Brigitte Schlegelberger

Inactivating mutations and overexpression of BCL10, a caspase recruitment domain-containing gene, in MALT lymphoma with t(1;14)(p22;q32)

Mucosa-associated lymphoid tissue (MALT) lymphomas most frequently involve the gastrointestinal tract and are the most common subset of extranodal non-Hodgkin lymphoma (NHL). Here we describe overexpression of BCL10 , a novel apoptotic signalling gene that encodes an amino-terminal caspase recruitment domain (CARD; ref. 2), in MALT lymphomas due to the recurrent t(1;14)(p22;q32) (ref. 3). BCL10 cDNAs from t(1;14)-positive MALT tumours contained a variety of mutations, most resulting in truncations either in or carboxy terminal to the CARD. Wild-type BCL10 activated NF-κB but induced apoptosis of MCF7 and 293 cells. CARD-truncation mutants were unable to induce cell death or activate NF-κB, whereas mutants with C-terminal truncations retained NF-κB activation but did not induce apoptosis. Mutant BCL10 overexpression might have a twofold lymphomagenic effect: loss of BCL10 pro-apoptosis may confer a survival advantage to MALT B-cells, and constitutive NF-κB activation may provide both anti-apoptotic and proliferative signals mediated via its transcriptional targets.

Patients with de novo acute myeloid leukaemia and complex karyotype aberrations show a poor prognosis despite intensive treatment: a study of 90 patients

The clinical significance of complex chromosome aberrations for adults with acute myeloid leukaemia (AML) was assessed in 920 patients with de novo AML who were karyotyped and treated within the German AML Cooperative Group (AMLCG) trials. Complex chromosome aberrations were defined as three or more numerical and/or structural chromosome aberrations excluding translocations t(8;21)(q22;q22), t(15;17)(q22;q11–q12) and inv(16)(p13q22). Complex chromosome anomalies were detected in 10% of all cases with a significantly higher incidence in patients  60 years of age (17·8% vs. 7·8%, P < 0·0001). Clinical follow‐up data were available for 90 patients. Forty‐five patients were < 60 years of age and were randomly assigned to double induction therapy with either TAD‐TAD [thioguanine, daunorubicin, cytosine arabinoside (AraC)] or TAD‐HAM (high‐dose AraC, mitoxantrone). Twenty‐one patients achieved complete remission (CR) (47%), 20 patients (44%) were non‐responders and 9% of patients died during aplasia (early death). The median overall survival (OS) was 7 months and the OS rate at 3 years was 12%. Patients receiving TAD‐HAM showed a significantly higher CR rate than patients receiving TAD‐TAD (56% vs. 23%, P = 0·04). Median event‐free survival was less than 1 month in the TAD‐TAD group and 2 months in the TAD‐HAM group, respectively (P = 0·04), with a median OS of 4·5 months vs. 7·6 months (P = 0·13) and an OS after 3 years of 7·6% vs. 19·6%. Forty‐five patients were  60 years of age: 28 of these patient were treated for induction using one or two TAD courses and 17 cases received TAD‐HAM with an age‐adjusted reduction of the AraC dose. The CR rate was 44%, 38% were non‐responders and 18% experienced early death. The median OS was 8 months and the OS rate at 3 years was 6%. In conclusion, complex chromosome aberrations in de novo AML predicted a dismal outcome, even when patients were treated with intensive chemotherapy. Patients under the age of 60 years with complex aberrant karyotypes may benefit from HAM treatment during induction. However, long‐term survival rates are low and alternative treatment strategies for remission induction and consolidation are urgently needed.

Classical and Molecular Cytogenetics of Tumor Cells

Cytogenetic findings are becoming increasingly important for the management of patients with malignant diseases, especially for those with hematologic neoplasias. The detection of aquired somatic mutations may help to establish the diagnosis of a neoplastic disorder and to rule out reactive changes due to toxic injury, vitamin deficiency or infections. Before, however, a chromosome aberration found in tumor cells can be taken as tumorassociated change it should be ruled out by chromosome analysis on PHA-stimulated blood lymphocytes that this chromosome aberration does not represent a constitutional abnormality. It is now clear that certain so-called primary chromosome abnormalities of tumor cells are associated with distinct clinico-histological disease entities. During tumor evolution additional chromosome aberrations appear and may determine the clinical course of the disease. Even these so-called secondary chromosome aberrations are non-randomly distributed throughout the genome. Therefore, cytogenetic studies are essential to make a specified diagnosis, to classify malignant disorders, to characterize the degree of neoplastic progression, to predict the prognosis, to test for remission, and to establish when relapse occurs. Thus, cytogenetic data can be of great help to select the appropriate treatment strategy.

