Axel Benner

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.

Mutations and Treatment Outcome in Cytogenetically Normal Acute Myeloid Leukemia

BACKGROUND Mutations occur in several genes in cytogenetically normal acute myeloid leukemia (AML) cells: the nucleophosmin gene (NPM1), the fms-related tyrosine kinase 3 gene (FLT3), the CCAAT/enhancer binding protein alpha gene (CEPBA), the myeloid-lymphoid or mixed-lineage leukemia gene (MLL), and the neuroblastoma RAS viral oncogene homolog (NRAS). We evaluated the associations of these mutations with clinical outcomes in patients. METHODS We compared the mutational status of the NPM1, FLT3, CEBPA, MLL, and NRAS genes in leukemia cells with the clinical outcome in 872 adults younger than 60 years of age with cytogenetically normal AML. Patients had been entered into one of four trials of therapy for AML. In each study, patients with an HLA-matched related donor were assigned to undergo stem-cell transplantation. RESULTS A total of 53% of patients had NPM1 mutations, 31% had FLT3 internal tandem duplications (ITDs), 11% had FLT3 tyrosine kinase-domain mutations, 13% had CEBPA mutations, 7% had MLL partial tandem duplications (PTDs), and 13% had NRAS mutations. The overall complete-remission rate was 77%. The genotype of mutant NPM1 without FLT3-ITD, the mutant CEBPA genotype, and younger age were each significantly associated with complete remission. Of the 663 patients who received postremission therapy, 150 underwent hematopoietic stem-cell transplantation from an HLA-matched related donor. Significant associations were found between the risk of relapse or the risk of death during complete remission and the leukemia genotype of mutant NPM1 without FLT3-ITD (hazard ratio, 0.44; 95% confidence interval [CI], 0.32 to 0.61), the mutant CEBPA genotype (hazard ratio, 0.48; 95% CI, 0.30 to 0.75), and the MLL-PTD genotype (hazard ratio, 1.56; 95% CI, 1.00 to 2.43), as well as receipt of a transplant from an HLA-matched related donor (hazard ratio, 0.60; 95% CI, 0.44 to 0.82). The benefit of the transplant was limited to the subgroup of patients with the prognostically adverse genotype FLT3-ITD or the genotype consisting of wild-type NPM1 and CEBPA without FLT3-ITD. CONCLUSIONS Genotypes defined by the mutational status of NPM1, FLT3, CEBPA, and MLL are associated with the outcome of treatment for patients with cytogenetically normal AML.

Matrix-based comparative genomic hybridization: Biochips to screen for genomic imbalances

Comparative genomic hybridization (CGH) to metaphase chromosomes has been widely used for the genome‐wide screening of genomic imbalances in tumor cells. Substitution of the chromosome targets by a matrix consisting of an ordered set of defined nucleic acid target sequences would greatly enhance the resolution and simplify the analysis procedure, both of which are prerequisites for a broad application of CGH as a diagnostic tool. However, hybridization of whole genomic human DNA to immobilized single‐copy DNA fragments with complexities below the megabase pair level has been hampered by the low probability of specific binding because of the high probe complexity. We developed a protocol that allows CGH to chips consisting of glass slides with immobilized target DNAs arrayed in small spots. High‐copy‐number amplifications contained in tumor cells were rapidly scored by use of target DNAs as small as a cosmid. Low‐copy‐number gains and losses were identified reliably by their ratios by use of chromosome‐specific DNA libraries or genomic fragments as small as 75 kb cloned in P1 or PAC vectors as targets, thus greatly improving the resolution achievable by chromosomal CGH. The ratios obtained for the same chromosomal imbalance by matrix CGH and by chromosomal CGH corresponded very well. The new matrix CGH protocol provides a basis for the development of automated diagnostic procedures with biochips designed to meet clinical needs. Genes Chromosomes Cancer 20:399–407, 1997.

CEBPA Mutations in Younger Adults With Acute Myeloid Leukemia and Normal Cytogenetics: Prognostic Relevance and Analysis of Cooperating Mutations

PURPOSE To assess the prognostic relevance of mutations in the CEBPA gene encoding CCAAT/enhancer binding protein alpha (C/EBP alpha) in a large prospective series of younger adults with acute myeloid leukemia (AML) and normal cytogenetics. PATIENTS AND METHODS The entire CEBPA coding region was sequenced in diagnostic samples from 236 AML patients 16 to 60 years of age with normal cytogenetics who were uniformly treated on two consecutive protocols of the AML Study Group Ulm, and CEBPA mutation status was correlated with clinical outcome. RESULTS CEBPA mutations were detected in 36 (15%) of 236 patients. Twenty-one (9%) of 236 patients had mutations predicted to result in loss of C/EBP alpha function. Remission duration and overall survival (OS) were significantly longer for the 36 patients with CEBPA mutations (P =.01 and P =.05, respectively). On multivariate analysis, wild-type CEBPA was an independent prognostic marker affecting remission duration (hazard ratio, 2.85; P =.01) and OS (hazard ratio, 1.87; P =.04). Analysis of cooperating mutations (both types of activating FLT3 mutations and MLL partial tandem duplications) showed that FLT3 mutations had no significant prognostic influence in patients with CEBPA mutations. Furthermore, there was no significant overlap between the subgroup of patients with CEBPA mutation with predicted loss of C/EBP alpha function and patients with FLT3 or MLL mutations, suggesting that CEBPA loss-of-function mutations define a distinct biologic subclass of AML with normal cytogenetics. CONCLUSION Mutant CEBPA predicts favorable prognosis and may improve risk stratification in AML patients with normal cytogenetics.

