Biomedical subjects

Karen Ernestus

Publications and source records attributed to Karen Ernestus.

12 recordsLinked to original sources

PubMed2006

A mycobacterial gene involved in synthesis of an outer cell envelope lipid is a key factor in prevention of phagosome maturation.

Virulent mycobacteria cause arrest of phagosome maturation as a part of their survival strategy in hosts. This process is mediated through multiple virulence factors, whose molecular nature remains elusive. Using Mycobacterium marinum as a model, we performed a genome-wide screen to identify mutants whose ability to inhibit phagosome maturation was impaired, and we succeeded in isolating a comprehensive set of mutants that were not able to occupy an early endosome-like phagosomal compartment in mammalian macrophages. Categorizing and ordering the multiple mutations according to their gene families demonstrated that the genes modulating the cell envelope are the principal factors in arresting phagosome maturation. In particular, we identified a novel gene, pmiA, which is capable of influencing the constitution of the cell envelope lipids, thereby leading to the phagosome maturation block. The pmiA mutant was not able to resist phagosome maturation and was severely attenuated in mice. Complementing the mutant with the wild-type gene restored the attenuated virulence to wild-type levels in mice.

Animals↗

PubMed2006

Customized oligonucleotide microarray gene expression-based classification of neuroblastoma patients outperforms current clinical risk stratification.

PURPOSE: To develop a gene expression-based classifier for neuroblastoma patients that reliably predicts courses of the disease. PATIENTS AND METHODS: Two hundred fifty-one neuroblastoma specimens were analyzed using a customized oligonucleotide microarray comprising 10,163 probes for transcripts with differential expression in clinical subgroups of the disease. Subsequently, the prediction analysis for microarrays (PAM) was applied to a first set of patients with maximally divergent clinical courses (n = 77). The classification accuracy was estimated by a complete 10-times-repeated 10-fold cross validation, and a 144-gene predictor was constructed from this set. This classifier's predictive power was evaluated in an independent second set (n = 174) by comparing results of the gene expression-based classification with those of risk stratification systems of current trials from Germany, Japan, and the United States. RESULTS: The first set of patients was accurately predicted by PAM (cross-validated accuracy, 99%). Within the second set, the PAM classifier significantly separated cohorts with distinct courses (3-year event-free survival [EFS] 0.86 +/- 0.03 [favorable; n = 115] v 0.52 +/- 0.07 [unfavorable; n = 59] and 3-year overall survival 0.99 +/- 0.01 v 0.84 +/- 0.05; both P < .0001) and separated risk groups of current neuroblastoma trials into subgroups with divergent outcome (NB2004: low-risk 3-year EFS 0.86 +/- 0.04 v 0.25 +/- 0.15, P < .0001; intermediate-risk 1.00 v 0.57 +/- 0.19, P = .018; high-risk 0.81 +/- 0.10 v 0.56 +/- 0.08, P = .06). In a multivariate Cox regression model, the PAM predictor classified patients of the second set more accurately than risk stratification of current trials from Germany, Japan, and the United States (P < .001; hazard ratio, 4.756 [95% CI, 2.544 to 8.893]). CONCLUSION: Integration of gene expression-based class prediction of neuroblastoma patients may improve risk estimation of current neuroblastoma trials.

Biomarkers, Tumor↗

PubMed2006

Differential expression of neuronal genes defines subtypes of disseminated neuroblastoma with favorable and unfavorable outcome.

PURPOSE: Identification of molecular characteristics of spontaneously regressing stage IVS and progressing stage IV neuroblastoma to improve discrimination of patients with metastatic disease following favorable and unfavorable clinical courses. EXPERIMENTAL DESIGN: Serial analysis of gene expression profiles were generated from five stage IVS and three stage IV neuroblastoma. Differential expression of candidate genes was evaluated by real-time quantitative reverse transcription-PCR in 76 pretreatment tumor samples (stage IVS n=27 and stage IV n=49). Gene expression-based outcome prediction was determined by Prediction Analysis for Microarrays using 38 tumors as a training set and 38 tumors as a test set. RESULTS: Comparison of serial analysis of gene expression profiles from stage IV and IVS neuroblastoma revealed approximately 500 differentially expressed transcripts. Genes related to neuronal differentiation were observed more frequently in stage IVS tumors as determined by associating transcripts to Gene Ontology annotations. Forty-one candidate genes were evaluated by quantitative reverse transcription-PCR and 18 were confirmed to be differentially expressed (P<or=0.001). Classification of patients according to expression patterns of these 18 genes using Prediction Analysis for Microarrays discriminated two subgroups with significantly differing event-free survival (96+/-6% versus 40+/-8% at 3 years; P<0.0001) and overall survival (100% versus 72+/-7% at 3 years; P=0.0003). This classifier was the only independent covariate marker in a multivariate analysis considering the variables stage, age, MYCN amplification, and gene signature. CONCLUSIONS: Spontaneously regressing and progressing metastatic neuroblastoma differ by specific gene expression patterns, indicating distinct levels of neuronal differentiation and allowing for an improved risk estimation of children with disseminated disease.