A 3-cM commonly deleted region in 6q21 in leukemias and lymphomas delineated by fluorescence in situ hybridization†

Deletions of the long arm of chromosome 6 (6q) are frequent chromosome aberrations in non‐Hodgkin lymphomas (NHLs) and acute lymphoblastic leukemias (ALLs). It is presumed that one or more tumor suppressor genes are localized on 6q. By means of fluorescence in situ hybridization (FISH), we attempted to detect and delineate deletions of 6q in leukemias and lymphomas. We performed FISH on 148 cases of lymphoma and acute leukemia using a panel of 36 YAC probes distributed from 6q12 to 6q27 and a centromeric probe of chromosome 6 as internal control. Deletions of 6q that included a 7‐cM commonly deleted region in 6q21 were detected in 59 patients who had B‐ and T‐cell low‐grade and high‐grade NHL and ALL. FISH with two YAC probes flanking this region was performed on an additional 97 cases of NHL and leukemia. Deletions in 6q21 were detected in an additional 21 cases. In five cases of high‐grade B‐ and T‐cell NHL and ALL, the deletion breakpoints were located within the commonly deleted region. To define the deletion breakpoints exactly and to narrow this region further, FISH was performed with six additional YAC probes that have been physically localized within this region. A 3‐cM (4–5 Mb) commonly deleted region in 6q21 was delineated. Our study suggests that this commonly deleted region harbors a putative tumor suppressor gene involved in the pathogenesis of both low‐grade and high‐grade NHL and ALL. Genes Chromosomes Cancer 27:52–58, 2000.

The significance of trisomy 8 in de novo acute myeloid leukaemia: the accompanying chromosome aberrations determine the prognosis

Trisomy 8 is the most frequent numerical chromosome aberration in acute myeloid leukaemia (AML). It occurs either as the sole anomaly or together with other clonal chromosome aberrations. We investigated whether accompanying chromosome anomalies influence the clinical outcome in patients with trisomy 8 and de novo AML. Since 1986, in 713 AML cases treated according to the protocols of the German AMLCG trials, chromosome analyses have been successfully performed. The overall incidence of trisomy 8 was 7.6%. Complete clinical follow‐up data were available for 51 patients who were divided into three different categories: group 1: trisomy 8 as the sole cytogenetic anomaly (n = 20); group 2: trisomy 8 in addition to favourable chromosome aberrations (t(8;21)(q22;q22), t(15;17)(q22;q21), inv(16)(p13q22)) (n = 10); and group 3: trisomy 8 accompanied by other anomalies, in most cases of complex type (n = 21). Complete remission (CR) rates were 70%, 90% and 67% for groups 1, 2 and 3, respectively. Event‐free survival (EFS) at 3 years differed significantly between patients with trisomy 8 only (37.5%), patients with trisomy 8 in combination with favourable aberrations (55.0%) and patients with trisomy 8 and other accompanying anomalies, mostly complex chromosome aberrations (9.0%) (group 1 v group 2: P = 0.12; group 1 v group 3: P = 0.005; group 2 v group 3: P = 0.05). In this study patients with +8 as the sole cytogenetic anomaly had an intermediate prognosis, patients with +8 in addition to favourable chromosome aberrations maintained a good clinical outcome, and patients with +8 in combination with other abnormalities showed the worst prognosis.

Frequent deletions of 6q23–24 in B-cell non-Hodgkin's lymphomas detected by fluorescence in situ hybridization

Deletions of the long arm of chromosome 6 (6q) are among the most frequent chromosome aberrations in malignant lymphomas and often occur as secondary changes in addition to typical translocations, such as t(14; 18). Using fluorescence in situ hybridization (FISH) with two YAC probes hybridizing to 6q23–24 and with the centromeric probe D6Z1 as internal control, we studied 31 cases of low‐grade and eight cases of high‐grade B‐cell lymphoma. Deletions of 6q23–24 were detected in 21 patients (56.8%) by FISH, compared to 13 patients (33.3%) by chromosome analysis. Deletions of 6q23–24 were found by FISH in 5 of 13 cases of small lymphocytic lymphoma, in 2 of 3 cases of mantle cell lymphoma, in 10 of 14 cases of t(14; 18) positive low‐grade follicular lymphoma, and in 4 of 8 cases of high‐grade follicular lymphoma. This study shows that deletions of 6q23–24 are more frequent in B‐cell lymphomas than previously suggested and that they can be detected more sensitively by FISH than by chromosome analysis. Contrary to previous reports indicating that the region 6q23–24 is preferentially deleted in low‐grade lymphomas without t(14; 18), our results indicate that deletions of 6q23–24 appear to be common in other pathological subsets of B‐cell lymphoma as well, especially in follicular lymphomas with t(14; 18). Genes Chromosom. Cancer 18:310–313, 1997.

