting the observation of increased MSC‐expansion in PL‐supplemented medium. In summary, we have established a GMP‐compatible protocol for safe and accelerated expansion of hMSC to be used in cell and tissue therapy. J. Cell. Physiol. 213: 18–26, 2007.

Essential role of MALT1 protease activity in activated B cell-like diffuse large B-cell lymphoma

A key element for the development of suitable anti-cancer drugs is the identification of cancer-specific enzymatic activities that can be therapeutically targeted. Mucosa-associated lymphoid tissue transformation protein 1 (MALT1) is a proto-oncogene that contributes to tumorigenesis in diffuse large B-cell lymphoma (DLBCL) of the activated B-cell (ABC) subtype, the least curable subtype of DLBCL. Recent data suggest that MALT1 has proteolytic activity, but it is unknown whether this activity is relevant for tumor growth. Here we report that MALT1 is constitutively active in DLBCL lines of the ABC but not the GCB subtype. Inhibition of the MALT1 proteolytic activity led to reduced expression of growth factors and apoptosis inhibitors, and specifically affected the growth and survival of ABC DLBCL lines. These results demonstrate a key role for the proteolytic activity of MALT1 in DLBCL of the ABC subtype, and provide a rationale for the development of pharmacological inhibitors of MALT1 in DLBCL therapy.

Characteristic pattern of chromosomal gains and losses in marginal zone B cell lymphoma detected by comparative genomic hybridization

Marginal zone B cell lymphoma (MZBCL) represents a distinct subtype of B cell non-Hodgkin’s lymphoma, which has been recently recognized and defined as a disease entity. We investigated 25 cases (18 at primary diagnosis and seven during the course of disease) of MZBCL by comparative genomic hybridization (CGH) and compared these results with cytogenetic, fluorescence in situ hybridization (FISH), and Southern blot data. Twenty of the 25 cases (80%) showed gains (total 49) or losses (total 15) of genetic material. In extranodal, nodal, and splenic MZBCL, material of chromosomes 3 (52% of cases), 18 (32%), X (24%), and 1q (16%) was most frequently gained, whereas losses predominantly involved chromosomes 17 (16%) and 9 (12%). High-level amplifications involving the regions 18q21-23 and 18q21-22, respectively, were detected in two cases. Gains of chromosomes 1q and 8q and losses of chromosome 17 or 17p occurred more frequently in relapsed or progressive lymphomas. For all of the frequently affected chromosomes, CGH allowed narrowing of the relevant subregions including 3q21-23, 3q25-29 and 18q21-23. By Southern blot analysis, the BCL2, BCL6, and CMYC proto-oncogenes were found to be a part of the over-represented regions in two cases, one case, and two cases, respectively, with gains involving 18q, 3q or 8q. In 13 cases, CGH revealed chromosomal imbalances which were not detected by cytogenetic analysis but could be confirmed by FISH or Southern blot analysis in all cases investigated. On the other hand, CGH failed to detect trisomy 3, trisomy 18, and deletion 7q in three cases with a low proportion of tumor cells bearing these abnormalities, as shown by interphase FISH. The characteristic pattern of chromosomal gains and losses detected in this study confirms the distinct nature of MZBCL and may point to chromosomal regions involved in the pathogenesis of these neoplasms.

Trisomy 3 in marginal zone B-cell lymphoma: A study based on cytogenetic analysis and fluorescence in situ hybridization

Trisomy 3 represents the most frequent and consistent chromosomal abnormality characterizing the recently defined entity marginal zone B‐cell lymphoma (MZBCL). By cytogenetic analysis and/or fluorescence in situ hybridization (FISH) on interphase nuclei we found an increased copy number of chromosome 3 in 22/36 (61%) successfully analysed cases, including 8/12 cases with extranodal MZBCL, 8/13 cases with nodal MZBCL, and 6/11 patients with splenic MZBCL. Sensitivity of interphase cytogenetics was somewhat higher than that of conventional cytogenetic investigation. Structural chromosomal changes involving at least one chromosome 3 were seen in 11/20 cases with an increased copy number of chromosome 3: +del(3)(p13) was demonstrated in three cases, and was the sole chromosomal abnormality in one of them; +i(3)(q10) was seen in two other patients; and rearrangements involving various breakpoints on the long arm of chromosome 3 were found in the remaining cases. FISH on metaphase spreads confirmed these structural abnormalities and additionally showed two unexpected translocations involving chromosome 3. We conclude that: (1) trisomy 3 occurs in a high proportion of extranodal, nodal and splenic MZBCL; (2) FISH on interphase nuclei is an additional and sensitive tool in detecting an increased copy number of chromosome 3 in MZBCL; (3) additional structural abnormalities involving the long arm of chromosome 3 are frequent but non‐recurrent and are perhaps secondary changes; and (4) abnormalities such as +del(3)(p13) and +i(3)(q10) suggest that genes located on the long arm of chromosome 3 are of particular importance in the pathogenesis of MZBCL.