Child, Preschool↗

PubMed2006

Oligonucleotide array-based comparative genomic hybridization (aCGH) of 90 neuroblastomas reveals aberration patterns closely associated with relapse pattern and outcome.

The study of genomic alterations in neuroblastoma is of particular importance since several cytogenetic markers proved to be closely associated with the clinical phenotype. To disclose patterns of gains and losses, we performed high-resolution oligonucleotide array-based comparative genomic hybridization (aCGH). A total cohort of 90 patients was classified into 6 subsets according to tumor stage and outcome: Stages 1-3+ (with event), Stage 1-3- (no event), Stage 4+/-, and Stage 4S+/-. The aberration patterns in Stages 1-3- and 4S- tumors differed from all other groups as they were predominantly characterized by losses (3, 4, 14, X) and gains (7, 17) of whole chromosomes. However, 59/65 (91%) tumors of Stages 1-3+ or Stage 4 revealed numerous structural copy number alterations (sCNA). While deletions in chromosomes 1, 3, and 11 discriminated outcome in Stage 4, there were no specific sCNA that distinguished tumor stage within the subgroup of unfavorable tumors. sCNA in 1p, 3p, 11q, 17q, or MYCN amplification (MNA) was seen among 22/24 patients who died, 10/12 with metastatic relapses, and 5/9 with local recurrences. Detailed breakpoint analyses on chromosomes 1, 3, 11, and 17 disclosed preferred breaking areas, although breakpoints were not identical. Amplifications were found in 18 patients and involved 2p24 (MYCN) and other segments of chromosome 2, as well as regions on chromosome arms 6q, 12q, and 17q. One single feature in 21q21.1 (BU678720, without known function yet) attracted particular attention since five patients showed a homozygous loss of this sequence.

Chromosome Aberrations↗

PubMed2006

Favorable outcome of triploid neuroblastomas: a contribution to the special oncogenesis of neuroblastoma.

There is a well-known association between patient outcome and tumor ploidy in neuroblastoma. To date, however, most clinical trials have not used this parameter for therapy stratification. Using conventional cytogenetics and fluorescence in situ hybridization (FISH), we investigated 36 tumors in terms of ploidy and chromosome 1 copy number (polysomy). In addition, interphase FISH for polysomy was performed on a second cohort of 440 neuroblastomas, together with the status of 1p, MYCN, and 11q. The main goals were as follows: (1) to assess the reliability of FISH to determine ploidy; (2) to illustrate associations between somy 1 and clinical/biologic factors; and (3) to investigate the role of somy 1 for predicting outcome. The comparison between karyotyping and FISH in the smaller cohort revealed 86% consistency between ploidy and polysomy (31/36). According to FISH, trisomic tumors in the second cohort showed structural chromosomal aberrations less frequently compared to di-/tetrasomic tumors (15 vs. 60%, P < 0.001). The portion of trisomic neuroblastomas was higher in stages 1, 2, and 4S versus stages 3 and 4 (55 vs. 24%, P < 0.001) and in children 18 months or younger versus those older than 18 months (55 vs. 19%, P < 0.001). Prognosis was significantly better for trisomic tumors versus di-/tetrasomic in the whole cohort [event-free (EFS) and overall survival (OS), P < 0.001]. In the subgroup without abnormalities of other molecular markers, EFS of trisomic neuroblastomas was better (P = 0.048), but was most likely due to an unequal stage distribution. In further subgroups, in terms of age and stage, significance between the somy groups was not reached, neither for EFS nor OS. The multivariate analyses including age, stage, chromosomal markers, and somy 1 confirmed the lack of independent prognostic power for the copy number of chromosome 1. This study demonstrates the following: (1) FISH is a practical alternative to other more labor-intensive techniques for determining ploidy; (2) trisomic tumors correlate with younger age at diagnosis, localized stage, and the lack of structural alterations; and (3) polysomy is not an independent prognostic marker. The sharp decline of trisomic tumors after the age of 18 months supports the idea of different genetic tumor entities.

