Biomedical subjects

Axel Benner

Publications and source records attributed to Axel Benner.

At least 37 records · Page 2Linked to original sources

PubMed2005

T25 repeat in the 3' untranslated region of the CASP2 gene: a sensitive and specific marker for microsatellite instability in colorectal cancer.

DNA mismatch repair deficiency is observed in about 10% to 15% of all colorectal carcinomas and in up to 90% of hereditary nonpolyposis colorectal cancer (HNPCC) patients. Tumors with mismatch repair defects acquire mutations in short repetitive DNA sequences, a phenomenon termed high-level microsatellite instability (MSI-H). The diagnosis of MSI-H in colon cancer is of increasing relevance, because MSI-H is an independent prognostic factor in colorectal cancer, seems to influence the efficacy of adjuvant chemotherapy, and is the most important molecular screening tool to identify HNPCC patients. To make MSI typing feasible for the routine pathology laboratory, highly reproducible and cost effective laboratory tests are required. Here, we describe a novel T25 mononucleotide marker in the 3'untranslated region of the CASP2 gene (CAT25) that displayed a quasimonomorphic repeat pattern in normal tissue of 200 unrelated individuals of Caucasian origin. In addition, CAT25 was monomorphic also in all tested donors of African and Asian origin (n = 102 and n = 79, respectively) and thus differs from the most commonly used markers BAT25 and BAT26. Without the analysis of corresponding normal tissue, CAT25 correctly detected 56 of 57 colorectal cancer specimens classified as MSI-H by using the standard National Cancer Institute/International Collaborative Group-HNPCC marker panel. Combined with the standard markers BAT25 and BAT26 in a multiplex PCR, all MSI-H colorectal cancer samples were typed correctly. No false-positive results were obtained in 60 non-MSI-H control colorectal cancer specimens. These data suggest that CAT25 should be included into novel marker panels for microsatellite testing thus allowing for a significant reduction of the complexity and costs of MSI typing. Moreover, CAT25 represents a highly promising marker for early detection of colorectal cancer in HNPCC germ line mutation carriers.

3' Untranslated Regions↗

PubMed2005

Immunoselective pressure and human leukocyte antigen class I antigen machinery defects in microsatellite unstable colorectal cancers.

In colorectal cancer, the immune response is particularly pronounced against tumors displaying the high microsatellite instability (MSI-H) phenotype. MSI-H tumors accumulate mutations affecting microsatellites located within protein encoding regions (coding microsatellites, cMS), which lead to translational shifts of the respective reading frames. Consequently, novel tumor-specific frameshift-derived neopeptides (FSP) are generated and presented by MSI-H tumor cells, thus eliciting effective cytotoxic immune responses. To analyze whether the immunoselective pressure was reflected by the phenotype of MSI-H colorectal cancer cells, we compared here the expression of antigen processing machinery (APM) components and human leukocyte antigen (HLA) class I antigen subunits in 20 MSI-H and 20 microsatellite-stable (MSS) colorectal cancer using a panel of newly developed APM component-specific monoclonal antibodies. In addition, we did a systematic analysis of mutations at cMS located within APM genes and beta2-microglobulin (beta2m). Total HLA class I antigen loss was observed in 12 (60.0%) of the 20 MSI-H lesions compared with only 6 (30.0%) of the 20 MSS colorectal cancer lesions. Moreover, total loss of membraneous HLA-A staining was significantly more frequent in MSI-H colorectal cancer (P = 0.0024). Mutations at cMS of beta2m and genes encoding APM components (TAP1 and TAP2) were detected in at least 7 (35.0%) of 20 MSI-H colorectal cancers but in none of the MSS colorectal cancers (P = 0.0002). These data show that defects of HLA class I antigen processing and presentation seem to be significantly more frequent in MSI-H than in MSS colorectal cancer, suggesting that in MSI-H colorectal cancer the immunoselective pressure leads to the outgrowth of cells with defects of antigen presentation.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2005

Preponderance of the oncogenic V599E and V599K mutations in B-raf kinase domain is enhanced in melanoma cutaneous/subcutaneous metastases.

