Potential of chromosomal and matrix-based comparative genomic hybridization for molecular diagnostics in lymphomas.
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Antineoplastic Combined Chemotherapy Protocols↗
Biomedical subjects
H Döhner
Publications and source records attributed to H Döhner.
Targeted bone marrow irradiation in the conditioning of high-risk leukaemia prior to stem cell transplantation.
Disease recurrence following stem cell transplantation (SCT) remains a major problem. Despite the sensitivity of leukaemias to chemotherapy and irradiation, conventional conditioning before SCT is limited by significant organ toxicity. Targeted irradiation of bone marrow and spleen by radioimmunotherapy may provide considerable dose escalation, with limited toxicity to non-target organs. In this study, 27 patients with high-risk or relapsing leukaemia were treated with rhenium-188-labelled CD66a,b,c,e radioimmunoconjugates (188Re-mAb) specific for normal bone marrow in addition to conventional conditioning with high-dose chemotherapy and 12 Gy total body irradiation prior to SCT. A mean activity of 10.2+/-2.1 (range 6.9-15.8) GBq 188Re-mAb was administered intravenously. Acute side-effects were assessed according to the CTC classification and patient outcome was determined. Mean radiation doses (Gy; range in parentheses) to relevant organs and whole body were as follows: 13.1 (6.5-22) to bone marrow, 11.6 (1.7-31.1) to spleen, 5.0 (2.0-11.7) to liver, 7.0 (2.3-11.6) to kidneys, 0.7 (0.3-1.3) to lungs and 1.4 (0.8-2.1) to the whole body. Stem cells engrafted in all patients within 9-18 days post SCT. Acute organ toxicity of grade II or less was observed. During follow-up for 25.4+/-5.3 (range 18-34) months, 4/27 (15%) patients died from relapse, and 9/27 (33%) from transplantation-related complications. Fourteen patients (52%) are still alive and in ongoing complete clinical remission. Radioimmunotherapy with the bone marrow-seeking 188Re-labelled CD66 mAb can double the dose to bone marrow and spleen without undue extramedullary acute organ toxicity, when given in addition to high-dose chemotherapy and 12 Gy TBI before allogeneic SCT. This intensified conditioning regimen may reduce the relapse rate of high-risk leukaemia.
Adolescent↗
[Information and counseling--a contribution to health promotion and health care].
Information and counselling are essential prerequisites for self-determination in old age. For the offers available the following differentiations are made: general oral, written or electronic information on supporting services, individual personal information and counselling, individual accompanying counselling and individual written information on the own health or disease situation. Models of good practice in Hamburg are presented as examples.
Activities of Daily Living↗
Mutation analysis of the origin recognition complex subunit 5 (ORC5L) gene in adult patients with myeloid leukemias exhibiting deletions of chromosome band 7q22.
The ORC5L gene encoding a subunit of the human origin recognition complex (ORC) maps to chromosome band 7q22, a region frequently deleted in adult acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Because of its localization within a region that is commonly deleted in patients with myeloid malignancies and because of the implication of its protein product in cell cycle control (DNA replication) and regulation of gene expression (transcriptional silencing), ORC5L appeared to be a candidate tumor suppressor gene for myeloid disorders associated with 7q22 deletions. Polymerase chain reaction amplification and sequencing analysis of the coding region of the remaining ORC5L allele has not revealed any mutations in nine patients with AML or MDS exhibiting 7q22 deletions. Allelic expression analysis indicates that ORC5L is not imprinted. These data suggest that ORC5L does not function as a tumor suppressor in patients with myeloid neoplasms.
Acute Disease↗
Fatal outcome in a patient developing Epstein-Barr virus-associated lymphoproliferative disorder (EBV-LPD) without measurable disease.
