The new polyomavirus (MCPyV) does not affect the clinical course in MCCs.

Since 2008, a new polyomavirus (MCPyV) in Merkel cell carcinomas (MCC) has been described, but little is known about its impact on the clinical course. The purpose of this study was to determine the presence of MCPyV in a large sample and to correlate the results with the clinical course of the disease. 59 samples from 44 patients were analysed for the presence of MCPyV using the primers LT3, VP1 and LT1. The clinical records of these patients were evaluated and correlated with the presence of MCPyV. 58% of specimens were positive for MCPyV. Of these, LT3 was positive in 53%, VP1 in 37% and LT1 in 10%. 57% of primary tumours and 53% of metastases were positive for LT3; the numbers for VP1 and LT1 were lower. There was no correlation between the detection of MCPyV in the primary tumour and the appearance of metastases. The survival time was statistically independent from the presence of MCPyV. There is a striking occurrence of MCPyV in MCC, but whether it affects the clinical course remains unclear.

[Cystic desmoplastic small round cell tumor: tumor development from cystic-"mesothelioblastic" areas?]. / Zystischer desmoplastischer klein- und rundzelliger Tumor: Tumorentwicklung aus zystisch-"mesothelioblastischen" Arealen?

A 7-cm cystic lesion in the upper left abdomen and additional smaller solid tumor nodules were diagnosed incidentally in a 15-year-old boy without tumor symptoms. The main tumorous cystic lesion showed a flattened single-cell tumor cell component in gradual transition to stratified, papillary and truly "invasive" typical desmoplastic areas of a desmoplastic small round-cell tumor (DSRCT). The Ki-67-proliferation index gradually increased within three histologic tumor patterns up to about 70% in the typical desmoplastic (infiltrating) component. Using microdissection techniques, EWS-WT1-gene fusion transcripts were detected in the cystic (single-cell-layered), the papillary and the solid tumor proliferations (exon 7 of EWS on chromosome 22 with exon 8 of WT1 on chromosome 11). The presented case illustrates a predominant cystic growth pattern of DSRCT, in which a stepwise development in the pathogenesis of DSRCT from cystic (-"mesothelioblastic") towards a more papillary proliferation and finally typical "infiltrative" desmoplastic tumor pattern might be discussed. The cystic pattern could represent an initial stage in the development of the neoplasia. The presence of specific EWS-WT1-gene fusion transcripts in all tumor growth patterns in this respect would indicate an early event in t(11;22)(p13;q12) translocation in the pathogenesis of DSRCT.

Biochip analysis: status quo.

Biochips are collections of miniaturized test sites (microarrays) arranged on a solid substrate onto which a large number of biomolecules are attached with high density. Like a computer chip performing millions of mathematical operations in a few split seconds, a biochip allows for simultaneous analyses of thousands of biological reactions, such as decoding genes, in a few seconds. Biochip technologies can be applied to numerous fields including genomic, proteomic, and glycomic research, as well as pharmacology and toxicology. However, one of the most common applications is in the determination of gene expression in human cells and tissues. Global gene expression analysis has helped to identify important genes and signalling pathways in human malignant tumors. And there is hope that microarrays will make the step from "the (laboratory) bench to the bedside (of the patient)". Recent studies have indeed revealed that analysis of differential gene expression by microarrays may help to identify subtypes of malignant tumors, that allow a risk stratification of the patients. However, there are several issues that need to be addressed before microarrays may become a tool for routine diagnostics, such as problems with bioinformatic analysis, construction of disease or tissue specific microarrays with only limited numbers of genes of interest, standard operation procedures for tissue preparation to prevent RNA degradation, etc.. In this article, an overview over of the multifarious biochip applications and technologies, its limitations, challenges and future developments is provided.

Establishment of an in vivo model for pediatric Ewing tumors by transplantation into NOD/scid mice.

