Martin Bentz

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.

Escalated-Dose BEACOPP in the Treatment of Patients With Advanced-Stage Hodgkin's Lymphoma: 10 Years of Follow-Up of the GHSG HD9 Study

PURPOSE The HD9 trial of the German Hodgkin Study Group compared two different doses (baseline and escalated) of the bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine, and prednisone (BEACOPP) chemotherapy regimen in 1,196 patients with advanced-stage Hodgkins lymphoma (HL). The previous analysis with 5 years median follow-up had indicated improved tumor control with BEACOPP escalated. Since the long-term safety and efficacy of this regimen has been debated, we report the 10-year follow-up. PATIENTS AND METHODS Patients received one of three chemotherapy regimens: eight cycles of cyclophosphamide, vincristine, procarbazine, and prednisone (COPP) alternating with doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD); eight cycles of BEACOPP baseline; or eight cycles of BEACOPP escalated. RESULTS Median follow-up was 111 months. At 10 years, freedom from treatment failure (FFTF) was 64%, 70%, and 82% with OS rates of 75%, 80%, and 86% for patients treated with COPP/ABVD (arm A), BEACOPP baseline (arm B), and BEACOPP escalated (arm C), respectively (P < .001). BEACOPP escalated was significantly better than BEACOPP baseline in terms of FFTF (P < .0001) and OS (P = .0053). A total of 74 second malignancies (6.2%) were documented, including acute myeloid leukemia (0.4%, 1.5%, and 3.0%), non-Hodgkins lymphoma (2.7%, 1.7%, and 1.0%), and solid tumors (2.7%, 3.4%, and 1.9%). The corresponding overall secondary malignancy rates were 5.7%, 6.6%, and 6.0%, respectively. CONCLUSION The 10-year follow-up of the HD9 trial demonstrates a stabilized significant improvement in long-term FFTF and OS for BEACOPP escalated in advanced-stage HL. These results challenge ABVD as standard of care for this patient population.

Chromosome aberrations in B-cell chronic lymphocytic leukemia : Reassessment based on molecular cytogenetic analysis

Abstract In B-cell chronic lymphocytic leukemia (B-CLL) clonal chromosome aberrations are detected in approximately 40–50% of tumors when using conventional chromosome banding analysis. Most studies find trisomy 12 to be the most frequent chromosome aberration, followed by structural aberrations of the long arm of chromosomes 13 and 14. Trisomy 12 and the ”14q+” marker are associated with shorter survival times, while the patients with 13q abnormalities have a favorable outcome, similar to those with a normal karyotype. The development of molecular cytogenetic techniques has greatly improved our ability to detect chromosome aberrations in tumor cells. Using fluorescence in situ hybridization, chromosome aberrations can be detected not only in dividing cells but also in interphase nuclei, an approach referred to as interphase cytogenetics. The prevalence of specific aberrations in B-CLL is currently being reassessed by interphase cytogenetics. By far the most frequent abnormality are deletions involving chromosome band 13q14, followed by deletions of the genomic region 11q22.3-q23.1, trisomy 12, deletions of 6q21-q23, and deletions/mutations of the TP53 tumor suppressor gene at 17p13. The evaluation of the true incidence of these aberrations now provides the basis for more accurate correlations with clinical characteristics and outcome. Deletions/mutations of the TP53 gene have been shown to be associated with resistance to treatment and to be an independent marker for poor survival. 11q deletions have been associated with extensive nodal involvement, rapid disease progression, and short survival times. Whether trisomy 12, 13q14, and 6q deletions have a prognostic impact awaits further study. The application of these molecular cytogenetic techniques will also contribute to the identification of the pathogenetically relevant genes that are affected by the chromosome aberrations in B-CLL.

Minimal sizes of deletions detected by comparative genomic hybridization

Comparative genomic hybridization (CGH) has been used widely for the molecular cytogenetic analysis of tumors. Until now, the spatial resolution of this technique for diagnosing deletions of chromosomal sequences has not been assessed in detail. In the present study, we performed CGH analyses on five DNA samples derived from B‐cell leukemias with 11q deletions, the sizes of which ranged from 3 Mbp to 14–18 Mbp. CGH experiments were evaluated by two established commercial analysis systems. Deletions down to a size of 10–12 Mbp were diagnosed based on a diagnostic threshold value of 0.8, if the vast majority of cells carried the deletion. For cases with smaller deletions, the ratio profiles were shifted toward underrepresentation at the respective chromosomal bands; however, the diagnostic threshold value was not reached. In all five cases, there was complete agreement between the two image analysis systems. Genes Chromosomes Cancer 21:172–175, 1998.