A genomewide screen for autism: Strong evidence for linkage to chromosomes 2q, 7q, and 16p

Autism is characterized by impairments in reciprocal communication and social interaction and by repetitive and stereotyped patterns of activities and interests. Evidence for a strong underlying genetic predisposition comes from twin and family studies, although susceptibility genes have not yet been identified. A whole-genome screen for linkage, using 83 sib pairs with autism, has been completed, and 119 markers have been genotyped in 13 candidate regions in a further 69 sib pairs. The addition of new families and markers provides further support for previous reports of linkages on chromosomes 7q and 16p. Two new regions of linkage have also been identified on chromosomes 2q and 17q. The most significant finding was a multipoint maximum LOD score (MLS) of 3.74 at marker D2S2188 on chromosome 2; this MLS increased to 4.80 when only sib pairs fulfilling strict diagnostic criteria were included. The susceptibility region on chromosome 7 was the next most significant, generating a multipoint MLS of 3.20 at marker D7S477. Chromosome 16 generated a multipoint MLS of 2.93 at D16S3102, whereas chromosome 17 generated a multipoint MLS of 2.34 at HTTINT2. With the addition of new families, there was no increased allele sharing at a number of other loci originally showing some evidence of linkage. These results support the continuing collection of multiplex sib-pair families to identify autism-susceptibility genes.

p16INK4a immunohistochemistry improves interobserver agreement in the diagnosis of cervical intraepithelial neoplasia.

It has been repeatedly shown that there is a substantial lack of interobserver reproducibility in the histologic diagnosis of cervical intraepithelial neoplasia (CIN), which might be improved by a more specific diagnostic biomarker. Cervical cancer and CIN, but not other cervical epithelia, express high levels of the cyclin-dependent kinase inhibitor p16INK4a, suggesting that staining for this marker could help to more precisely identify CIN in tissue sections and therefore reduce variation in interpretation of cervical lesions. To test this hypothesis, 194 cervical cone biopsy samples were selected from a routine histopathology laboratory. Two consecutive sections from each biopsy were stained with hematoxylin and eosin and with a p16INK4a-specific monoclonal antibody, respectively. Five experienced cervical pathologists examined the slides. The agreement in the diagnosis between pairs or groups of observers was calculated by kappa statistics. Significant discrepancies were observed in the diagnostic interpretation of hematoxylin and eosin-stained slides, particularly for low-grade lesions (kappa value 0.60 [95% confidence interval 0.58–0.63]). There was significantly better agreement in the interpretation of p16INK4a expression (kappa value 0.91 [95% confidence interval 0.84–0.99]). Expression of p16INK4a was restricted to CIN 2/CIN 3, CIN 1 associated with high-risk human papillomavirus, or cervical cancer. p16INK4a immunostaining allowed precise identification of even small CIN or cervical cancer lesions in biopsy sections and helped to reduce interobserver variation in the histopathologic interpretation of cervical biopsy specimens. Thus, p16INK4a immunohistochemistry can reduce false-negative and false-positive biopsy interpretation and thereby significantly improve cervical (pre)-cancer diagnosis.

Prognostic Significance of Partial Tandem Duplications of the MLL Gene in Adult Patients 16 to 60 Years Old With Acute Myeloid Leukemia and Normal Cytogenetics: A Study of the Acute Myeloid Leukemia Study Group Ulm

PURPOSE To evaluate the incidence and clinical significance of partial tandem duplications (PTDs) of the mixed lineage leukemia (MLL) gene in a large series of newly diagnosed adult patients (16 to 60 years old) with acute myeloid leukemia (AML) intensively treated within the multicenter treatment trials AML-HD93 and AML-HD98A. PATIENTS AND METHODS Identification of MLL PTD was performed centrally using Southern blot analysis in pretreatment samples from 525 of 683 assessable patients. PTD was confirmed by polymerase chain reaction (PCR) and sequencing of the PCR products. RESULTS MLL PTD was identified in none of the 129 patients with t(8;21), inv(16), and t(15;17); in 19 (7.7%) of 247 patients with normal karyotype; and in 10 (8.5%) of 119 patients with all other abnormalities, with 30 cases of t(11q23) excluded. In the group of patients with a normal karyotype, there was no difference in the presenting clinical features between the PTD-positive and the PTD-negative cases. Sixteen (89%) of the 18 assessable PTD-positive patients and 158 (78%) of the 203 PTD-negative patients achieved a complete remission. After a median follow-up time of 19 months, 11 of the 16 PTD-positive patients relapsed compared with 54 of the 158 PTD-negative patients; the median remission durations of the PTD-positive and the PTD-negative groups were 7.75 months and 19 months, respectively (P <.001). Multivariate analysis identified the MLL PTD status as the single prognostic factor for remission duration. CONCLUSION Within the subgroup of young adult AML patients with normal karyotype, MLL PTD is associated with short remission duration.