Ewing tumors are a clinically heterogeneous group of childhood sarcomas that represent a paradigm for understanding solid tumor biology, as they are the first group of sarcomas for which a chromosome translocation has been characterized at the molecular level. However, the biologic organization of the tumor, especially the processes that govern proliferation, differentiation, and metastasis of primitive tumor stem cells is poorly understood. Therefore, to develop a biologically relevant in vivo model, five different Ewing tumor cell lines and primary tumor cells from three patients were transplanted into immune-deficient mice via intravenous injection. NOD/scid mice that carry a complex immune deficiency and thus nearly completely lack the ability to reject human cells were used as recipients. Overall, 26 of 52 mice (50%) transplanted with VH-64, WE-68, CADO-ES1, TC-71, and RM-82 cells and 4 of 10 mice (40%) transplanted with primary tumor cells engrafted. Moreover, primary cells that did not grow in vitro proliferated in mice. The pattern of metastasis was similar to that in patients with frequent metastases in lungs (62%), bone marrow (30%), and bone (23%). Using limiting dilution experiments, the frequency of the engraftment unit was estimated at 1 Ewing tumor-initiating cell in 3 x 10(5) VH-64 cells. These data demonstrate that we have been able to establish an in vivo model that recapitulates many aspects of growth and progression of human Ewing tumors. For the first time, this model provides the opportunity to identify and characterize primitive in vivo clonogenic solid tumor stem cells. This model will, therefore, be instrumental in studying many aspects of tumor cell biology, including organ-selective metastasis and tumor angiogenesis.

Cytogenetic analysis of multifocal bladder cancer supports a monoclonal origin and intraepithelial spread of tumor cells.

Bladder cancer is often characterized by a multifocal growth pattern. This observation has given rise to the hypothesis of "field cancerization," predicting a polyclonal origin of multiple tumors rising from an area of independently transformed mucosa cells. On the other hand, genetic studies suggested a monoclonal origin. To address these contradictory hypotheses, we performed comparative genomic hybridization (CGH) on 32 tumors originating from six bladder cystectomy specimens. All tumors derived from the same patient showed a set of 7-13 identical chromosomal aberrations and additional individual alterations. Most striking were the findings of 17p losses in all (32 of 32) tumors of the six cystectomy specimens and 20p gains in all tumors of four bladders, as well as an unexpected high number of chromosomal changes (20.4 alterations per tumor on average). To clarify a possible role of the TP53 tumor suppressor gene on 17p13, we applied immunohistochemistry and sequence analysis on the tumors and additional 52 mucosa samples. Identical TP53 mutations and protein overexpression was found in individual tumors only as well as in mucosa samples from continuous areas. Our results not only provide further evidence for a monoclonal origin of multifocal bladder cancer but also point at intraepithelial migration of tumor cells carrying specific chromosomal aberrations.

Quality assurance in RT-PCR-based BCR/ABL diagnostics--results of an interlaboratory test and a standardization approach.

Here we describe the results of an interlaboratory test for RT-PCR-based BCR/ABL analysis. The test was organized in two parts. The number of participating laboratories in the first and second part was 27 and 20, respectively. In the first part samples containing various concentrations of plasmids with the ela2, b2a2 or b3a2 BCR/ABL transcripts were analyzed by PCR. In the second part of the test, cell samples containing various concentrations of BCR/ABL-positive cells were analyzed by RT-PCR. Overall PCR sensitivity was sufficient in approximately 90% of the tests, but a significant number of false positive results were obtained. There were significant differences in sensitivity in the cell-based analysis between the various participants. The results are discussed, and proposals are made regarding the choice of primers, controls, conditions for RNA extraction and reverse transcription.

Genetic relation of lobular carcinoma in situ, ductal carcinoma in situ, and associated invasive carcinoma of the breast.