A 51-year-old female patient in the first chronic phase of CML received an allogeneic PBSCT from a matched unrelated donor. The transplant was manipulated by CD34+ cell selection. On day +193 after transplantation the patient was readmitted to the hospital with recurrent fever of unknown origin and cough. Clinical, radiographic and sonographic evaluation revealed no characteristic findings besides a mild splenomegaly. Screening for EBV, CMV, RSV and HSV did not indicate an active infection. On day +203 the patient developed generalized seizures, respiratory failure and died within 24 h in multiorgan failure. The macroscopic postmortem was still not enlightening; the histological examination however, demonstrated diffuse organ infiltration by monoclonal lymphoblastoid cells due to EBV-LPD.
Fatal Outcome↗
Quantification of CBFbeta/MYH11 fusion transcript by real time RT-PCR in patients with INV(16) acute myeloid leukemia.
Amplification of the CBFbeta/MYH11 fusion transcript by a qualitative reverse transcription-polymerase chain reaction (RT-PCR) has been used to detect minimal residual disease (MRD) and assess the risk for disease relapse in inv(16)(p13q22) acute myeloid leukemia (AML). This strategy has, however, produced conflicting results and because of an uncertain predictive value, its use in the clinical setting cannot be recommended. The objective of the current study was to evaluate if quantification by Real Time RT-PCR could be useful to determine levels of CBFbeta/MYH11 fusion transcripts predictive of clinical outcome in inv(16)(p13q22) AML at diagnosis or during remission. Bone marrow (BM) samples from 16 patients with inv(16) AML enrolled on a German multicenter trial (AML HD93) were analyzed for levels of CBFbeta/MYH11 fusion transcripts by Real Time RT-PCR at diagnosis (n= 14), during remission (n= 10) and at relapse (n=6). The CBFbeta/MYH11 transcript copy number in each sample was normalized to copies of an internal control housekeeping transcript (ie 18S). The copy number measured at diagnosis or relapse were 3 to 4 log higher that those measured during remission, following completion of induction treatment. A high CBFbeta/MYH11 transcript copy number at diagnosis had a significant correlation with a high percentage of BM blasts (Spearman's coefficient = -0.66; P= 0.03), and a borderline correlation with a short complete remission (CR) duration (Spearman's coefficient = -0.51; P= 0.07). No difference in levels of CBFbeta/MYH11 fusion transcripts measured during intensification therapy was found between patients destined to relapse and those who continued in CCR (P= 0.75). Following completion of the entire chemotherapy program, patients that during CR showed a CBFbeta/MYH11 fusion transcript copy number >10 had a significantly shorter CR duration (P= 0.002) and higher risk for disease relapse (P= 0.05) than patients with a CBFbeta/MYH11 fusion transcript copy number <10. The results of the current study, therefore, suggest that it is possible to determine in remission samples a threshold of CBFbeta/MYH11 transcript copy number above which relapse occurs and below which continuous CR is likely.
Adolescent↗
Fludarabine plus cyclophosphamide is an efficient treatment for advanced chronic lymphocytic leukaemia (CLL): results of a phase II study of the German CLL Study Group.
The efficacy and toxicity of a combination of fludarabine and cyclophosphamide (FC) was evaluated in patients with B-cell chronic lymphocytic leukaemia (CLL). Between April 1997 and July 1998, 36 patients with CLL (median age 59 years) received a regimen that consisted of fludarabine 30 mg/m(2) in a 30-min IV infusion, d 1-3, and cyclophosphamide 250 mg/m(2) in a 30-min IV infusion on d 1-3. Cycles were repeated every 28 d. Twenty-one patients had received between one and three different treatment regimens prior to the study, while 15 patients had received no prior therapy. The median Eastern Cooperative Oncology Group performance score was 1. One patient was at Binet stage A, 18 were stage B and 17 patients were stage C. Objective responses, assessed according to the revised guidelines of the National Cancer Institute-sponsored Working Group, were recorded in 29 out of 32 assessable patients (90.6%). Twenty-four partial remissions and five complete remissions were observed. Two patients showed no change and one patient showed disease progression. At February 2000, three of the responders had relapsed. Severe neutropenia, anaemia and thrombocytopenia (Common Toxicity Criteria grade 3 and 4) were observed in 25, six and six patients (69.4%, 16.7% and 16.7%) respectively. Other side-effects were uncommon. No treatment-related deaths and no grade 3 or 4 infections occurred. We conclude that the combination of fludarabine and cyclophosphamide showed significant activity in patients with CLL. Myelosuppression was the major side-effect. These results warrant further study on the FC combination in randomized trials.