Delineation of two clinically and molecularly distinct subgroups of posterior fossa ependymoma.

Despite the histological similarity of ependymomas from throughout the neuroaxis, the disease likely comprises multiple independent entities, each with a distinct molecular pathogenesis. Transcriptional profiling of two large independent cohorts of ependymoma reveals the existence of two demographically, transcriptionally, genetically, and clinically distinct groups of posterior fossa (PF) ependymomas. Group A patients are younger, have laterally located tumors with a balanced genome, and are much more likely to exhibit recurrence, metastasis at recurrence, and death compared with Group B patients. Identification and optimization of immunohistochemical (IHC) markers for PF ependymoma subgroups allowed validation of our findings on a third independent cohort, using a human ependymoma tissue microarray, and provides a tool for prospective prognostication and stratification of PF ependymoma patients.

Outcome Prediction in Pediatric Medulloblastoma Based on DNA Copy-Number Aberrations of Chromosomes 6q and 17q and the MYC and MYCN Loci

PURPOSE Medulloblastoma is the most common malignant brain tumor in children. Current treatment decisions are based on clinical variables. Novel tumor-derived biomarkers may improve the risk stratification of medulloblastoma patients. PATIENTS AND METHODS A model for the molecular risk stratification was proposed from an array-based comparative genomic hybridization (array-CGH) screen (n = 80). Fluorescence in situ hybridization (FISH) analyses for chromosome arms 6q, 17p, and 17q and the MYC and MYCN loci were performed in an independent validation set (n = 260). Copy number aberrations were correlated with clinical, histologic, and survival data. RESULTS Gain of 6q and 17q and genomic amplification of MYC or MYCN were each associated with poor outcome in the array-CGH study (n = 80). In contrast, all patients with 6q-deleted tumors survived. Given these findings, the following hierarchical molecular staging system was defined: (1) MYC/MYCN amplification, (2) 6q gain, (3) 17q gain, (4) 6q and 17q balanced, and (5) 6q deletion. The prognostic value of this staging system was investigated by FISH analysis (n = 260). The addition of molecular markers to clinical risk factors resulted in the identification of a large proportion of patients (72 of 260 patients; 30%) at high risk for relapse and death who would be considered standard risk by application of clinical variables alone. CONCLUSION Genomic aberrations in medulloblastoma are powerful independent markers of disease progression and survival. By adding genomic markers to established clinical and histologic variables, outcome prediction can be substantially improved. Because the analyses can be conducted on routine paraffin-embedded material, it will be especially feasible to use this novel molecular staging system in large multicenter clinical trials.

Individual Patient Data–Based Meta-Analysis of Patients Aged 16 to 60 Years With Core Binding Factor Acute Myeloid Leukemia: A Survey of the German Acute Myeloid Leukemia Intergroup

PURPOSE To evaluate prognostic factors for relapse-free survival (RFS) and overall survival (OS) and to assess the impact of different postremission therapies in adult patients with core binding factor (CBF) acute myeloid leukemias (AML). PATIENTS AND METHODS Individual patient data-based meta-analysis was performed on 392 adults (median age, 42 years; range, 16 to 60 years) with CBF AML (t(8;21), n = 191; inv(16), n = 201) treated between 1993 and 2002 in prospective German AML treatment trials. RESULTS RFS was 60% and 58% and OS was 65% and 74% in the t(8;21) and inv(16) groups after 3 years, respectively. For postremission therapy, intention-to-treat analysis revealed no difference between intensive chemotherapy and autologous transplantation in the t(8;21) group and between chemotherapy, autologous, and allogeneic transplantation in the inv(16) group. In the t(8;21) group, significant prognostic variables for longer RFS and OS were lower WBC and higher platelet counts; loss of the Y chromosome in male patients was prognostic for shorter OS. In the inv(16) group, trisomy 22 was a significant prognostic variable for longer RFS. For patients who experienced relapse, second complete remission rate was significantly lower in patients with t(8;21), resulting in a significantly inferior survival duration after relapse compared with patients with inv(16). CONCLUSION We provide novel prognostic factors for CBF AML and show that patients with t(8;21) who experience relapse have an inferior survival duration.