Age of Onset↗

PubMed2005

AIDS-related B-cell lymphoma (ARL): correlation of prognosis with differentiation profiles assessed by immunophenotyping.

This study was undertaken to analyze the differentiation profiles assessed by immunophenotyping in AIDS-related B-cell lymphoma (ARL) and their relation to the clinical course. Paraffin-embedded sections of 89 ARL cases during 1989 to 2004 were stained immunohistochemically with antibodies to CD3, CD10, CD20, CD38, CD138/Syndecan-1 (Syn-1), multiple myeloma-1/interferon regulatory factor-4 (MUM1/IRF4), B-cell lymphoma protein-2 (BCL-2), BCL-6, latent membrane protein-1 (LMP-1), and Ki-67. Expression of CD10 and CD20 were associated with better overall survival (OS; P = .009 and P = .04, respectively). Expression of CD20 was associated with longer disease-free survival (DFS; P = .03), whereas expression of CD138/Syn-1 was associated with shorter DFS (P = .03). OS and DFS were worse in patients with immunophenotypic profiles related to post-germinal center (GC) differentiation (BCL-6 and CD10 negative, MUM1/IRF4 and/or CD138/Syn-1 positive) when compared with GC differentiation (P = .01). When controlled for age-adjusted International Prognostic Index (IPI), prior AIDS-defining illness (ADI), and year of ARL diagnosis, a post-GC differentiation remained significantly associated with poor OS and DFS. Expression of CD10 was associated with a preserved immunocompetence, whereas CD20 was less frequent in patients developing ARL while on highly active antiretroviral therapy (P = .04). In summary, lack of CD20 or CD10 expression and a post-germinal center signature are associated with a worse prognosis in ARL.

Adult↗

PubMed2005

Lacking immunocytological GD2 expression in neuroblastoma: report of 3 cases.

Immunocytological bone marrow assessment for contamination with neuroblastoma cells is based on their characteristic GD2 surface staining. Neuroblastoma without GD2 expression have been rarely and only after antibody therapy reported. Conventional cytology was performed using Pappenheim staining. For immunocytology, the APAAP method was utilized with the 14G2a anti-GD2 mouse monoclonal antibody. 7 x 10(5) cells on cytospin preparations were investigated. In 2003, 288 bone marrow samples from 191 neuroblastoma patients were investigated by cytology and immunocytology. Three cases demonstrated GD2 negativity on cytologically unambiguous neuroblastoma cells. Two female cases (94 and 37 months of age) with stage 4 neuroblastoma had GD2 expressing neuroblastoma cells in bone marrow at diagnosis. At 2nd relapse 25 and 23 months after diagnosis and 8 months and 12 months after anti-GD2 antibody treatment (ch14.18), the bone marrow infiltrating neuroblastoma cells lacked GD2 staining. The third patient, a 63-month-old girl with bone marrow replacement by neuroblastoma cells showed at diagnosis a mixture of GD2-unstained tumor clumps and very weakly stained neuroblastoma cells. Neuroblastoma cells may lack GD2 expression at diagnosis and at recurrence. This observation has diagnostic and therapeutic implications.

Antibodies, Monoclonal↗

PubMed2004

Expression of MHC class I, MHC class II, and cancer germline antigens in neuroblastoma.

BACKGROUND: Neuroblastoma is the most common solid extracranial tumor in childhood, still with poor survival rates for metastatic disease. Neuroblastoma cells are of neuroectodermal origin and express a number of cancer germline (CG) antigens. These CG antigens may represent a potential target for immunotherapy such as peptide-based vaccination strategies. OBJECTIVE: The purpose of this study was to analyze the presence of MAGE-A1, MAGE-A3/A6, and NY-ESO-1 on an mRNA and protein level and to determine the expression of MHC class I and MHC class II antigens within the same tumor specimens. METHODS: A total of 68 tumors were available for RT-PCR, and 19/68 tumors were available for immunohistochemical (IHC) analysis of MAGE-A1, MAGE-A3/A6, and NY-ESO-1. In parallel, the same tumors were stained with a panel of antibodies for MHC class I and MHC class II molecules. RESULTS: Screening of 68 tumor specimens by RT-PCR revealed expression of MAGE-A1 in 44%, MAGE-A3/A6 in 21%, and NY-ESO-1 in 28% of cases. Immunohistochemistry for CG antigens of selected tumors showed good agreement between protein and gene expression. However, staining revealed a heterogeneous expression of CG antigens. None of the selected tumors showed MHC class I or MHC class II expression. CONCLUSIONS: mRNA expression of MAGE-A1, MAGE-A3/A6, and NY-ESO-1 is congruent with the protein expression as determined by immunohistochemistry. The heterogeneous CG-antigen expression and the lack of MHC class I and II molecules may have implications for T-cell-mediated immunotherapy in neuroblastoma.