Adult↗
Chronic lymphocytic leukemia: case-based session.
Drs. Hartmut Döhner, Michael J. Keating, Kanti R. Rai and Emili Montserrat form the panel to review chronic lymphocytic leukemia (CLL) while focusing on the clinical features of a particular patient. The pace of progress in CLL has accelerated in the past decade. The pathophysiological nature of this disease, as had been known in the past, was based largely on the intuitive and empiric notions of two leaders in hematology, William Dameshek and David Galton. Now the works of a new generation of leaders are providing us with the scientific explanations of why CLL is a heterogeneous disease, perhaps consisting of at least two separate entities. In one form of CLL, the leukemic lymphocytes have a surface immunoglobulin (Ig) variable region gene that has undergone somatic mutations, with tell-tale markers suggesting that these cells had previously traversed the germinal centers. Such patients have a distinctly superior prognosis than their counterparts whose leukemic lymphocytes IgV genes have no mutations (these are indeed immunologically naive cells), who have a worse prognosis. The introduction of fluorescence in situ hybridization (FISH) technique has provided us with new insights into the diverse chromosomal abnormalities that can occur in CLL, and which have significant impact on the clinical behavior and prognosis of patients with this disease. Major advances in therapeutics of CLL also have occurred during the past decade. Two monoclonal antibodies, Campath-1H (anti-CD52) and rituximab (anti-CD20), and one nucleoside analogue, fludarabine, have emerged as three agents of most promise in the front-line treatment of this disease. Studies currently in progress reflect our attempts to find the most effective manner of combining these agents to improve the overall survival statistics for CLL patients. As in many other hematological malignancies, high dose chemotherapy followed by autologous or HLA-compatible allogeneic stem cells rescue strategies are under study as a salvage treatment for a relatively younger age group of CLL patients with poor prognosis characteristics.
Cytogenetic Analysis↗
Molecular aspects of B-cell lymphomas of the gastrointestinal tract.
The B-cell lymphomas of the gastrointestinal (GI) tract have represented a field of extensive research ever since a close association was shown with such chronic inflammatory processes as Helicobacter pylori infection. Evidence suggested that the mucosa-associated lymphoid tissue induced by inflammation and autoimmune processes is the environment that gives rise to the small-cell lymphomas of the GI tract (eg, extranodal marginal zone B-cell lymphoma according to Revised European-American Classification of Lymphoid Neoplasms and the World Health Organization Classification of Neoplastic Diseases of the Hematopoietic and Lymphoid Tissue). The small B-cell lymphoma may then progress to highly malignant variants. The B-cell lymphomas of the GI tract may present a stepwise model for lymphomagenesis and progression. This review covers molecular biology and molecular cytogenetic aspects that lead to new insights into the biology of GI lymphomas and potential prognostic factors.
Cell Transformation, Neoplastic↗
Genomic aberrations and survival in chronic lymphocytic leukemia.