Disclosure of Candidate Genes in Acute Myeloid Leukemia With Complex Karyotypes Using Microarray-Based Molecular Characterization

PURPOSE To identify novel genomic regions of interest in acute myeloid leukemia (AML) with complex karyotypes, we applied comparative genomic hybridization to microarrays (array-CGH), allowing high-resolution genome-wide screening of genomic imbalances. PATIENTS AND METHODS Sixty AML cases with complex karyotypes were analyzed using array-CGH; parallel analysis of gene expression was performed in a subset of cases. RESULTS Genomic losses were found more frequently than gains. The most frequent losses affected 5q (77%), 17p (55%), and 7q (45%), and the most frequent genomic gains 11q (40%) and 8q (38%). Critical segments could be delineated to genomic fragments of only 0.8 to a few megabase-pairs of DNA. In lost/gained regions, gene expression profiling detected a gene dosage effect with significant lower/higher average gene expression levels across the genes located in the respective regions. Furthermore, high-level DNA amplifications were identified in several regions: 11q23.3-q24.1 (n = 7), 21q22 (n = 6), 11q23.3 (n = 5), 13q12 (n = 3), 8q24 (n = 3), 9p24 (n = 2), 12p13 (n = 2), and 20q11 (n = 2). Parallel analysis of gene expression in critical amplicons displayed overexpressed candidate genes (eg, C8FW and MYC in 8q24). CONCLUSION In conclusion, a large spectrum of genomic imbalances, including novel recurring changes in AML with complex karyotypes, was identified using array-CGH. In addition, the combined analysis of array-CGH data with gene expression profiles allowed the detection of candidate genes involved in the pathogenesis of AML.

t(11;14)-positive mantle cell lymphomas exhibit complex karyotypes and share similarities with B-cell chronic lymphocytic leukemia.

Until now, few data on additional chromosomal aberrations in t(11;14)‐positive mantle cell lymphomas (MCLs) have been published. We analyzed 39 t(11;14)‐positive MCLs by either comparative genomic hybridization (CGH; n = 8), fluorescence in situ hybridization (FISH) with a set of DNA probes detecting the most frequent aberrations in B‐cell neoplasms (n = 12), or both techniques (n = 19). The t(11;14) was present in all cases. In 37 of 39 cases, chromosomal imbalances were found. In 27 cases, complex karyotypes, i.e., ≥ 3 aberrations, were identified. The most frequent aberrations were losses of 13q14–21 or 13q32–34 (27 cases), 9p21 (16 cases), and 11q22–23 (12 cases) and gains of 3q26–29 (19 cases), 8q22–24 (11 cases), and 18q21–22 (9 cases). In 26% of cases (7 of 27) analyzed by CGH, a total of 10 high‐level DNA amplifications were identified. Although in comparison with B‐cell chronic lymphopcytic leukemia (B‐CLL) MCL is characterized by a much higher complexity of chromosomal aberrations, there are striking similarities: 13q14 deletions were identified in more than 50% of both MCL and B‐CLL cases. In contrast, in our CGH database containing 293 B‐cell lymphomas, this aberration was found in only 11% of other nodal lymphomas. Even more strikingly, 11q deletions, which are present in 20%–30 % of MCL and B‐CLL, were found very rarely in other nodal B‐cell lymphomas (CGH: 1 of 208 cases; FISH: 1 of 69 cases). These data show that MCL is characterized by specific secondary aberrations and that there may be similarities in the pathogenesis of MCL and B‐CLL. Genes Chromosomes Cancer 27:285–294, 2000.

Deletions below 10 megabasepairs are detected in comparative genomic hybridization by standard reference intervals

Comparative genomic hybridization (CGH) is a widely used technique for studying chromosomal imbalances. The sensitivity of the technique is, however, relatively low. Deletions down to a size of 10–12 Mbp have been detected by the use of fixed diagnostic thresholds. In this study, we applied standard reference intervals as detection criteria on a number of deletions in the range of 3 Mbp to 14–18 Mbp. All deletions were detected. Thus, detection by standard reference intervals confers a considerably higher sensitivity to CGH analysis compared to fixed diagnostic thresholds. Genes Chromosomes Cancer 25:410–413, 1999.