Successful use of the same slide for consecutive fluorescence in situ hybridization experiments

The feasibility of using the same slide repeatedly for fluorescence in situ hybridization (FISH) experiments was systematically evaluated by applying standard procedures and various combinations of direct‐ and indirect‐labeled probes to slides from patients with hematologic malignancies. Specific and distinct hybridization signals along with weak background signals and chromosome morphology of good to moderate quality could be obtained in up to three experiments performed consecutively on the same slide. Signals related to biotin‐ or digoxigenin‐labeled probes applied in previous hybridizations were still visible with variable intensity, but interpretation problems that may result from this signal noise can be avoided by using adequate probes, detection systems and fluorochromes, and sequence of experiments. Genes Chromosom Cancer 16:261–264 (1996).

High level PSMA expression is associated with early PSA recurrence in surgically treated prostate cancer.

Prostate specific membrane antigen (PSMA) is a suggested target for antibody‐based therapy of prostate cancer potentially involved in the regulation of cell migration. This study was undertaken, to gain more insight on the role of PSMA in early prostate cancer and its distribution in various normal tissues.

Translocations t(11;18)(q21;q21) and t(14;18)(q32;q21) are the main chromosomal abnormalities involving MLT/MALT1 in MALT lymphomas.

The recently discovered MLT/MALT1 gene is fused with the API2 gene in the t(11;18)(q21;q21), which characterizes about one-third of MALT lymphomas. In order to screen for variant translocations and amplifications of MLT/MALT1, we have developed a novel, undirected two-color interphase fluorescence in situ hybridization (FISH) assay with two PAC clones flanking MLT/MALT1. This assay was applied to 108 marginal zone B-cell lymphomas (MZBCLs), including 72 extranodal MALT lymphomas, 17 nodal, and 19 splenic MZBCL. In 19 MALT lymphomas (26%), but in none of the nodal or splenic MZBCL, separated hybridization signals of the MLT/MALT1 flanking probes, were found. Further FISH analyses showed that 12 of these 19 cases displayed the classical t(11;18) and the remaining seven cases revealed the novel t(14;18)(q32;q21), involving the MLT/MALT1 and IGH genes. The frequency at which these translocations occurred varied significantly with the primary location of disease. The t(11;18) was mainly detected in gastrointestinal MALT lymphomas, whereas the t(14;18) occurred in MALT lymphomas of the parotid gland and the conjunctiva. Amplification of MLT/MALT1 was not observed in any of the lymphomas analyzed. We conclude that the translocations t(11;18)(q21;q21) and t(14;18)(q21;q32) represent the main structural aberrations involving MLT/MALT1 in MALT lymphomas, whereas true amplifications of MLT/MALT1 occur rarely in MZBCL.

Risk factors for therapy-related myelodysplastic syndrome and acute myeloid leukemia treated with allogeneic stem cell transplantation