BACKGROUND: Fluorescence in situ hybridization has improved the detection of genomic aberrations in chronic lymphocytic leukemia. We used this method to identify chromosomal abnormalities in patients with chronic lymphocytic leukemia and assessed their prognostic implications. METHODS: Mononuclear cells from the blood of 325 patients with chronic lymphocytic leukemia were analyzed by fluorescence in situ hybridization for deletions in chromosome bands 6q21, 11q22-23, 13q14, and 17p13; trisomy of bands 3q26, 8q24, and 12q13; and translocations involving band 14q32. Molecular cytogenetic data were correlated with clinical findings. RESULTS: Chromosomal aberrations were detected in 268 of 325 cases (82 percent). The most frequent changes were a deletion in 13q (55 percent), a deletion in 11q (18 percent), trisomy of 12q (16 percent), a deletion in 17p (7 percent), and a deletion in 6q (7 percent). Five categories were defined with a statistical model: 17p deletion, 11q deletion, 12q trisomy, normal karyotype, and 13q deletion as the sole abnormality; the median survival times for patients in these groups were 32, 79, 114, 111, and 133 months, respectively. Patients in the 17p- and 11q-deletion groups had more advanced disease than those in the other three groups. Patients with 17p deletions had the shortest median treatment-free interval (9 months), and those with 13q deletions had the longest (92 months). In multivariate analysis, the presence or absence of a 17p deletion, the presence or absence of an 11q deletion, age, Binet stage, the serum lactate dehydrogenase level, and the white-cell count gave significant prognostic information. CONCLUSIONS: Genomic aberrations in chronic lymphocytic leukemia are important independent predictors of disease progression and survival. These findings have implications for the design of risk-adapted treatment strategies.
Adult↗
BCMSUN, a candidate gene for B-cell chronic lymphocytic leukemia and mantle-cell lymphoma, has an independently expressed homolog on 1p22-p31, BCMSUN-like.
The most frequent chromosomal imbalance in B-cell chronic lymphocytic leukemia (B-CLL) and mantle-cell lymphoma (MCL) is loss of material from 13q14.3. BCMSUN (previously Leu2, t4, cDNA 1B4) is one of 3 proposed candidate genes isolated from the minimally deleted region. We identified a homolog of BCMSUN, termed BCMSUNL (for BCMSUN-like). Radiation-hybrid mapping with a PCR-amplified fragment and fluorescence in situ hybridization with 2 PAC clones containing coding information for BCMSUNL revealed its localization at 1p22-p31. Interphase fluorescence in situ hybridization, however, revealed that the BCMSUNL gene locus is not part of the critical deletion region of 1p22 in MCLs. Analysis of DNA sequences derived from the respective PAC clones and available in public databases uncovered an intronless structure of BCMSUNL. Compared to BCMSUN, the new gene lacks exon 2 and shows 90.3% homology on the nucleic acid level. Both genes are expressed in peripheral blood lymphocytes from healthy donors as well as B-CLL and MCL tumors, with retention of genetic material at 13q14.3. Therefore, analysis of the candidate tumor-suppressor gene BCMSUN at 13q14.3 must be based on assays that distinguish between the 2 homologous genes.
Base Sequence↗
Mantle cell lymphoma is characterized by inactivation of the ATM gene.
In mantle cell lymphoma (MCL), the translocation t(11;14) is considered the cytogenetic hallmark of the disease. Recently, however, deletion of the chromosomal region 11q22-q23 has been identified as a frequent event in this type of cancer, indicating the existence of a pathogenically relevant tumor suppressor gene in this region. The deleted segment contains the ATM (ataxia telangiectasia mutated) gene. ATM is an interesting candidate as a tumor suppressor gene because constitutive inactivation of the gene predisposes ataxia telangiectasia patients to lymphoid malignancies. To assess the potential involvement of the gene in MCL lymphomagenesis, we performed mutation analysis of ATM in 12 sporadic cases of MCL, 7 of them with a deletion of one ATM gene copy, by using single-strand conformation polymorphism analysis of reverse transcription-PCR-amplified mRNA and subsequent DNA sequencing. In all seven cases containing a deletion of one ATM allele, a point mutation in the remaining allele was detected, which resulted in aberrant transcript splicing, truncation, or alteration of the protein. In addition, biallelic ATM mutations were identified in two MCLs that did not contain 11q deletions. Interestingly, in three cases analyzed, the ATM mutations detected in the tumor cells were not present in nonmalignant cells, demonstrating their somatic rather than germ-line origin. The inactivation of both alleles of the ATM gene by deletion and deleterious point mutation in the majority of cases analyzed indicates that ATM plays a role in the initiation and/or progression of MCL.