BACKGROUND: Downstream of Ras, the serine/threonine kinase B-raf has been reported to be mutated, among other carcinomas, in a substantial subset of primary melanomas with a preponderance of mutations within the kinase domain including the activating V599E and V599K transitions. METHODS: We here investigated a representative series of 60 resection specimens of cutaneous and subcutaneous melanoma metastases for the presence of mutations within the activation segment (exon 15) of the B-raf kinase domain by polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) gel electrophoresis. RESULTS: Sequencing of cloned PCR-SSCP amplicons resulted in 24 (40%) samples harbouring somatic mutations which is not exceeding the mutation frequency in recently investigated primary melanomas. The activating mutation T1796A was present in 24/60 (40%) resection specimens, followed in frequency by the oncogenic g1795A mutation in 8/60 (13%) cases. As to the B-raf protein sequence, the acidic amino acid transitions V599E and V599K were predicted in 19/60 (32%) and 6/60 (10%) cases, respectively, but were not associated with enhanced risk for subsequent metastasis in patients' follow up. In comparison to the primary melanomas that we recently investigated, the spectrum of predicted B-raf protein mutations narrowed significantly in the cutaneous/subcutaneous metastases. Unexpectedly, V599 and V599E mutations were absent in cutaneous/subcutaneous metastases derived from acrolentiginous melanomas as preceding primary tumours. CONCLUSION: During transition from primary melanomas towards cutaneous/subcutaneous metastases, the spectrum of predicted B-raf mutations narrows significantly. Focusing on the V599E and V599K, these oncogenic mutations are likely to affect melanocyte-specific pathways controlling proliferation and differentiation.

Cell Differentiation↗

PubMed2005

Evidence for distinct pathomechanisms in genetic subgroups of chronic lymphocytic leukemia revealed by quantitative expression analysis of cell cycle, activation, and apoptosis-associated genes.

PURPOSE: In patients with chronic lymphocytic leukemia (CLL), the VH mutation status and genomic aberrations (13q-, +12q, 11q-, 17p-) identify distinct prognostic subgroups. The aim was to elucidate biologic mechanisms through which these genetic markers may exert their pathogenic influence. PATIENTS AND METHODS: Twenty-four genes involved in apoptosis, cell cycle, B-cell activation, and B-cell receptor (BCR) signaling were analyzed by real-time quantitative reverse transcription polymerase chain reaction (RQ-PCR) in 82 CLL cases constituting prototypic genetic CLL subgroups as defined by the VH mutation status and the genomic aberrations 13q-, +12, 11q-, and 17p-. RESULTS: The VH mutation subgroups were characterized by a differential expression of the BCR associated genes ZAP70 and PI3K. Among the subgroups defined by genomic aberrations, there was a deregulation of candidate genes from the affected critical genomic regions such as CDK4 (up), ATM (down), and TP53 (down) in the groups +12, 11q-, and 17p-, respectively. Additionally, the genomic subgroups were characterized by a significant deregulation of cell cycle and apoptosis regulators: AKT (up) in 13q, E2F1 (up) in +12, MYC (up) and BCL-2 (down) in 17p-, and CCND3 (down) in 11q- as well as 17p-. The 17p- subgroup showed an additional down-regulation of BCR-associated genes such as SYK and PI3K. CONCLUSION: The characteristic gene expression patterns observed implicate a differential regulation of cell cycle, apoptosis, and BCR signaling in the genetic subgroups illustrating distinct pathomechanisms and are evidence for a gene dosage effect being operative in CLL. These findings link the biologic diversity and clinical heterogeneity of CLL.

Apoptosis↗

PubMed2005

Duplication of chromosome arms 9q and 11q: evidence for a novel, 14q32 translocation-independent pathogenetic pathway in multiple myeloma.

14q32 translocations [t(14q)] represent critical but not universal events in multiple myeloma (MM). Gains of chromosome arms 1q, 9q, and 11q (+1q, +9q, and +11q) have recently been identified as frequent aberrations in this disease, but their pathogenetic significance remains unclear. We studied a series of 108 MM patients using fluorescence in situ hybridization and DNA probes mapping to chromosome bands 1q21, 9q34, 11q25, 13q14, and 14q32. Three subsets of tumors were defined: (1) MM+/+ (detection of +9q and +11q; 43.5% of cases), (2) MM+/- (+9q or +11q; 21.3%), and (3) MM-/- (neither +9q nor +11q; 35.2%). The incidence of t(14q) was significantly different in these subgroups: 23% in MM+/+, 56% in MM+/-, and 89% in MM-/-. Deletion of 13q (13q-) also was significantly less frequent in MM+/+ (23%) than in MM+/- and MM-/- (36% and 63%, respectively). The nonrandom distribution of chromosomal aberrations in the present series of MM tumors points to a novel, 14q32 translocation-independent pathogenetic pathway in plasma cell neoplasms.

