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PubMed2005

Ph-Chromosome-positive chronic myeloid leukemia with associated bone marrow mastocytosis.

The concurrent development of chronic myeloid (CML) or myelomonocytic (CMML) leukemia in patients with systemic mastocytosis (SM) is a well recognized phenomenon. Although the leukemia often resembles CML in morphological and in clinical terms, a Ph-Chromosome-positive variant has not been reported in SM so far. We here describe a 43-year-old female patient with typical Ph-Chromosome-positive CML in whom a co-existing bone marrow mastocytosis, a special subvariant of SM, was diagnosed. RT-PCR analysis revealed the typical p210 kDa form of BCR/ABL in leukemic cells. The diagnosis SM was based on the typical focal aggregates of spindle-shaped mast cells (MC) in the bone marrow, expression of CD25 in MC, and the c-kit mutation D816V, which was detectable in microdissected bone marrow MC, but not in microdissected leukemic cells, suggesting the presence of two different (sub)clones of neoplastic cells. Therapy with the BCR/ABL-targeting drug Imatinib (STI571) resulted in complete cytogenetic remission of CML. Together, our case provides further evidence for the biologic diversity of leukemias that may occur in patients with SM. The exact knowledge of the pathology and target-profile of the associated leukemias in SM have important therapeutic implications.

Adult↗
PubMed2005

Bradykinin B2 type receptor activation regulates fluid and electrolyte transport in the rabbit kidney.

Bradykinin is an important autacoid produced in the kidney, regulating both renal function and blood pressure. In vivo studies in anesthetized rabbits, revealed that BK induced diuresis (UV), natriuresis (U(Na)V) and was not associated with renal hemodynamic changes. These diuretic and natriuretic effects were blocked by the BK-B2 antagonist HOE-140. BK also inhibits vasopressin (AVP)-stimulated water flow (L(p)) in microperfused rabbit cortical collecting ducts (rCCD), in a concentration-dependent fashion, consistent with its in vivo diuretic effects. BK-B1 antagonist Leu8-des-Arg9-BK did not alter the effect of BK on Lp, but HOE-140 completely blocked the inhibitory effects of BK on Lp. While BK did not increase [Ca2+]i in fura-2 loaded freshly microdissected rCCD, BK increased [Ca2+]i in immortalized cultured rCCD cells demonstrating different signaling mechanisms are activated by BK in microdissected versus cultured rCCD. In microperfused rCCD, neither the protein kinase C inhibitor staurosporine nor the phospholipase C (PLC) inhibitor U-73,122 attenuated the BK response arguing against activation of PLC/PKC by BK in rCCD. We conclude: (1) BK induces UV and U(Na)V by a BK-B(2) receptor; (2) BK inhibits AVP-stimulated Lp by a BK-B2 receptor suggesting that its effects on Lp are not via a PLC/PKC; (3) finally, BK raises [Ca2+]i in rCCD cells by a BK-B2 receptor mechanism.

Animals↗
PubMed2004

Regulation of apoptosis by peroxisome proliferators.