AIMS: The mutual relation of lobular carcinoma in situ (LCIS) and ductal carcinoma in situ (DCIS) of the breast, as accepted precursor lesions of invasive breast cancer, is controversial. Because they display genetic heterogeneity, it is not clear how genetically advanced these entities are and what causes the transition to an invasive carcinoma. METHODS: Six cases of LCIS, four of them with associated lobular invasive carcinoma, four cases of intermediately differentiated DCIS with an associated invasive lobular carcinoma, and nine cases of intermediately and poorly differentiated DCIS with associated ductal invasive carcinoma were investigated by means of comparative genomic hybridisation (CGH) after microdissection and immunohistochemical staining of E-cadherin. RESULTS: LCIS was characterised by a low average rate of copy number changes, no evidence of amplifications, and a high rate of gains and losses of chromosomal material at 1q and 16q, respectively. A high degree of genetic homology with well differentiated DCIS was obvious, as reported previously. The cases of intermediately differentiated DCIS with associated lobular invasive components and lobular differentiation revealed striking homologies, and a significant difference of E-cadherin expression. The comparison of preinvasive and invasive breast lesions, irrespective of differentiation within the same patient, revealed no specific alteration that might be associated with invasion. Genetic alterations seen in invasive carcinoma were not necessarily seen in the adjacent precursor lesions. CONCLUSIONS: These results provide strong evidence that invasive breast cancer is a disease with multiple cytogenetic subclones already present in preinvasive lesions. Moreover, specific CGH alterations associated with invasion were not observed. Furthermore, the close genetic association between well differentiated and a subgroup of intermediately differentiated DCIS and LCIS led to the hypothesis that LCIS and a subgroup of DCIS are different phenotypic forms of a common genotype.

Temporary remission of an alveolar rhabdomyosarcoma diagnosed and treated as acute leukemia.

A 29-year-old man with alveolar rhabdomyosarcoma was considered to be suffering from acute leukemia. A bone marrow aspirate had revealed extensive infiltration by atypical blast-like cells which were interpreted as acute lymphoblastic leukemia. Although there was no confirmation of this diagnosis by immunophenotyping chemotherapy with a protocol suited for the treatment of acute lymphoblastic leukemia was started prior to histological analysis and resulted in a complete temporary remission after the first cycle. Histological analysis of a bone marrow biopsy revealed an alveolar rhabdomyosarcoma, as further confirmed by molecular genetic analysis. Two months after the end of chemotherapy, there was an extensive recurrence and the patient died one year after initial diagnosis with chemotherapy refractory disease. In conclusion, rhabdomyosarcoma should always be included in the differential diagnosis of systemic diseases with extensive bone marrow infiltration by tumor cells which could otherwise be misinterpreted as a haematological malignancy.

Telomerase is a highly sensitive and specific molecular marker in fine-needle aspirates of breast lesions.

PURPOSE: Telomerase has been detected in a majority of human malignant tumors, making telomerase activity (TA) one key difference between mortal and immortal cells. In this study, we evaluated in blind-trial fashion the association of TA with cytologic and final clinical/pathologic diagnosis in fine-needle aspirates (FNAs) of breast lesions. MATERIALS AND METHODS: In 172 FNAs, including 80 samples that were cytologically malignant, 18 that were atypical but not diagnostic for malignancy, and 74 that were cytologically benign, TA was determined by a modified nonradioactive telomeric repeat amplification protocol (TRAP) assay. Final diagnosis was made by pathologic examination of follow-up surgical material available for all the cytologically malignant samples, a majority of the cytologically atypical samples, and a portion of the cytologically benign samples. RESULTS: TA was detected in 85 of 172 samples. Comparison of the cytologic and histologic diagnoses with TA showed that 80 of 87 samples from patients with breast cancer were telomerase-positive, resulting in a sensitivity of 92%. TA was found in four of five FNAs from carcinomas that were considered cytologically atypical but not diagnostic for malignancy. Eighty of 85 samples from patients with benign breast lesions were telomerase-negative, revealing a specificity of 94%. The five positive cases in this group were all fibroadenomas with low TA. Among the 18 cases with a cytologic diagnosis of atypia, there was a strong positive relationship between TRAP findings and histologic diagnosis. CONCLUSION: The detection of TA in FNAs of breast lesions is a highly sensitive and specific marker of malignancy and may be used as an adjunct in cases with an equivocal cytologic diagnosis.

Telomerase activity distinguishes between neuroblastomas with good and poor prognosis.