Chromosome Aberrations↗

PubMed2005

Delineation of distinct subgroups of multiple myeloma and a model for clonal evolution based on interphase cytogenetics.

To delineate multiple myeloma (MM) subgroups and their clonal evolution, we analyzed 81 newly diagnosed patients by interphase fluorescence in situ hybridization using a comprehensive probe set for 10 chromosomes and two IGH rearrangements. A median of 5 probes per patient displayed aberrant signal numbers (range, 1-10). Additional copies most frequently found were for 15q22, 19q13, 9q34, 11q23, and 1q21. Losses commonly observed were of 13q14.3, 17p13, and 22q11. Predominance of gain or loss was quantified by a copy number score (CS) for each patient. Two peaks (CS = +3 and CS = 0) were found by plotting patient copy number scores over CS values corresponding to hyperdiploid and nonhyperdiploid MM. Cluster analysis revealed four major branches: (i) gain of 9q, 15q, 19q, and/or 11q; (ii) deletion of 13q and t(4;14); (iii) t(11;14); and (iv) gain of 1q. Statistical modeling of an oncogenetic tree indicated that early independent events were gain of 15q/9q and/or 11q, t(11;14); deletion of 13q followed by t(4;14); and gain of 1q. Aberrations of 17p13, 22q11, 8p12, and 6q21 were found as subsequent events. MM with gain of 1q was delineated as a subentity with significantly higher beta-2-microglobulin and lower hemoglobin levels, indicating a poor prognosis. From our results, we propose a model of MM for clonal evolution.

Evolution, Molecular↗

PubMed2005

Diagnostics of rectal cancer: endorectal ultrasound.

In rectal cancer, accurate preoperative staging is essential to adequately select patients for different therapeutic regimes. Endosonography has been proven to be an accurate staging modality in multiple prospective studies. A recent large retrospective study, however, has cast doubt on the actual accuracy of endorectal ultrasound for staging rectal cancer in everyday clinical routines. The results of endosonographic staging of rectal tumours over a period of 10 years at the Department of Surgery of the University of Heidelberg are presented. In a first time period, 424 patients with rectal cancer were staged by endosonography and the data recorded prospectively. The examinations were exclusively done by four surgeons with high experience and scientific interest in endosonography. The second time period comprises 332 patients with rectal tumours (including adenomas) having undergone endosonography by six different examiners after introduction of this staging method into the clinical routine. The data here were analysed retrospectively. Accuracy, sensitivity, specificity, and positive and negative predictive values were calculated for the T and N classifications for both series. In the second series, eight factors which have been postulated to influence staging accuracy in the literature were included in a regression analysis in order to identify relevant factors for staging inaccuracies. Accuracy for staging of the T classification was 81% in the first series versus 71.7% in the second series. In the regression analysis of the second series, status post-chemoradiation proved to be the most significant factor for staging inaccuracy (p < 0.0002). When excluding all patients having undergone chemoradiation, the accuracy for staging of the T classification rose to 76%. A major problem of endosonography in this second series was overstaging; the T category was overestimated in 76 cases (22.9% of patients). The main error here was overstaging of adenomas as cancerous lesions (45.5% of all adenomas) and T2-cancers as more advanced cancers (42.2% of all T2-cancers). When excluding the adenomas from this analysis, the accuracy increased to 73.5%. Accuracy for staging of the N classification was 76% in the first series versus 71% in the second series. Status post-chemoradiation again was a relevant factor (p < 0.0003); when excluding these patients the accuracy increased to 73%. The accuracy of endosonography for rectal tumours decreases after introduction of the method into the everyday clinical routine. Nonetheless, apart from magnetic resonance imaging with an endorectal coil, rectal endosonography is still the most accurate staging modality for rectal tumours and allows adequate selection of patients for different therapeutic regimes. As the major problem of rectal endosonography is overstaging, more patients are likely to undergo overtreatment rather than undertreatment. Endosonography is inaccurate in staging patients having undergone chemoradiation.

Endosonography↗

PubMed2005

Preponderance of the oncogenic V599E and V599K mutations in the B-raf kinase domain is enhanced in melanoma lymph node metastases.