Peroxisome proliferators (PPs) constitute a large and chemically diverse family of non-genotoxic rodent hepatocarcinogens that activate the PP-activated receptor alpha (PPARalpha). In order to investigate the hypothesis that PPs elicit their carcinogenic effects through the suppression of apoptosis, we established an in vitro assay for apoptosis using both primary rat hepatocytes and the FaO rat hepatoma cell line. Apoptosis was induced by transforming growth factor beta1 (TGFbeta1), the physiological negative regulator of liver growth. In this system, PPs could suppress both spontaneous and TGFbeta1-induced apoptosis. In order to understand the mechanisms of this regulation of apoptosis, we conducted microarray analysis followed by pathway-specific gene clustering in TGFbeta1-treated cells. After treatment, 76 genes were up-regulated and 185 were down-regulated more than 1.5-fold. Cluster analysis of up-regulated genes revealed three clusters, A-C. Cluster A (4h) was associated with 12% apoptosis and consisted of genes mainly of the cytoskeleton and extracellular matrix such as troponin and the proteoglycan SDC4. In cluster B (8h; 25% apoptosis), there were many pro- and anti-apoptotic genes such as XIAP, BAK1 and BAD, whereas at 16h (40% apoptosis) the regulated genes were mainly those of the cellular stress pathways such as the genes implicated in the activation of the transcription factor NFkappab. Genes found down-regulated in response to TGFbeta1 were mainly those associated with oxidative stress and several genes implicated in glutathione production and maintenance. Thus, TGFbeta1 may induce apoptosis via a down regulation of oxidant defence leading to the generation of reactive oxygen species. The ability of PPs to impact on these apoptosis pathways remains to be determined. To approach this question, we have developed a technique using laser capture microdissection of livers treated with the PP, clofibric acid coupled with gene expression array analysis. Results show that some of the key steps of the LCM process had an impact on the gene profiles generated. However, this did not preclude accurate determination of a PP-specific molecular signature. Thus, the choice of appropriate controls will ensure that meaningful gene expression analyses can be performed on tissue microdissected from the foci generated in clofibric acid treated livers. These data will allow the identification of specific genes that are regulated by PPs leading to changes in apoptosis and ultimately to tumours.

Animals↗
PubMed2006

Aging may adversely affect testicular sperm recovery in patients with Klinefelter syndrome.

OBJECTIVES: To investigate the clinical parameters or laboratory analyses that may have a predictive value for successful sperm retrieval in Klinefelter syndrome (KS). METHODS: A total of 74 patients with nonmosaic KS were included in this study. All patients were azoospermic and underwent microdissection testicular sperm extraction for sperm recovery. The predictive values of patient age, testicular volume, serum follicle-stimulating hormone, luteinizing hormone, and testosterone levels were assessed for successful sperm recovery. RESULTS: Testicular sperm recovery was successful in 42 (56.7%) of 74 men. The serum follicle-stimulating hormone, luteinizing hormone, and total testosterone levels did not show any difference between the patients with successful and those with unsuccessful sperm recovery. However, the patients with successful sperm recovery were significantly younger (31.6 +/- 4.3 years) than those with failed attempts (35 +/- 5.1 years, P = 0.002). In the receiver operating characteristics curve analysis, a cutoff of 30.5 years of age had a sensitivity of 78% and a specificity of 48% for successful sperm retrieval. Logistic regression analysis showed that sperm recovery was inversely related to patient age (odds ratio 0.854, 95% confidence interval 0.76 to 0.95). CONCLUSIONS: Microdissection testicular sperm extraction is an effective sperm recovery technique in patients with KS. Our data have demonstrated that aging might adversely affect the sperm recovery rate in men with KS. These results suggest that earlier infertility assessment and testicular sperm extraction in men with KS might play a critical role in their treatment.

Adult↗
PubMed2004

High-accuracy amplification of nanogram total RNA amounts for gene profiling.

Microarray-based gene profiling of laser-assisted microdissected tissues or clinical biopsies is still a challenge since the amount of total RNA in such samples is limited and amplification of RNA is mandatory. Representative amplification of mRNA is highly dependent on the reverse transcription reaction, which is error prone, and on the number of amplification cycles. To improve the accuracy of RNA amplification, we optimized, combined, and tested different amplification strategies for Affymetrix oligonucleotide array hybridization. We demonstrate that different protocols differ significantly in quality of mRNA amplification. To demonstrate the accuracy and reproducibility of our optimized protocol in a clinical setting, we analyzed total RNAs from laser-assisted, microdissected cells of human prostate tissues. On the basis of these results, we recommend a standard reverse transcription reaction for small-sample-transcriptome profiling experiments as part of the Minimal Information about a Microarray Experiment (MIAME) set of standards.

Animals↗
PubMed2005

High-resolution array CGH increases heterogeneity tolerance in the analysis of clinical samples.