Which compartments are involved in Philadelphia-chromosome positive chronic myeloid leukaemia? An answer at the single cell level by combining May-Grünwald-Giemsa staining and fluorescence in situ hybridization techniques

Chronic myeloid leukaemia (CML) is believed to represent a stem cell disorder involving all three cell lineages. The typical chromosomal aberration, the Philadelphia chromosome, is the translocation (9;22)(q34;q11). Several studies with cytogenetics, fluorescence in situ hybridization (FISH), or polymerase chain reaction have investigated the presence of the t(9;22) in different cell compartments. However, questions still remain. In six cases of CML we combined the standard May‐Grünwald‐Giemsa staining with FISH at the single‐cell level and were able to demonstrate that not only all maturation stages of granulopoiesis, erythropoiesis, and megakaryocytes, but also plasma cells, eosinophils, basophils and monocytes carried the Philadelphia chromosome in 53–98% of samples. Using immunological identification of single cells we were able to demonstrate that the t(9;22) is detectable in 34% of CD3‐positive T lymphocytes, in 32% of CD19‐positive B lymphocytes, and in 82% of CD34‐positive precursor cells. The results give new insight into the biology of CML and may have implications for future therapeutic strategies.

Recurrent chromosome abnormalities in peripheral T-cell lymphomas

Cytogenetic findings in 45 cases of peripheral T-cell lymphomas (PTL) diagnosed according to the updated Kiel classification are reported. Recurrent numerical chromosome aberrations comprised -X, -Y, -13, +X, +3, +5 and +7. Recurrent structural aberrations included t/del(1)(p31-32), t(2;5)(p23;q35), dup(5)(q23q31-32), t/dup(6q), t/del(6q), trisomy 7q, and trisomy 8q, mostly due to i(8)(q10), and changes in 14q11 and 14q32.1, mostly due to inv(14)(q11q32.1), t/del(13)(q14), t(6;7)(q13;q13), and t(13;17)(q11-13;p11). All deletions in 6q involved band 6q21 and all partial trisomies of 7q led to an amplification of band 7q21. Further studies are needed to ascertain whether these cytogenetic findings in PTL are of clinical and prognostic significance.

No evidence for deletions of the NBS1 gene in lymphomas

Patients with Nijmegen breakage syndrome (NBS) have a high risk to develop malignant diseases, most frequently B-cell lymphomas. The NBS gene product, nibrin, is involved in DNA recombination repair, a function shared with known tumor suppressor genes like BRCA1 and BRCA2. This led us to investigate whether NBS acts as tumor suppressor gene in the development of non-Hodgkin lymphomas. Therefore, we performed fluorescence in situ hybridization analysis using a BAC clone containing the entire NBS1 region on eight B-cell and eight T-cell lymphomas, including one B-cell and two T-cell lymphomas with structural abnormalities of 8q. None of the tumors showed a deletion of the NBS1 gene, demonstrating that deletion of the NBS1 gene is not a major cause or a primary event in tumorigenesis of human B- and T-cell lymphomas.

Cell lineage specific involvement in acute promyelocytic leukaemia (APL) using a combination of May-Grünwald-Giemsa staining and fluorescence in situ hybridization techniques for the detection of the translocation t(15;17)(q22;q12)

Acute promyelocytic leukaemia (APL) is strongly associated with the translocation t(15;17) which therefore provides a reliable marker to assess the potential involvement of different cell lineages. Six cases with morphologically, cytogenetically and molecularly proven APL were analysed at diagnosis or relapse by combining fluorescence in situ hybridization (FISH) with standard May‐Grünwald‐Giemsa (MGG) staining at the single cell level on bone marrow and blood smears. With the FICTION technique, combining immunophenotyping with FISH, haemopoietic precursor cells were identified using monoclonal antibodies against CD34, B‐ and T‐lymphocytes could be identified with CD19 and CD3. In addition, HLA‐DR‐positive cells were studied for the presence of t(15;17).