Downstream of Ras, the serine/threonine kinase B-raf has been reported to be mutated, amongst other carcinomas, in a substantial subset of primary melanomas, with a preponderance of mutations within the kinase domain, including the activating V599E and V599K transitions. We investigated a representative series of 54 resection specimens of melanoma lymph node metastases for the presence of mutations within the activation segment (exon 15) of the B-raf kinase domain by polymerase chain reaction (PCR) and single-strand conformational polymorphism (SSCP) gel electrophoresis. Sequencing of cloned PCR-SSCP amplicons resulted in 24 (44%) samples harbouring somatic mutations, which is not significantly different from the mutation frequency found in recently investigated primary cutaneous melanomas (Deichmann M, Thome M, Benner A, Näher H. B-raf exon 15 mutations are common in primary melanoma resection specimens but not associated with clinical outcome. Oncology 2004; 66:411-419). The activating mutation T1796A was present in 20 (83%) of these resection specimens, followed in frequency by the oncogenic g1795A mutation in five (21%) cases. With regard to the B-raf protein sequence, the acidic amino acid transitions V599E and V599K were predicted in 15 (62%) and five (21%) of the 24 positive metastases, respectively. The detection of mutations at this hot spot codon was significantly associated with subsequent visceral metastasis (P=0.03; Fisher's exact test). During the transition from primary melanomas (see reference above) to lymph node metastases, the spectrum of B-raf mutations narrows significantly (P=0.00047). The oncogenic B-raf mutations V599E and V599K, as early events in melanocyte transformation, persist throughout metastasis with important prognostic implications.

Exons↗

PubMed2004

Microarray-based screening for molecular markers in medulloblastoma revealed STK15 as independent predictor for survival.

Medulloblastoma, a primitive neuroectodermal tumor of the cerebellum, is one of the most common central nervous system malignancies of childhood. Despite aggressive multimodal therapy, including surgery, irradiation, and chemotherapy, 5-year survival rates have only approached 50-60%. To identify potential candidate genes that predict for overall survival (OS), we performed a gene expression profiling analysis in 35 newly diagnosed medulloblastoma neoplasms. Subsequently, the nine most promising candidate genes were analyzed by immunohistochemistry and fluorescence in situ hybridization on tumor tissue microarrays representing a series of 180 tumors. We found 54 genes in which expression levels predicted for unfavorable survival in medulloblastoma. In line with the gene expression profiling analysis, a positive staining for STK15 (P = 0.0006), stathmin 1 (P = 0.001), and cyclin D1 (P = 0.03) was associated with an unfavorable OS, whereas cyclin B1, DAXX, Ki-67, MYC, NRAS, and p53 showed no statistical significant effect. In comparison to clinically defined parameters such as gender, age, metastatic stage, extent of tumor resection, application of chemotherapy, and tumor grade, positive staining for STK15 was identified as an independent prognostic factor for OS (P = 0.026). Moreover, additional gene copy numbers of MYC (P = 0.003) and STK15 (P = 0.05) predicted for poor survival. The combination of gene expression profiling with tissue microarray experiments allowed the identification of a series of candidate genes that predicts for survival in medulloblastoma. Of the results highlighted by the various data analysis procedures, genes associated with cell proliferation (cyclin D1), transcription (MYC), and especially mitosis (stathmin 1, STK15) appear particularly intriguing with respect to medulloblastoma pathomechanism.

Adolescent↗

PubMed2004

Bagging survival trees.

Predicted survival probability functions of censored event free survival are improved by bagging survival trees. We suggest a new method to aggregate survival trees in order to obtain better predictions for breast cancer and lymphoma patients. A set of survival trees based on B bootstrap samples is computed. We define the aggregated Kaplan-Meier curve of a new observation by the Kaplan-Meier curve of all observations identified by the B leaves containing the new observation. The integrated Brier score is used for the evaluation of predictive models. We analyse data of a large trial on node positive breast cancer patients conducted by the German Breast Cancer Study Group and a smaller 'pilot' study on diffuse large B-cell lymphoma, where prognostic factors are derived from microarray expression values. In addition, simulation experiments underline the predictive power of our proposal.

Breast Neoplasms↗

PubMed2004

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.

Acute Disease↗

PubMed2004

Glutathione S-transferase M1 or T1 null genotype as a risk factor for developing multiple primary neoplasms in the upper aero-digestive tract, in Indian males using tobacco.