Genomic DNA-Chip Hybridization Reveals a Higher Incidence of Genomic Amplifications in Pancreatic Cancer than Conventional Comparative Genomic Hybridization and Leads to the Identification of Novel Candidate Genes

Genomic analyses aimed at the detection of high-level DNA amplifications were performed on 13 widely used pancreatic cancer cell lines and 6 pancreatic tumor specimens. For these analyses, array-based comparative genomic hybridization (Matrix-CGH) onto dedicated microarrays was used. In comparison with chromosomal CGH (eight amplifications), a >3-fold number of DNA amplifications was detected (n = 29). The most frequent amplifications mapped to 7p12.3 (three pancreatic cancer cell lines and three pancreatic tumor specimens), 8q24 (four pancreatic cancer cell lines and one pancreatic tumor specimen), 11q13 (three pancreatic cancer cell lines and three pancreatic tumor specimens), and 20q13 (four pancreatic cancer cell lines and three pancreatic tumor specimens). Genes contained in the consensus regions were MYC (8q24), EGFR (7p12.3), and FGF3 (11q13). In six of seven pancreatic cancer cell lines and pancreatic tumor specimens with 20q13 amplifications, the novel candidate gene NFAT C2, which plays a role in the activation of cytokines, was amplified. Other amplifications also affected genes for which a pathogenetic role in pancreatic carcinoma has not been described, such as BCL10 and BCL6, two members of the BCL family. A subset of amplified genes was checked for overexpression by means of real-time PCR, revealing the highest expression levels for BCL6 and BCL10. Thus, Matrix-CGH allows the detection of a high number of amplifications, resulting in the identification of novel candidate genes in pancreatic cancer.

Clinical, molecular, and prognostic significance of WHO type inv(3)(q21q26.2)/t(3;3)(q21;q26.2) and various other 3q abnormalities in acute myeloid leukemia.

PURPOSE Acute myeloid leukemia (AML) with inv(3)(q21q26.2)/t(3;3)(q21;q26.2) [inv(3)/t(3;3)] is recognized as a distinctive entity in the WHO classification. Risk assignment and clinical and genetic characterization of AML with chromosome 3q abnormalities other than inv(3)/t(3;3) remain largely unresolved. PATIENTS AND METHODS Cytogenetics, molecular genetics, therapy response, and outcome analysis were performed in 6,515 newly diagnosed adult AML patients. Patients were treated on Dutch-Belgian Hemato-Oncology Cooperative Group/Swiss Group for Clinical Cancer Research (HOVON/SAKK; n = 3,501) and German-Austrian Acute Myeloid Leukemia Study Group (AMLSG; n = 3,014) protocols. EVI1 and MDS1/EVI1 expression was determined by real-time quantitative polymerase chain reaction. RESULTS 3q abnormalities were detected in 4.4% of AML patients (288 of 6,515). Four distinct groups were defined: A: inv(3)/t(3;3), 32%; B: balanced t(3q26), 18%; C: balanced t(3q21), 7%; and D: other 3q abnormalities, 43%. Monosomy 7 was the most common additional aberration in groups (A), 66%; (B), 31%; and (D), 37%. N-RAS mutations and dissociate EVI1 versus MDS1/EVI1 overexpression were associated with inv(3)/t(3;3). Patients with inv(3)/t(3;3) and balanced t(3q21) at diagnosis presented with higher WBC and platelet counts. In multivariable analysis, only inv(3)/t(3;3), but not t(3q26) and t(3q21), predicted reduced relapse-free survival (hazard ratio [HR], 1.99; P < .001) and overall survival (HR, 1.4; P = .006). This adverse prognostic impact of inv(3)/t(3;3) was enhanced by additional monosomy 7. Group D 3q aberrant AML also had a poor outcome related to the coexistence of complex and/or monosomal karyotypes and cryptic inv(3)/t(3;3). CONCLUSION Various categories of 3q abnormalities in AML can be distinguished according to their clinical, hematologic, and genetic features. AML with inv(3)/t(3;3) represents a distinctive subgroup with unfavorable prognosis.

Comparative genomic hybridization: uses and limitations.

Comparative genomic hybridization (CGH) has contributed significantly to the current knowledge of genomic alterations in hematologic malignancies. Characteristic patterns of genomic imbalances not only have confirmed recent classification schemes in non-Hodgkins lymphoma, but they provide a basis for the successful identification of genes with previously unrecognized pathogenic roles in the development of different lymphomas. Based on its technical limitations, there is little reason to apply CGH to chromosomes of metaphase cells in routine diagnostic settings. However, the new approach of CGH to DNA microarrays, a procedure termed matrix-CGH, overcomes most of the limitations and opens new approaches for diagnostics and identification of genetically defined leukemia and lymphoma subgroups. Current efforts to develop leukemia specific matrix-CGH DNA chips, which are designed to meet the clinical needs, are presented and discussed.