Recent advances in array comparative genomic hybridization (array CGH) technology are revolutionizing our understanding of tumor genomes. Marker-based arrays enable rapid survey at megabase intervals, while tiling path arrays examine the entire genome in unprecedented detail. Tumor biopsies are typically small and contain infiltrating stromal cells, requiring tedious microdissection. Tissue heterogeneity is a major barrier to high-throughput profiling of tumor genomes and is also an important consideration for the introduction of array CGH to clinical settings. We propose that increasing array resolution will enhance detection sensitivity in mixed tissues and as a result significantly reduce microdissection requirements. In this study, we first simulated normal cell contamination to determine the heterogeneity tolerance of array CGH and then validated this detection sensitivity model on cancer specimens using the newly developed submegabase resolution tiling-set (SMRT) array, which spans the human genome with 32,433 overlapping BAC clones.

Biomarkers, Tumor↗
PubMed2004

Allelic imbalances in endometrial stromal neoplasms: frequent genetic alterations in the nontumorous normal-appearing endometrial and myometrial tissues.

OBJECTIVES: Endometrial stromal sarcoma (ESS) is among the rarest primary malignant tumors of the uterus. The aim of this study was to examine the possibility of loss of heterozygosity (LOH) and microsatellite instability (MIS) in different tissue components of ESS. METHODS AND MATERIALS: Using PCR, we examined DNA extracts from microdissected tissues of 27 uterus samples containing malignant stromal cells of ESS (20 low grade and 3 high grade sarcomas), benign tumor cells of endometrial stromal nodules (ESN, 4 cases) as well as tumor-free myometrial and endometrial tissues close to and distant from the tumors. Normal cervical tissues (epithelial cells, stroma cells) were also microdissected and analyzed. Fifteen polymorphic DNA markers (chromosomes 2p, 3p, 5q, 10q, 11q, 13q, and 17p) were tested to identify possible genetic alterations. Samples from 10 women with prolapsed uteri without any histopathologic abnormalities were also selected as controls. RESULTS: While no genetic alterations could be identified in 12 (44.5%) ESS cases, 15 (55.5%) revealed LOH with at least one polymorphic DNA marker. LOH were found in 3 (100%) high-grade sarcomas, 10 (50%) low-grade ESS, and 2 (50%) benign ESN. Although LOH was found more often in the neoplastic stromal cells, several cases showed concurrent and independent LOH in the tumor-free myometrial or endometrial tissues either close to or distant from the tumors. The most common genetic abnormality (LOH) was observed at PTEN, a tumor suppressor gene located on chromosome 10q. No tumor was associated with microsatellite instability (MSI). The control group without any histologic abnormalities did not show LOH or MSI. CONCLUSIONS: The frequent occurrence of LOH and the lack of MSI suggest that loss of function(s) of tumor suppressor genes and not mismatch repair deficiency plays a key role in the pathogenesis of endometrial stromal neoplasms. The concurrent and independent occurrence of LOH in the stromal tumor cells and the tumor-free and normal-appearing myometrial and endometrial tissues strongly support the concept of genetic alterations in microenvironmental tissues and the interaction(s) between different tissue components in the development and progression of endometrial stromal neoplasms.

Endometrial Neoplasms↗
PubMed2005

Clinical applications of microarray technology: creatine kinase B is an up-regulated gene in epithelial ovarian cancer and shows promise as a serum marker.