In this study conducted amongst Indian male tobacco users with upper aero-digestive tract (UADT) squamous carcinoma, 30 patients with multiple primary neoplasms (MPN) were compared with 28 age and sex matched patients with a single primary neoplasm (SPN) for various environmental factors (form of tobacco use, alcohol, radiotherapy for index cancer) and genetic parameters (family history of UADT cancers and GSTT1/GSTM1 genotype). The GSTM1/T1 null genotype, seen in 60% patients with MPN versus 33% patients with SPN (P=0.03) had an odds ratio of 3.7 [CI=1.14-11.99; P=0.03] for developing MPN. Tobacco use in the form of smoking with or without chewing, as opposed to only chewing, and regular alcohol intake were the two other factors with almost three fold increased risk for the development of MPN, although, the effect was not statistically significant. All three patients with a family history of UADT cancer developed MPN, suggesting an inherited predisposition.

Adult↗

PubMed2004

BRAF mutations distinguish anorectal from cutaneous melanoma at the molecular level.

BACKGROUND & AIMS: Anorectal melanoma (AM) is a rare but highly malignant tumor, displaying histologic and immunohistochemical features very similar to cutaneous melanoma (CM). Because BRAF mutations were recently identified in the majority of CM and nevi, we investigated AM for BRAF mutations and mutations of NRAS , an additional component of the MAPK-signalling pathway. METHODS: DNA from formalin-fixed and paraffin-embedded AM was PCR amplified and sequenced. RESULTS: We detected BRAF mutations in 2 of 19 cases and NRAS mutations in none of the cases. Mutations in exon 15 of BRAF were present in only 1 tumor (1 of 19 cases). The A1800T base exchange represented a novel mutation and resulted in a K600N transition in an AM from a 96-year-old white man who presented with rectal bleeding and painful sitting of a few weeks' duration. The second positive AM case, a 69-year-old white man who presented with painless rectal bleeding and clinical symptoms of an intestinal constipation showed a novel missense mutation (C1327T leading to R443W conversion) in BRAF exon 11. None of the AM cases displayed the oncogenic V599E mutation preponderating in CM. CONCLUSIONS: With regard to the frequency of V599E BRAF mutations, AM significantly differs from CM (P < or = .0001), which suggests that BRAF mutations distinguish anorectal from cutaneous melanoma at the molecular level.

Aged↗

PubMed2004

Combined effect of recombinant CD19 x CD16 diabody and thalidomide in a preclinical model of human B cell lymphoma.

Combining different treatment strategies offers the possibility of improving treatment results for cancer patients. The aim of our study was therefore to investigate the combination of treatment of established s.c. human B non-Hodgkin's lymphoma in severe immune deficient mice using a recombinant bispecific CD19 x CD16 diabody (targeting natural killer cells to CD19 cells) and the angiogenesis inhibitor thalidomide. Monotherapy with either thalidomide or diabody caused an approximate 50% reduction in tumor growth rate. The combined treatment showed evidence for a synergistic effect resulting in a 74% reduction in median tumor size. In the combined treatment group, two of five animals had complete remissions of their s.c. tumor. These results suggest that a combination treatment with recombinant diabodies and angiogenesis inhibition represents a useful approach in cancer therapy.

Animals↗

PubMed2004

Post-transplantation tumour load in bone marrow, as assessed by quantitative ASO-PCR, is a prognostic parameter in multiple myeloma.

High-dose therapy (HDT) and autologous transplantation prolongs remission duration and survival in multiple myeloma (MM), but relapse still occurs at a median of 2 years post-HDT. In order to investigate whether the number of residual tumour cells in the bone marrow (BM) after transplantation can predict the duration of response, a quantitative allele-specific oligonucleotide polymerase chain reaction (qASO-PCR) assay was used to measure tumour load in BM at 3-6 months post-HDT in 67 patients. The method of maximally selected log-rank statistics was used to test for the existence of a cut-off value in the BM tumour load data set. A cut-off value with respect to progression-free survival (PFS) was identified (P = 0.001). The estimated threshold for placing patients into a "good" or "bad" prognostic group was 0.015% (n = 22 and 38 respectively) with a median PFS of 64 months vs. 16. Multivariate analysis showed grouping by PCR result to be an independent prognostic factor for PFS (estimated hazard ratio after shrinkage, 3.91). This study identifies for the first time a threshold of the post-HDT tumour load with prognostic significance for PFS in MM. Quantitative molecular assessment thus may help to identify those patients who are in need of further treatment early after autologous transplantation.