BACKGROUND: Treatment of neuroblastoma has remained a major challenge in pediatric oncology because the assessment of the individual prognosis, particularly in disseminated disease is still obscure. Previous studies have correlated clinical outcome with activity levels of telomerase, a cellular reverse transcriptase which has been detected in the majority of human malignant tumors. PATIENTS AND METHODS: In this blind-trial study, a non-radioactive telomeric repeat amplification protocol (TRAP) with an internal telomerase-assay standard was used on an automated laser fluorescence sequencer for the detection and semiquantitative analysis of telomerase activity (TA) in 67 neuroblastomas of all clinical stages from the German Neuroblastoma Trial and 2 ganglioneuromas. TA levels were correlated with event-free and overall survival rates and established prognostic markers such as MYCN. RESULTS: TA was present in 14 of 69 (20%) samples, including 3 of 22 stage IVS, 8 of 14 stage IV, 1 of 10 stage III, 1 of 7 stage II and 1 of 14 stage I neuroblastomas and 0 of 2 ganglioneuromas. We found a strong statistical correlation between the presence of TA and poor clinical prognosis with regard to all tumor stages. Multivariate analysis revealed TA as an independent prognostic marker. In particular, the analysis of TA in IVS neuroblastomas distinguished two different prognostic groups. CONCLUSIONS: Our data suggest that TA is an independent prognostic marker in neuroblastoma which, in combination with other markers such as MYCN, may proof useful in assessing the individual patient's prognosis.

Different genetic pathways in the evolution of invasive breast cancer are associated with distinct morphological subtypes.

Invasive breast cancer shows a wide range of morphological differentiation, associated with differences in prognosis, but as yet, the underlying genetic mechanisms cannot be accounted for. In order to establish a model of the possible progression from the different subtypes of ductal carcinoma in situ (DCIS) to invasive breast cancer, 77 selected cases of invasive breast cancer representing distinct morphological subtypes were investigated by means of comparative genomic hybridization (CGH). There was a high degree of genetic homology between tubular and tubulo-lobular carcinoma and well-differentiated DCIS, and between ductal invasive carcinoma G3 and poorly differentiated DCIS. Highly differentiated invasive breast cancers were characterized by a loss of 16q and a low average number of aberrations per case. In high-grade tumours, losses of this chromosomal region were seen with a much lower frequency in cases with evidence of an aneuploid tumour status. These data demonstrate the close genetic similarity of well-, intermediately, and poorly differentiated DCIS and distinct morphological types of invasive breast carcinoma, providing further evidence that DCIS is a direct precursor lesion of invasive breast cancer and that various evolutionary genetic pathways exist.

PCR based detection of mycobacteria in paraffin wax embedded material routinely processed for morphological examination.

BACKGROUND: The incidence of mycobacterial infections has increased during the past five years. A prompt diagnosis is indispensable for initiating appropriate treatment. Because culturing of mycobacteria takes three to six weeks and sensitivity of microscopic detection of acid fast bacilli is low, amplification methods provide promising possibilities. Recently, the polymerase chain reaction (PCR) has been shown to be useful for confirming a mycobacterial infection, especially in cases with unexpected histological findings or lack of suitable material for culturing. AIMS: To evaluate the impact of PCR based techniques in the detection of mycobacterial infections in uncultured routine histological specimens as an alternative to surgical pathology. METHODS: Two hundred and twenty nine formalin fixed and paraffin wax embedded samples from 141 patients with clinical or histological suspicion of a mycobacterial infection were investigated using three different PCR assays and Southern blotting. PCR results were compared with histology and culture and the patients' clinical findings. RESULTS: When using culture as the reference method, the sensitivity for the detection of mycobacteria of the tuberculosis complex was 90%, specificity was 92%, the positive predictive value was 81%, and the negative predictive value was 96%. The sensitivity for the detection of nontuberculous mycobacteria was 100% and specificity was 78%, the positive predictive value was 26%, and the negative predictive value was 100%. The patients' clinical findings supported the PCR positive results, indicating a mycobacterial infection in 11 of 18 initially culture negative cases and in 21 of 35 PCR positive cases without culture results. CONCLUSIONS: These results indicate that PCR based techniques are sensitive, specific, and rapid methods for the detection of mycobacteria in routinely processed paraffin wax embedded and formalin fixed histological samples.