After successful treatment of malignant diseases, therapy-related myelodysplastic syndrome and acute myeloid leukemia have emerged as significant problems. This study shows that allogeneic stem cell transplantation can cure a significant portion of these patients. See related perspective article on page 542. Background After successful treatment of malignant diseases, therapy-related myelodysplastic syndrome and acute myeloid leukemia have emerged as significant problems. Design and Methods The aim of this study was to investigate outcome and risk factors in patients with therapy-related myelodysplastic syndrome or acute myeloid leukemia who underwent allogeneic stem cell transplantation. Between 1981 and 2006, 461 patients with therapy-related myelodysplastic syndrome or acute myeloid, a median age of 40 years and a history of solid tumor (n=163), malignant lymphoma (n=133), or other hematologic diseases (n=57) underwent stem cell transplantation and their data were reported to the European Group for Blood and Marrow Transplantation. Results The cumulative incidence of non-relapse mortality and relapse at 3 years was 37% and 31%, respectively. In a multivariate analysis significant factors for relapse were not being in complete remission at the time of transplantation (p=0.002), abnormal cytogenetics (p=0.005), higher patients’ age (p=0.03) and therapy-related myelodysplastic syndrome (p=0.04), while higher non-relapse mortality was influenced by higher patients’ age. Furthermore, there was a marked reduction in non-relapse mortality per calendar year during the study period (p<0.001). The 3-year relapse-free and overall survival rates were 33% and 35%, respectively. In a multivariate analysis significant higher overall survival rates were seen per calendar year (p<0.001), for younger age (<40 years) and normal cytogenetics (p=0.05). Using age (<40 years), abnormal cytogenetics and not being in complete remission at the time of transplantation as risk factors, three different risk groups with overall survival rates of 62%, 33% and 24% could be easily distinguished. Conclusions Allogeneic stem cell transplantation can cure patients with therapy-related myelodysplastic syndrome and acute myeloid leukemia and has markedly improved over time. Non-complete remission, abnormal cytogenetics and higher patients’ age are the most significant factors predicting survival.

Genetic abnormalities in chronic lymphocytic leukemia and their clinical and prognostic implications

Clonal chromosome abnormalities can be detected in approximately 50% of patients with chronic lymphocytic leukemia (CLL). The most common changes are trisomy 12, followed by structural abnormalities of 13q, 11q, 6q, and 14q. By fluorescence in situ hybridization (FISH), these aberrations can be demonstrated even in cases with insufficient mitotic yield or a normal karyotype. The biologic consequences of trisomy 12 are unknown, but a gene dosage effect is suspected and studies on partial trisomy 12 indicate that the region 12q13 to 12q22 might be of particular pathogenetic importance. Trisomy 12 is strongly associated with atypical lymphocyte morphology and seems to be a secondary event in leukemogenesis, as shown by combined immunophenotyping and interphase FISH. Structural abnormalities of 13q frequently involve hetero- and homozygous deletions of a region in 13q14, distal to the retinoblastoma gene, which may be the site of a tumor suppressor gene. In contrast to a normal karyotype or structural changes of 13q, complex karyotypic abnormalities, high percentage of abnormal metaphases, trisomy 12 and structural changes involving the P53 tumor suppressor gene on 17p13 are adverse prognostic indicators. Cytogenetic and molecular findings provide important diagnostic, clinical, and prognostic information which can contribute to treatment decisions and follow-up of CLL patients.

Genetic abnormalities in marginal zone B-cell lymphoma.

Marginal zone B‐cell lymphoma (MZBCL) including extranodal mucosa‐associated lymphoid tissue (MALT)‐type lymphoma, nodal, and splenic MZBCL represents a distinct subtype of B‐non‐Hodgkins lymphoma. Recently, important progress in the elucidation of the genetic mechanisms underlying the pathogenesis and disease progression of these lymphomas has been made. The API2 gene, an inhibitor of apoptosis, and the novel MLT gene have been found to be altered by the t(11;18)(q21;21), which represents the most frequent structural chromosomal abnormality in extranodal low‐grade MALT lymphoma. Another gene involved in the regulation of apoptosis, the BCL10 gene, has been cloned from a MALT lymphoma cytogenetically characterized by the t(1;14)(p22;q32). Along the same lines, inactivating mutations of the proapoptotic FAS gene have been detected in a relatively high proportion of extranodal MZBCLs. Considering these data and the fact that at least some MALT lymphomas show low levels of apoptosis and seem to escape from FAS‐mediated apoptosis one may speculate that abrogation of apoptosis constitutes a central pathogenetic mechanism in the development of these lymphomas. The pathogenetic role of trisomy 3, the most frequent numerical chromosomal change of MZBCL, is not known. The minimal overrepresented region has been delineated to 3q21‐23 and 3q25‐29 using comparative genomic hybridization. The BCL6 proto‐oncogene, located on 3q27, which is rearranged in some MZBCL and a high proportion of large cell B‐cell lymphomas with extranodal localization, represents one of the candidate genes residing in these critical regions. Copyright