Antigens, Neoplasm↗

PubMed2003

Gain of distal chromosome arm 17q is not associated with poor prognosis in neuroblastoma.

PURPOSE: In several studies, gain of the distal long arm of chromosome 17 was shown to be a frequent and prognostically relevant factor in neuroblastoma, in addition to MYCN amplification (MNA) or 1p deletion. We asked whether this observation could be confirmed in a German cohort. EXPERIMENTAL DESIGN: To evaluate the frequency and prognostic impact of 17q gain, we investigated tissue samples from 193 neuroblastoma patients by the use of fluorescence in situ hybridization. The DNA probe (MPO) was located in distal 17q in the region of interest as used by several groups. To analyze the association of patients' outcome with the breakpoint position within 17q, we used the more proximal DNA probe ERBB2 in 17q21 on a selected number of cases. Gain was defined as an excess of 17q material compared with the chromosome 17 centromere in at least 50% of the analyzed tumor cells. In addition, alterations in chromosomes 1p, 3p, and 11q, as well as MYCN status, were determined to describe the interrelationship between the different parameters and to evaluate an independent prognostic influence. RESULTS: Gain of 17q was found in 61% of the investigated tumors. An additional 23% displayed an excess of 17q in less than half of all cells. Gain correlated with stage 4 disease (P = 0.003) and with other chromosomal alterations, such as 1p (P < 0.001), 3p (P = 0.01), 11q (P = 0.001), and MNA (P = 0.016), as well as with increased patient age (P = 0.01). Outcome was not different between patients with 17q gain compared with those without, however. A prognostic influence could not be delineated in all stages or in localized or in stage 4 subgroups or in the MYCN nonamplified patient cohort. Outcome did not differ between patients with additional 17q material in <10% of the cells or in >70%. Patients showing a breakpoint in the more proximal part of 17q could not be described as having a more favorable outcome compared with patients with a more distal breakpoint. Prognosis was poor in patients with MNA and/or 11q loss either with or without 17q gain. A multivariate analysis including the chromosomal parameters 17q, 11q, and MYCN status, as well as stage, showed MYCN and 11q status (P < 0.001), but not 17q or stage as significant prognostic factors. CONCLUSION: Although the most frequent aberration in neuroblastoma to date, we found no association between 17q gain and an inferior prognosis in our cohort; the status of MYCN and 11q, however, allowed reliable prediction of patients' outcomes.

Chromosome Aberrations↗

PubMed2003

FISH analyses for alterations in chromosomes 1, 2, 3, and 11 define high-risk groups in neuroblastoma.

BACKGROUND: The prognostic chromosomal markers 1p loss and MYCN amplification (MNA) are only present in a subgroup of approximately 30% of neuroblastomas. To further characterize high and low risk subsets we investigated alterations in chromosome arms 3p and 11q, additional changes in 1p and MYCN as well as the somy-status of chromosome 1 in the same sample. PROCEDURE: Fluorescence in situ hybridization (FISH) was used as an alternative technique to PCR/LOH- or comparative genomic hybridization (CGH) analyses. Alterations in chromosomes 3p and 11q were investigated in 182 unselected tumors, 1p loss and MNA in 174 and 179 of these, respectively. The somy-status of chromosome 1 was determined in 165 tumors as it highly correlates with the tumor ploidy. RESULTS: Alterations in the four chromosomal regions were found in the following frequencies: 3p26: 19%, 11q23: 29%, 1p36: 29%, MNA: 19%. Fifty-two percent of all cases displayed structural aberrations in at least one chromosomal region, 83% in stage 4 and 30% in stages 1-3, 4s. All aberrations were thus correlated with stage 4 disease but were also present in a substantial subset of localized and 4s tumors. Trisomy of chromosome 1 was found in 38% of the tumors, disomy or tetrasomy in 62%. Patients with alterations in any of the four chromosomes and di/tetrasomy 1 showed a significantly increased age at diagnosis. Loss in 1p and MNA were closely associated with each other, as well as 3p and 11q aberrations but not the groups 1p/MNA versus 3p/11q. Only a small portion of trisomic tumors showed aberrations in at least one of the four chromosomal regions (14%) in contrast to the majority of the di/tetrasomic cases (74%). As already known the MYCN status discriminated between good and poor outcome in localized and metastatic stage 4 tumors. In addition alterations in 1p or 11q, deletion in 3p and di/tetrasomy 1 were associated with an unfavorable prognosis in MYCN single copy tumors of stages 1-3, 4s. Multivariate analysis revealed 11q alterations and MNA as the most important chromosomal prognostic factors in all stages. CONCLUSION: FISH analyses for chromosomal alterations in 3p and 11q as well as in 1p and MYCN allows to define different groups with an increased risk for disease progression.