Alleles↗
Risk of false positive results in comparative genomic hybridization.
Comparative genomic hybridization, a widely used method for screening for genomic imbalances, suffers from a lack of standardized evaluation. In order to compare a recently proposed data-driven procedure with the commonly used fixed cutoff values, we tested 257 events by both procedures as well as by fluorescence in situ hybridization using selected probes. With the data-driven procedure, a much higher fraction (42/218 vs. 8/218) of false positive results was obtained, whereas a higher sensitivity with respect to the detection of imbalances (30/39 vs. 19/39) was reached. Based on the significantly higher positive likelihood ratios and the positive predictive value, we strongly recommend the use of fixed diagnostic thresholds, because the alternative procedure generates an unacceptably high portion of incorrectly scored chromosomal imbalances. Genes Chromosomes Cancer 28:353-357, 2000.
Chromosome Aberrations↗
Primary gastric apoptosis-rich T-cell lymphoma co-expressing CD4, CD8, and cytotoxic molecules.
In contrast to primary gastric lymphomas of B-cell type, little is known about primary gastric T-cell lymphomas. We describe three cases with remarkably similar features: diffuse growth, epitheliotropism, medium too large cell size, high apoptotic rates, and a CD3+, CD4+, CD8+, CD45RO+ immunophenotype. Clonal TCRgamma gene rearrangement was shown in two cases. Epstein-Barr virus infection was excluded in two cases. Taking advantage of fresh-frozen material, we analyzed two cases further, revealing CD5-, CD16+, CD56-, CD57-, CD25+, CD30+, CD103 (alphaEbeta7)+, bcl-2 protein+, CD95+, CD95 ligand(L)-. CD95L, however, was detected in histiocytic and fibroblastoid by stander cells. The lymphomas expressed granzyme B, perforin, and the TIA-1 antigen in various combinations. All three cases had a very unfavorable clinical course characterized by local recurrence and/or dissemination to other epithelial sites, leading to death within 6-12 months after the initial diagnosis despite surgery and aggressive antineoplastic treatment. These data suggest a novel variant of peripheral T-cell lymphoma operationally characterized as primary gastric, apoptosis-rich, CD103+, EBV-, T-cell lymphoma co-expressing CD4, CD8, CD16 and cytotoxic molecules.
Adult↗
Deletion of the multidrug resistance-associated protein (MRP1) gene in acute myeloid leukemia with inversion of chromosome 16 has no prognostic impact.
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Acute Disease↗
BCL10 is not the gene inactivated by mutation in the 1p22 deletion region in mantle cell lymphoma.
The BCL10 gene has recently been cloned from the 1p22 breakpoint of the translocation t(1 ;14)(p22;q32) observed in mucosa-associated lymphoid tissue (MALT) lymphoma. BCL10 was shown to be a proapoptotic-signaling gene encoding a protein that contains an amino-terminal caspase recruitment domain (CARD). Mutations within the BCL10 coding region resulting in truncated BCL10 proteins with loss of their proapoptotic function and preservation of their NF-kappaB activating function were detected in MALT lymphoma. Based on these findings it was proposed that BCL10 might have tumor suppressor function. Deletions involving 1p22 are commonly observed in mantle cell lymphoma (MCL). To investigate its role in MCL we have analyzed a series of 15 MCL for deletion and mutation of BCL10. Monoallelic 1p22 deletions were detected by fluorescence in situ hybridization in five of the 15 cases and were shown to affect BCL10 in all cases. BCL10 was screened for mutations by DNA sequencing of RT-PCR amplified transcripts. In none of the 15 MCL cases studied were mutations found in the BCL10 coding region. A previously reported polymorphism exhibiting a silent 24C > G substitution was found in eight MCL cases and in four healthy probands. A missense mutation 13G >T resulting in a substitution of a serine by an alanine was seen in one of the controls. Our results strongly suggest that BCL10 is not the candidate tumor suppressor gene inactivated by deletion or mutation in band 1p22 in MCL.