OBJECTIVE: (1) To identify and (2) validate genes that are up-regulated in ovarian cancer, and (3) to investigate whether the activity of a candidate gene, creatine kinase B (CKB) is elevated in pre-operative sera from ovarian cancer patients compared to patients with benign pelvic masses and normal controls. METHODS: MICROMAX cDNA microarray system and RNA derived from pooled ovarian cancer cell lines and normal ovary surface epithelial cells (HOSE) were used to identify differentially expressed genes. Using RNA from both cell lines and from tissue obtained through laser capture microdissection (LCM), we performed quantitative PCR in order to validate up-regulation of one of these genes, creatine kinase B (CKB). Using a commercially available enzyme assay, CKB activity was measured in pre-operative serum samples obtained from 45 ovarian cancer patients, 49 patients with a benign pelvic mass, as well as 37 normal controls. Statistical analysis was preformed using an unpaired Student's t test. RESULTS: Microarray technology revealed that CKB gene expression had a cancer to HOSE ratio of 18. RNA levels of CKB, measured by real-time PCR, were elevated a mean (and standard error) of 36-fold (8.4) in cancer cell lines compared with HOSE cells and 22.75-fold (10.45) in microdissected ovarian cancer epithelial cells compared with normal ovarian epithelial cells. In serum, the mean (+/-standard error) of CKB enzyme activity in cancer cases was 24.7 U/L units (+/- 5.1) compared to 9.6 U/L (+/- 1.6) for benign mass cases (P = 0.0088) and to 8.5 U/L (1.7) for normal controls (P = 0.0096). CONCLUSIONS: Microarray technology offers a method to identify tumor biomarkers with potential clinical usefulness. Our data indicated that CKB gene expression is up-regulated in ovarian cancer cells in vitro and in vivo and that CKB enzyme activity is significantly elevated in sera from ovarian cancer patients, including those with stage I disease. These findings suggest a potential role for CKB as a marker for early diagnosis.

Adult↗
PubMed2000

Rearrangement of immunoglobulin gene in metastatic Waldenström macroglobulinemia to the vitreous.

PURPOSE: To report metastatic Waldenström macroglobulinemia cells with immunoglobulin heavy chain gene rearrangement in the vitreous and the blood. METHODS: A 58-year-old man with Waldenström macroglobulinemia developed bilateral vitreitis. Diagnostic vitrectomy was performed on the left eye. The vitreous cells and the peripheral blood lymphocytes were analyzed using microdissection and polymerase chain reaction amplification. RESULTS: Vitrectomy specimen of the left eye contained a few degenerated cells. Molecular analysis showed immunoglobulin heavy chain gene rearrangement at the third complementary determining region of the vitreal infiltrating cells and peripheral blood lymphocytes. CONCLUSIONS: Waldenström macroglobulinemia rarely metastasizes to the vitreous. Molecular detection of the immunoglobulin heavy chain gene third complementary determining region rearrangement is helpful in the diagnosis of the malignancy. Microdissection combined with polymerase chain reaction is a useful and innovative tool for molecular pathological investigation.

B-Lymphocytes↗
PubMed2001

Persistent p53 mutations in single cells from normal human skin.

Epidermal clones of p53-mutated keratinocytes are abundant in chronically sun-exposed skin and may play an important role in early development of skin cancer. Advanced laser capture microdissection enables genetic analysis of targeted cells from tissue sections without contamination from neighboring cells. In this study p53 gene mutations were characterized in single cells from normal, chronically sun-exposed skin. Biopsies were obtained from skin subjected to daily summer sun and skin totally protected from the sun by blue denim fabric. Using laser capture microdissection, 172 single-cell samples were retrieved from four biopsies and analyzed using single-cell polymerase chain reaction and direct DNA sequencing. A total of 14 different mutations were identified in 26 of 99 keratinocytes from which the p53 gene could be amplified. Mutations displayed a typical UV signature and were detected in both scattered keratinocytes and in a small cluster of p53-immunoreactive keratinocytes. This minute epidermal p53 clone had a diameter of 10 to 15 basal cells. Two missense mutations were found in all layers of epidermis within the p53 clone. The presented data show that p53 mutations are common in normal skin and that a clone of keratinocytes with a mutated p53 gene prevailed despite 2 months of total protection from ultraviolet light.

Adult↗
PubMed2003

Identical allelic losses in mature teratoma and other histologic components of malignant mixed germ cell tumors of the testis.