Adult↗

PubMed2004

Significant thrombocytopenia associated with the addition of rituximab to a combination of fludarabine and cyclophosphamide in the treatment of relapsed follicular lymphoma.

Fludarabine in combination with cyclophosphamide is an effective treatment for newly diagnosed as well as relapsed follicular lymphoma. The anti-CD20 antibody rituximab has been employed successfully for the same indications. No such data were available on a combined use of these agents. Therefore, we conducted a phase II study to evaluate the safety and efficacy of a combination of rituximab (375 mg/m2), fludarabine (4 x 25 mg/m2) and cyclophosphamide (1 x 750 mg/m2), for the treatment of relapsed follicular lymphoma. An unexpected, severe hematologic toxicity with significant, prolonged thrombocytopenias WHO grade III/IV in 6 (35%) of 17 patients treated in total occurred, leading to early termination of the trial. Cytologic and serologic analyses point toward a direct toxic effect. Older patients (mean age 64.7 vs. 56.5 yr) were significantly (P = 0.02) more likely to suffer from this toxicity, whereas no other clinical or hematologic parameter differed statistically between the patients suffering from thrombocytopenia and those who did not. The addition of rituximab to fludarabine/cyclophosphamide employed at doses given above in relapsed follicular lymphoma may have led to this increase in thrombocytopenias. Therefore, caution should be exercised when combining these drugs for the treatment of patients with relapsed follicular lymphoma, especially when treating older patients.

Adult↗

PubMed2004

B-raf exon 15 mutations are common in primary melanoma resection specimens but not associated with clinical outcome.

Downstream of Ras, the serine/threonine kinase B-raf has recently been reported to be mutated, among other carcinomas, in a majority of melanoma cell lines with a preponderance of mutations within the kinase domain including the activating V599E transition. We therefore investigated a representative number of 50 primary melanoma resection specimens for the presence of mutations within the activation segment (exon 15) of the B-raf kinase domain. Applying polymerase chain reaction and single-strand conformation polymorphism gel electrophoresis, followed by DNA cloning and sequencing, we found 19 cases (38%) to harbor somatic B-raf exon 15 mutations. With respect to the B-raf protein sequence, the V599E mutation was predicted in 63% of these positive melanomas, followed in frequency by the V599K transition (31%). Detection of B-raf exon 15 mutations or prediction of the activating mutation V599E were not statistically associated with the risk for subsequent metastasis in the follow-up of patients. Altogether, the B-raf oncogene is affected in a substantial subset of melanoma resection specimens. As B-raf alterations possibly affect melanocyte-specific pathways controlling proliferation and differentiation, activation of this oncogene may contribute to the development of melanoma.

Cloning, Molecular↗

PubMed2004

Somatic mitochondrial mutations in melanoma resection specimens.

As epidemiological data suggest ultraviolet (UV) radiation to be the major environmental factor for the development of melanoma, we screened this malignancy for UV-specific C right curved arrow T and CC right curved arrow TT DNA mutations as described in squamous and basal cell carcinomas of the skin. Mitochondrial (mt) DNA was addressed which is known to be highly more susceptible for mutations than nuclear DNA. In the mt genome we chose part of the non-coding displacement-loop (D-loop) containing two hypermutable (C)n tracts especially prone for C right curved arrow T and CC right curved arrow TT changes. A total of 69 melanoma resection specimens was investigated by polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) electrophoresis, followed by DNA cloning and sequencing. We detected alterations of the mt D-loop fragment in 4/34 (12%) melanoma primary tumours and in 7/35 (20%) cutaneous and subcutaneous metastases which was no statistically higher proportion upon Fisher's exact test (p=0.17). Among these 11 positive cases, 9 exhibited alterations in the (C)n microsatellite sequences indicating microsatellite instability (MSI) of mtDNA (C)n tracts. Besides 7 insertions and 2 deletions of nucleotides, 11 mutations occurred including only 4 UV-specific C right curved arrow T mutations in a nodular melanoma of the skin and in two subcutaneous metastases. CC right curved arrow TT mutations were not detected in any tissue sample. As (C)n tract alterations occurred in 2/9 primary melanomas with subsequent metastasis, whereas all 20 non-metastasizing cutaneous melanomas were not affected, these somatic changes may reflect genomic imbalance during tumour progression and may be useful for the assessment of patients' prognosis.

Base Sequence↗