Comparative genomic hybridization and telomerase activity analysis identify two biologically different groups of 4s neuroblastomas.

Chromosomal aberrations of 20 stage 4s neuroblastomas were analysed by comparative genomic hybridization (CGH). In a subset of 13/20 tumours, telomerase activity was evaluated by the telomeric repeat amplification protocol (TRAP). The CGH data were compared with the CGH results of ten stage 1 and 2 (stage 1/2) and 22 stage 3 and 4 (stage 3/4) neuroblastomas. A total of 17/20 stage 4s neuroblastomas did not progress clinically, whereas tumour progression with lethal outcome occurred in 3/20 cases. The CGH data of clinically non-progressing stage 4s tumours revealed a high rate of whole-chromosome aberrations (73.4%) with an overrepresentation of mainly chromosomes 2, 6, 7, 12, 13, 17, 18 and an underrepresentation of mainly chromosomes 3, 4, 11, 14. MYCN amplification or 1p deletion was observed in only 1/27 or 2/17 clinically non-progressing stage 4s tumours respectively, whereas all three progressive stage 4s neuroblastomas showed MYCN amplification, 1p deletion and, in 2/3 cases, distal 17q gains. Except for one case, telomerase activity was not observed in non-progressing stage 4s neuroblastomas. In contrast, 4s tumours with lethal outcome revealed elevated telomerase activity levels. Our data suggest that stage 4s neuroblastomas belong to two biologically different groups, one of which displays the genetic features of localized stage 1/2 tumours, whereas the other mimics advanced stage 3/4 neuroblastomas.

Telomerase activity in human proliferative breast lesions.

Telomerase, a cellular reverse transcriptase, has been detected in the majority of human malignant tumors, where it provides an escape mechanism from proliferative limitations due to progressive telomere erosion with each cell division. In this study, we used a non-radioactive telomeric repeat amplification protocol (TRAP) with an internal telomerase assay standard for the detection and semiquantitative analysis of 98 single frozen sections of normal breast tissue and benign and malignant breast lesions on an automated laser-fluorescence sequencer. Telomerase activity was detected in 36 of 40 (90%) infiltrating breast carcinomas, whereas no activity was found in nonmalignant breast tissues including blunt duct adenosis, papilloma, ductal hyperplasia and atypical ductal hyperplasia. However, telomerase activity was detected in 59% of ductal in situ carcinomas, suggesting that telomerase reactivation is an early event in breast carcinogenesis. We found a positive correlation between telomerase activity levels and cell proliferation determined by MIB1 immunostaining. No correlation, however, could be demonstrated between telomerase activity and other known breast cancer prognostic indicators. Telomerase activity was also detected in 60% of fibroadenomas indicating that careful interpretation of analysis of telomerase activity in fine needle aspirates is required, since low telomerase activity may not necessarily be an indicator of malignancy in breast tissue.

[Evidence of genetic alterations in chromosome 11 in embryonal and alveolar rhabdomyosarcoma]. / Nachweis genetischer Veränderungen auf Chromosom 11 bei embryonalen und alveolären Rhabdomyosarkomen.