Child↗

PubMed2003

Deletions in chromosome arms 3p and 11q are new prognostic markers in localized and 4s neuroblastoma.

PURPOSE: To find new nonrandom chromosomal changes in neuroblastoma (NB) with a potential to forecast the patient's outcome, alterations in chromosome arms 3p and 11q were investigated. EXPERIMENTAL DESIGN: Frequency and prognostic potential of 3p and 11q alterations in 144 NBs were analyzed using interphase fluorescence in situ hybridization with DNA probes for 3p26 and 11q23. Aberrations were defined as deletion (monosomy of a specific region) or imbalance (at least two intact and additional 3p26- or 11q23-deleted chromosomes). RESULTS: Forty-two of 144 cases (29%) displayed 11q alterations (21% deletions, 8% imbalances). Most aberrations were associated with stage 4 disease (28 of 59, 47%) but were also present in localized and 4s tumors (14 of 85, 16%; P = 0.007). Patients with 11q deletion/imbalance were significantly older at diagnosis (P < 0.001). Changes in 3p were detected in 26 of 144 (18%) samples (15% deletions, 3% imbalances). These alterations were also associated with stage 4 [20 of 59 (34%) versus 6 of 85 (7%) in stages 1-3 and 4s, P = 0.007], and the median age was increased (P < 0.001). Aberrations in both chromosomes were highly associated with each other (P < 0.001). MYCN amplification (MNA) was detected in 10% and 12% of tumors with 11q and 3p alterations, and changes in 1p36 occurred in 13% and 26% of the 3p- and 11q-aberrant tumors. MYCN amplification and 11q deletion/imbalance tended to show an inverse correlation (P = 0.07) as well as 1p and 3p deletion/imbalance (P = 0.07). Patients with 3p and 11q abnormalities in localized/4s tumors showed an inferior outcome compared with those without these alterations (P = 0.002 and P = 0.0027, respectively), in particular in MYCN single copy tumors (P < 0.0001 and P = 0.0006, respectively). CONCLUSION: Alterations in 3p and 11q are frequent nonrandom aberrations in NB and define a new high-risk subgroup in MYCN single copy stage 1-3 and 4s disease.

Adolescent↗

PubMed2002

Fluorescence in situ hybridization analyses of chromosome band 1p36 in neuroblastoma detect two classes of alterations.

Chromosomal alterations in 1p36 were investigated in 196 neuroblastoma tumors using fluorescence in situ hybridization. Additionally, by using the same technique, it was determined whether MYCN was amplified in 149 of these. The most frequent finding was a deletion in 1p36, leading to monosomy of this region (29 cases, 15%). Furthermore, we found tumors with at least two intact copies of chromosome 1 and additional 1p36-deleted copies. Altogether, 21 tumors (11%) displayed this imbalance of 1p36. Similar to the cases with deletion, imbalances were predominantly found in stage 4 tumors (81%), and they were significantly associated with an increased patient age (P = 0.01). Nearly all 1p-deleted tumors showed amplification of MYCN (24/27 analyzed samples, 89%), whereas only 8 of 21 (38%) with imbalance did. Eight cases with imbalance were investigated for loss of heterozygosity (LOH) using microsatellite markers in 1p35-36. Only 4 displayed 1p36 LOH, whereas the remaining 4 were heterozygous. Both patients with deletion of 1p and with imbalance had a poor outcome [3-year rate of event-free-survival (EFS): 33 +/- 15% and 41 +/- 15%], which was significantly worse compared to the outcome of patients without 1p alterations (3-year EFS: 70 +/- 5%; P = 0.01 and P = 0.0059). We conclude that besides monosomic short arm deletions, imbalance of 1p36 is a strong marker of a poor prognosis in neuroblastoma and not necessarily associated with MYCN amplification and LOH.

Adult↗