Adaptor Proteins, Signal Transducing↗
Heterogeneity of the API2-MALT1 gene rearrangement in MALT-type lymphoma.
The translocation t(11;18)(q21;q21), which is the most frequent chromosomal aberration in extranodal marginal zone B cell lymphomas of MALT-type, was characterised in a series of 34 biopsies, including 18 gastric non-Hodgkin's lymphomas (NHL) of MALT-type, six MALT-type NHL of extragastral origin and 10 extranodal large B cell lymphomas (LBL). Based on fluorescence in situ hybridisation, STS-PCR analysis and screening of genomic PAC libraries, a physical map of contiguous DNA probes on chromosome 11 was constructed containing the anti-apoptotic genes API2 and API1 adjacent to the translocation breakpoint. RACE-PCR experiments revealed MALT1 the chromosome 18-derived fusion partner of API2, which has also been reported recently by other groups. RT-PCR analysis and DNA sequencing demonstrated the expression of an API2-MALT1 fusion transcript in 18/24 gastral and extragastral MALT-type lymphomas. In none of 10 LBLs was a translocation specific RT-PCR product detected. Five variants of the fusion transcript were identified and in all instances the open reading frame of the fused portion of the MALT1 gene was maintained. The molecular analysis of these variants allowed the design of optimised assays for the diagnosis of the API2-MALT1 gene rearrangement.
Caspases↗
Genetic features of B-cell chronic lymphocytic leukemia.
The genetic features of B-cell chronic lymphocytic leukemia (CLL) are currently being reassessed by molecular cytogenetic techniques such as fluorescence in situ hybridization (FISH). Conventional cytogenetic studies by chromosome banding are difficult in CLL mainly because of the low in vitro mitotic activity of the tumor cells, which leads to poor quantity and quality of metaphase spreads. Molecular genetic analyses are limited because candidate genes are known for only a few chromosomal aberrations that are observed in CLL. FISH was found to be a powerful tool for the genetic analysis of CLL as it overcomes both the low mitotic activity of the CLL cells and the lack of suitable candidate genes for analysis. Using FISH, the detection of chromosomal aberrations can be performed at the single cell level in both dividing and non-dividing cells, thus circumventing the need of metaphase preparations from tumor cells. Probes for the detection of trisomies, deletions and translocation breakpoints can be applied to the regions of interest with the growing number of clones available from genome-wide libraries. Using the interphase cytogenetic FISH approach with a disease specific set of probes, chromosome aberrations can be found in more than 80% of CLL cases. The most frequently observed abnormalities are losses of chromosomal material, with deletions in band 13q14 being the most common, followed by deletions in 11q22-q23, deletions in 17p13 and deletions in 6q21. The most common gains of chromosomal material are trisomies 12q, 8q and 3q. Translocation breakpoints, in particular involving the immunoglobulin heavy chain locus at 14q32, which are frequently observed in other types of non-Hodgkin's lymphoma, are rare events in CLL. Genes affected by common chromosome aberrations in CLL appear to be p53 in cases with 17p deletion and ataxia telangiectasia mutated (ATM), which is mutated in a subset of cases with 11q22-q23 aberrations. However, for the other frequently affected genomic regions, the search for candidate genes is ongoing. In parallel, the accurate evaluation of the incidence of chromosome aberrations in CLL by FISH allows the correlation of genetic abnormalities with clinical disease manifestations and outcome. In particular, 17p abnormalities and deletions in 11q22-q23 have already been shown to be among the most important independent prognostic factors identifying subgroups of patients with rapid disease progression and short survival. In addition, deletion 17p has been associated with resistance to treatment with purine analogs. Therefore, genetic abnormalities may allow a risk assessment for individual patients at the time of diagnosis, thus giving the opportunity for a risk-adapted management.
Chromosome Aberrations↗