Teratomas of the testis in post-pubertal patients are histologically diverse tumors that often coexist with other types of germ cell tumors. Using laser capture microdissection and loss of heterozygosity analysis, we investigated the clonality of mature teratoma and its relationship to other components of malignant mixed germ cell tumors to gain potential insight into the histogenetic relationship of teratoma with other germ cell tumor components. All 16 patients had mature teratoma as one component of their mixed germ cell tumors. The other histological subtypes included immature teratoma, seminoma, embryonal carcinoma, yolk sac tumor, and choriocarcinoma. Laser-assisted microdissection was performed on the formalin-fixed, paraffin-embedded tissue. Polymerase chain reaction was used to amplify genomic DNA at specific loci on chromosome 1p36.2 (D1S508), 2q22-32 (D2S156), 9p21-22 (D9S162), 11p13 (D11S903), 12q22-23 (D12S1051), and 18q21 (D18S46). Fourteen of 16 (88%) cases showed allelic loss in one or more components of the mixed germ cell tumors. Fourteen of 16 mature teratomas showed allelic loss in at least one of six microsatellite polymorphic markers analyzed. The frequency of allelic loss in mature teratoma was 50% (7 of 14) with D1S508, 33% (5 of 15) with D2S156, 58% (7 of 12) with D9S162, 43% (6 of 14) with D11S903, 20% (3 of 15) with D12S1051, and 33% (5 of 15) with D18S46. Completely concordant allelic loss patterns between mature teratoma and all of the other germ cell tumor components were seen in 10 of 14 tumors in which mature teratoma showed loss of heterozygosity. Our data support the common clonal origin of mature teratoma with other components of malignant mixed germ cell tumors of the testis.

Adolescent↗
PubMed2003

Identification of genes up-regulated in urothelial tumors: the 67-kd laminin receptor and tumor-associated trypsin inhibitor.

Studies investigating changes in gene expression in urothelial carcinoma have generally compared tumors of different stages and grades but comparisons between low-grade, noninvasive tumors and normal urothelium are needed to identify genes involved in early tumor development. We isolated the urothelium from a low-grade tumor and corresponding normal mucosa by laser capture microdissection on frozen sections. The RNA extracted was amplified to generate suppressive subtractive cDNA libraries. Random sequencing of cDNA clones identified approximately 100 unique species. Of these 83% were known genes, 15% had homology to genes with an unknown function in humans, and 2% did not show homology to any published gene sequence. Two of the known genes, the 67-kd laminin receptor (67LR) and tumor-associated trypsin inhibitor (TATI), had previously been associated with metastatic progression in many tumor types, although 67LR has not been investigated in urothelial tumors. Immunolabeling of the original tissue with antibodies against these two genes confirmed overexpression, validating our strategy: 67LR was not expressed in the normal urothelium but was present in the tumor, whereas TATI expression was confined to umbrella cells in the normal urothelium, but extended to all cell layers in the tumor. We investigated both markers further in a separate series of tumors of different stages and grades. TATI was more consistently overexpressed than 67LR in all tumor grades and stages. Levels of secreted TATI were significantly higher in urine samples from patients with tumors compared to controls. Our strategy, combining laser capture microdissection and cDNA library construction, has identified genes that may be involved in the early phases of urothelial tumor development rather than with disease progression, highlighting the importance of comparing tumor with normal rather than just tumors of different stages and grades.

Carcinoma↗
PubMed2004

Dynamic regulation of estrogen receptor-beta expression by DNA methylation during prostate cancer development and metastasis.

Estrogen receptor (ER)-beta is thought to exert anti-proliferative effects in the normal prostate but supports prostate cancer (PCa) cell survival. We previously reported that the receptor's expression declined as PCa developed in the gland but reappeared in lymph node and bone metastases. To investigate whether hypermethylation was the underlying mechanism for these phenomena, we first identified two CpG islands (CGIs) encompassing 41 CpG dinucleotides, located separately in the untranslated exon 0N and the promoter region of ER-beta. Using immunostained, laser capture-microdissected samples from 56 clinical specimens, we demonstrated an inverse relationship exists between the extent of ER-beta CGI methylation and receptor expression in normal, hyperplastic, premalignant, and malignant foci of the prostate and in lymph node and bone metastases. Treatment of PCa cell lines (LNCaP and DU145), that express little ER-beta mRNA, with a demethylating agent increased levels of receptor expression thus corroborating our in vivo findings that methylation is involved in ER-beta silencing. Methylation centers in the promoter region and exon 0N were identified by hierarchical cluster analysis of bisulfite sequencing data obtained from 710 alleles. Methylation at these centers was insignificant in normal epithelium, reached 80 to 90% in grade 4/5 PCa, but declined to less than 20% in bone metastases. In addition, progressive methylation spreading from the exonic CGI to the promoter CGI, which correlated with loss of ER-beta expression, was detected in microdissected samples and in cell cultures. Using a new class of methylated oligonucleotides that mediate sequence-specific methylation in cellulo, we demonstrated that methylation of the promoter CGI, but not the exonic CGIs, led to transcriptional inactivation of ER-beta. Our results present the first evidence that epigenetic regulation of ER-beta is a reversible and tumor stage-specific process and that gene silencing via methylated oligonucleotides may have therapeutic potential in the treatment of advanced PCa.