Various chromosome 11 alterations have been described in rhabdomyosarcoma (RMS). Allelic losses of 11p15.5 are characteristic of embryonal RMS (eRMS), whereas an increase in the expression of the Igf2 gene located on 11p15.5 has regularly been observed in eRMS and alveolar RMS (aRMS). The aim of our study was to analyse chromosome 11 alterations of RMS by combining different molecular-genetic and cytogenetic methods. 16 RMSs (7 aRMS with proven 2;13 or 1;13 translocations, 9 eRMS) were studied by a PCR-based microsatellite analysis of loci 11p15.5 and 11q23. Comparative genomic hybridization (CGH) was performed in 8/16 cases. The ploidy status of chromosome 11 was evaluated using the FISH technique. A RT-PCR analysis of Igf2 gene region was performed to evaluate the imprinting status. Allelic losses (LOH) of 11p15.5 were observed in 4/7 aRMS and 8/9 eRMS. These losses were accompanied by LOH of 11q23 in 2/7 aRMS and 5/9 eRMS, respectively. CGH of all the eRMSs and one aRMS studied revealed gains of genetic material mostly involving the entire length of chromosome 11. One aRMS showed a loss of chromosome 11 material, both in CGH and LOH analysis. In 2/9 aRMS, which neither in CGH nor in LOH analysis had exhibited chromosome 11 alterations, biallelic IGF-II expression was revealed. Our results show that chromosome 11 alterations play a major role in the biology of both alveolar and eRMS. Combining the data of our study, we demonstrated that three different chromosome 11 alterations are involved in the tumorigenesis of RMS: 1) LOH resulting in hemizygosity of chromosome 11. 2) LOH with simultaneous gains of chromosome 11 material due to uniparental polysomy. 3) loss of imprinting for the Igf2 gene in the absence of gross chromosome 11 alterations.

[The demonstration of the radiation-induced production of tumor necrosis factor-alpha in Ewing's sarcoma RM 82 in vitro and in vivo]. / Nachweis einer strahleninduzierten Produktion von Tumor-Nekrose-Faktor alpha im Ewing-Sarkom RM 82 in vitro und in vivo.

AIM: The expression of cytokines plays an important role in the transmission of the effects of ionizing radiation to tumor cells and normal tissue. Tumor necrosis factor alpha (TNF alpha), a pleiotropic monokine, is of special interest because of its cytotoxic effect on tumor cells and the induction of hemorrhagic necrosis in tumors. We examined the influence of ionizing radiation on TNF alpha production in a human Ewing's sarcoma cell line in vitro and in vivo. METHODS: The protein and mRNA levels of the Ewing's sarcoma cell line RM 82 were examined in vitro with "Enhanced Amplified Sensitivity Immunoassay" (EASIA) and semiquantitative RT-PCR before and after treatment with single doses of 2 to 40 Gy, 1 to 72 hours after irradiation. After successful transplantation to nude mice, the time and dose correlation of TNF alpha mRNA production was examined in vivo. RESULTS: In vitro, RM 82 had a basal protein level of TNF alpha of 20.1 +/- 4.3 pg/ml/10(6) cells. We observed a time- and dose-dependent increase of TNF alpha expression with a maximum of 125 pg/ml/10(6) (5.9 fold) 24 hours after irradiation with 20 Gy. At the mRNA level, the maximal up-regulation occurred 6 to 12 hours after 10 Gy. In vivo, the xenograft tumor maintained the capacity of TNF alpha expression. Time- and dose-dependency in mRNA production showed a maximum increase 6 hours after treatment with 10 Gy. CONCLUSIONS: The presented experiments show in vitro a dose- and time-dependent up-regulation of TNF alpha in the Ewing's sarcoma cell line RM 82 on protein and mRNA level. For the first time this phenomenon was also observed in vivo in a human xenograft tumor. This tumor model could be used for further experiments to examine the role of TNF alpha as a biologic radiation response modifier in human tumors.

Assessment of molecular genetic detection of chromosome translocations in the differential diagnosis of pediatric sarcomas.