Base Sequence↗
PubMed2002

Enrichment, immunomorphological, and genetic characterization of fetal cells circulating in maternal blood.

Fetal cells circulating in the peripheral blood of pregnant women are a potential target for noninvasive genetic analyses. They include epithelial (trophoblastic) cells, which are larger than peripheral blood leukocytes. We enriched circulating trophoblastic cells using the isolation by size of epithelial tumor cells (ISET) method. Peripheral blood was obtained at 11 to 12 weeks of pregnancy. Cells isolated by ISET were stained by hematoxylin and eosin or by immunohistochemistry. Large epithelial cells were microdissected and fetal cell identification was obtained by polymerase chain reaction with short tandem repeats and/or Y-specific primers. By analyzing only 2 ml of blood, we found a variable number (n = 1 to 7) of Y-positive cells (overall 15 of 23) in all of the six mothers carrying a male fetus. In contrast, none of the 26 cells isolated from seven mothers carrying a female fetus scored positive. Eleven cells were analyzed by using short tandem repeat-specific markers: six of them showed a fetal profile and five showed a maternal profile consistently with Y-specific results. Only one-fifth of the single cell DNA was used for fetal cell assessment, leaving enough material for further genetic tests. We also show that the ISET approach allows the performance of fluorescence in situ hybridization analyses and the detection of DNA point mutations in single microdissected cells. We conclude that this is a powerful approach to enrich circulating fetal cells and prove their fetal origin, and that it may have implications for noninvasive prenatal diagnosis of genetic disorders.

Cell Separation↗
PubMed2001

Correlation between dysplasia and mutations of six tumor suppressor genes in Barrett's esophagus.

BACKGROUND: Barrett's esophagus (BE) may progress to adenocarcinoma through dysplastic progression. Classification of dysplasia in BE has significant interobserver variability. Our objective was to determine whether genetic alterations in BE correlate with degrees of histologic dysplasia. METHODS: Fixed tissue from 37 patients with BE and adenocarcinoma was studied for six tumor suppressor genes. Tissues were microdissected and analyzed for loss of heterozygosity. Microdissection of individual crypts showing metaplasia and dysplasia were performed and analyzed for 23 of the 37 patients whose tumors were heterozygous for at least four of the six genes studied. RESULTS: Frequency of alterations for MXI1, hOGG1, p53, MTS1, DCC, and APC were 7 of 32 (22%), 12 of 35 (34%), 12 of 26 (46%), 17 of 30 (57%), 17 of 27 (63%), and 23 of 36 (64%), respectively. Analysis of BE demonstrated that crypts with metaplasia, low-grade dysplasia, and high-grade dysplasia strongly correlated with alterations in tumor suppressor genes (p < 0.0001). CONCLUSIONS: This pilot study demonstrates that genetic analysis can be performed on individual crypts in patients with BE, and that alterations may facilitate objective classification of the severity of dysplasia.

Adenocarcinoma↗
PubMed2001

Single cell analysis of CAG repeat in brains of dentatorubral-pallidoluysian atrophy (DRPLA).

Somatic mosaicism of an expanded repeat is present in tissues of patients with triplet repeat diseases. Of the spinocerebellar ataxias associated with triplet repeat expansion, the most prominent heterogeneity of the expanded repeat is seen in dentatorubral-pallidoluysian atrophy (DRPLA). The common feature of this somatic mosaicism is the difference in the repeat numbers found in the cerebellum as compared to other tissues. The expanded allele in the cerebellum shows a smaller degree of expansion. We previously showed by microdissection analysis that the expanded allele in the granular layer in DRPLA cerebellum has less expansion than expanded alleles in the molecular layer and white matter. Whether this feature of lesser expansion in granule cells is common to other types of neurons is yet to be clarified. We used a newly developed excimer laser microdissection system to analyze somatic mosaicism in the brains of two patients, one with early- and another with late-onset DRPLA, and used single cell PCR to observe the cell-to-cell differences in repeat numbers. In the late onset patient, repeat expansion was more prominent in Purkinje cells than in granule cells, but less than that in the glial cells. In the early onset patient, repeat expansion in Purkinje cells was greater than in granule cells but did not differ from that in glial cells. These findings suggest that there is a difference in repeat expansion among neuronal subgroups and that the number of cell division cycles is not the only determinant of somatic mosaicism.

DNA Mutational Analysis↗
PubMed2002

Loss of heterozygosity on chromosome 5p13-12 predicts adverse prognosis in advanced bladder cancer independent of tumor stage and grade.

PURPOSE: The individual prognosis in patients with bladder cancer is only partially predicted by tumor stage and grade. Various molecular markers have been shown to correlate with disease progression and prognosis but few add some predictive capacity beyond that offered by standard clinical and pathological parameters. MATERIALS AND METHODS: A total of 191 urothelial carcinomas from 157 patients were analyzed. Paired normal and tumor cells were microdissected by manual and microbeam-microdissection of membrane mounted native tissue, and analyzed for loss of heterozygosity (LOH) at a critical region of LOH on chromosome 5p13-12 that is involved in bladder cancer progression. LOH data were correlated with progression-free and tumor specific survival by multivariate analysis. RESULTS: Informativity of the assay was 60.7%. Log rank analysis of 100 evaluable patients showed a progression-free survival benefit without LOH at 5p13-12 of more than 3 years (p <0.01). Mean followup was 36 months. Multivariate analysis revealed that this benefit was not predicted by tumor stage and grade alone in advanced disease (stages T3/4 and/or N+/M+, stages III/IV). CONCLUSIONS: LOH at the critical region on chromosome 5p13-12 is a molecular marker of adverse prognosis in advanced bladder carcinoma independent of tumor stage and grade.

Carcinoma, Transitional Cell↗
PubMed1998

Paragangliomas of the head and neck region show complete loss of heterozygosity at 11q22-q23 in chief cells and the flow-sorted DNA aneuploid fraction.

Nonchromaffin paragangliomas of the head and neck region, also known as glomus tumors, are usually benign neoplasms consisting of clusters of chief cells surrounded by sustentacular cells arranged in so-called 'Zellballen.' Most of the patients have a familial background. In a previous study, examining all chromosome arms, we found loss of heterozygosity (LOH) predominantly at the chromosome 11q22-q23 region, where the disease causing gene PGL1 has been located by linkage analysis. However, all tumors showed only partial loss of allele signal intensities, and it was not clear whether this represented allelic imbalance or cellular heterogeneity. In the current study, we have performed LOH analysis for the 11q22-q23 region on DNA-aneuploid tumor cells, enriched by flow sorting, and on purified chief cell fractions obtained by single-cell microdissection. Complete LOH was found for two markers (D11S560 and CD3D) in the flow-sorted aneuploid fractions, whereas no LOH was found in the diploid fractions of three tumors. The microdissected chief cells from two of these tumors also showed complete LOH for both markers, indicating that the chief cells are clonal proliferated tumor cells. These results indicate that the PGL1 gene is likely to be a tumor suppressor gene, which is inactivated according to the two-hit model of Knudson. Furthermore, it shows that chief cells are a major if not the sole neoplastic component of paragangliomas.

Chromosomes, Human, Pair 11↗