BACKGROUND: Recent studies have shown that many types of soft-tissue sarcomas are characterized by specific chromosomal translocations, which are likely to be of etiologic significance. In order to evaluate their diagnostic impact, a panel of 129 sarcomas comprising 78 Ewing's tumors (ET), 19 rhabdomyosarcomas (RMS), 20 neuroblastomas (NB), 9 synovialsarcomas, 2 esthesioneuroblastomas, and 1 desmoplastic small-round-cell tumor (DSRCT) were analysed for the occurrence of the major recurrent translocations, such as t(11;22)(q24;q12), t(21;22)(q22;q12), t(11;22)(p13;q12), t(2;13)(q35;q14), t(1;13)(p36;q14), and t(X;18)(p11;q11). METHODS: Nitrogen-frozen tissue material was analysed by means of Reverse Transcription followed by PCR (Polymerase-Chain Reaction) and nested PCR (RT-PCR). Specificity of the PCR products obtained was confirmed by non-isotopic Southern-Blot analysis with gene-specific probes and/or automated direct sequence analysis. RESULTS: 75 ETs have been shown to carry either a t(11;22) or t(21;22) translocation by identification of chimeric EWS-FLI-1 or EWS-ERG gene-fusion transcripts respectively. 3 ETs were lacking EWS/FLI-1 or EWS-ERG fusion products. 2 of these tumors were shown on review to have unusual morphological features for ETs. 8/19 RMS were initially diagnosed as alveolar RMS. These tumours were shown to carry either a t(2;13) translocation exhibiting chimeric PAX3-FKHR fusion transcripts or a t(1;13) translocation with PAX7-FKHR chimeric gene products. One RMS of the embryonal group also carried a t(1;13) translocation. Reevaluation demonstrated a partly alveolar morphology. In 8/9 synovial sarcomas a t(X;18) translocation was identified. Expression of a EWS-WTI gene-fusion product associated with a t(11;22) translocation was found in the DSRCT. None of these rearrangements were detected in the NBs and 2 esthesioneuroblastomas. CONCLUSIONS: Our results support the concept that the major recurrent translocations are histogenetically specific for a subset of sarcomas. Thus, the detection of tumor type-specific translocations represents an extremely useful diagnostic modality as an adjunct to surgical pathology.

A new germline TP53 gene mutation in a family with Li-Fraumeni syndrome.

This report describes an unusual clinical presentation of Li-Fraumeni syndrome. Family history revealed a mild aggregation of adult cancers in one generation, and an unusual clustering of brain tumours of early childhood in the following generation. In order to evaluate the genetic basis for cancer predisposition in this family, molecular genetic analysis for the occurrence of germline TP53 tumour suppressor gene mutations was performed on 12 siblings of two generations. Indirect mutation analysis was performed by the single-strand conformation polymorphism (SSCP) technique. Alterations were characterised by automated direct fluorescence sequencing analysis. Tumour material was also examined for p53 protein accumulation by immunohistochemistry. Initially, a TP53 gene germline missense mutation was detected in an 11-year-old kindred with acute myeloid leukaemia (AML) following intensive treatment of a brain tumour. In peripheral blood and bone marrow samples of this proband, a reduction to hemizygosity occurred. During AML treatment, detection of LOH of 17p was used as a marker for clonality and treatment control. The mutation was found to be inherited from the proband's mother, who was diagnosed with breast cancer at the age of 48 years. Further, three siblings were carriers, and two are apparently healthy at the age of 21 and 23 years. Knowledge of germline mutations may allow accurate DNA-based carrier diagnosis which is of important clinical significance for treatment strategy and control. Furthermore, the occurrence of unaffected carriers in this family raises questions about appropriate methods of cancer surveillance and counselling for these people.

Discordance, in a malignant hyperthermia pedigree, between in vitro contracture-test phenotypes and haplotypes for the MHS1 region on chromosome 19q12-13.2, comprising the C1840T transition in the RYR1 gene.

A point mutation in the gene encoding the skeletal muscle calcium release channel (RYR1) has been proposed as the probable cause of malignant hyperthermia (MH) in swine, where it segregates with the disease in all MH-prone strains investigated. The same C-to-T exchange in nucleotide position 1840 of the human RYR1 cDNA sequence was found in a few human MH pedigrees. We report a German MH pedigree where in vitro contracture test (IVCT) results and haplotypes of markers for the MHS1/RYR1 region including this base transition have yielded several discrepancies. The MH-susceptible phenotype was defined by IVCT performed according to the European standard protocol. Haplotypes were constructed for markers for the MHS1/RYR1 region on chromosome 19 and include the C1840T base exchange. Discussing the probabilities for a number of hypotheses to explain these data, we suggest that our results may challenge the causative role of this mutation--and possibly the role of the RYR1 gene itself--in human MH susceptibility, at least in some cases.