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PubMed2002

Proteomic analysis and identification of new biomarkers and therapeutic targets for invasive ovarian cancer.

Epithelial ovarian cancer kills almost 16 000 women each year in part due to late stage of presentation and lack of reliable biomarkers for disease detection. CA-125, the currently accepted serum marker, alone lacks the sensitivity for early stage diagnosis, as only 50% of early stage cases are detected with this marker. Although more early stage cases may be detected by lysophosphatidic acid, this marker is also elevated in other cancers. One major objective of the NCI-FDA Tissue Proteomics Initiative has been to combine the technique of laser capture microdissection (LCM) of epithelial tumor cells in human tissue specimens with two-dimensional gel electrophoresis (2-D PAGE) to identify proteins that may serve as invasive ovarian cancer-specific biomarkers for early detection and/or new therapeutic targets. We performed 2-D PAGE on lysates from five microdissected ovarian tumors (three invasive ovarian cancers and two noninvasive, low malignant potential (LMP) ovarian tumors). We then compared silver stained 2-D gels created from microdissected lysates with SYPRO-Ruby stained 2-D PAGE profiles of the patient-matched undissected bulk tumor lysates from all five patients. Twenty-three proteins were consistently differentially expressed between both the LMP and three invasive ovarian tumors in the limited study set. Thirteen were uniquely present in all three of the invasive ovarian cancer cases and absent or underexpressed in the two LMP cases. Ten were uniquely present in the LMP cases but absent or underexpressed in all invasive ovarian cancer cases. Credentialing and preliminary target validation of the mass spectrometry identified proteins cut from the Ruby-red stained gels was performed by LCM coupled Western blot and reverse-phase array technology in a study set of six cases (the aforementioned five cases used in the 2-D PAGE profiling component of the study plus one additional LMP case). The analysis revealed that the 52 kDa FK506 binding protein, Rho G-protein dissociation inhibitor (RhoGDI), and glyoxalase I are found to be uniquely overexpressed in invasive human ovarian cancer when compared to the LMP form of this cancer. The direct comparison of LCM generated proteomic profiles of invasive vs. LMP ovarian cancer may more directly generate important markers for early detection and/or therapeutic targets unique to the invasive phenotype.

Biomarkers, Tumor↗
PubMed2001

Allelic deletion mapping on chromosome 6q and X chromosome inactivation clonality patterns in cervical intraepithelial neoplasia and invasive carcinoma.

OBJECTIVE: Loss of heterozygosity (LOH) profiles and X chromosome inactivation patterns are analyzed in 42 patients with cervical intraepithelial neoplasias (CIN), including low-grade (CIN1) and high-grade (CIN2, CIN3) lesions, and 22 patients with invasive cervical carcinomas. METHOD: Laser capture microdissection was utilized to procure pure matched normal and lesional cells from each case. Sixteen microsatellite markers on four chromosomal arms, 6q21-q25.1, 8p21, 13q12.3--q13, and 17q12--q21, were amplified for LOH, as well as the HUMARA locus for X chromosome inactivation analysis. Eight additional markers spanning the long arm of chromosome 6 were utilized in all cases showing LOH on this arm and in which further tissue material was available for microdissection. RESULTS: Fifty-five percent of carcinomas showed deletions on chromosome bands 6q21--q25.1, 43% on 13q12.3--q13, and 40% on 17q12--q21. Deletions on 6q were identified in CIN3 (40%), CIN2 (37%), and CIN1 (10%), on 13q in CIN3 (33%) and CIN2 (33%), and rarely on chromosomal arm 17q. Finer 6q mapping revealed that marker D6S310 (q22) represented the centromeric and marker D6S255 (q25--q16) the telomeric boundary of deletion. A second, telomeric area of deletion at marker D6S281 (q27) was also identified. Monoclonal X chromosome inactivation patterns were identified in 12/13 cancers, 13/14 CIN3, 5/10 CIN2, and 0/6 CIN1. CONCLUSIONS: Two areas of deletion on chromosome 6q were identified in cervical tumors, suggesting the presence of tumor suppressor gene(s) inactivated in this neoplasia. LOH on this arm were identified early during cervical tumor progression. LOH on 13q and 17q also occur in cervical cancers. X chromosome inactivation patterns suggest that CIN develops into a monoclonal lesion during progression from CIN1 to CIN3.

Chromosome Mapping↗
PubMed1997

Loss of heterozygosity of the retinoblastoma gene in liver cirrhosis accompanying hepatocellular carcinoma.

Carcinogenesis is a multistep process. Most hepatocellular carcinoma (HCC) is preceded by liver cirrhosis, but the genetic changes involved in cirrhosis are not known well. The present study was conducted to evaluate aberration of the retinoblastoma (RB) gene in HCC and adjacent non-tumorous liver using 22 patients with chronic liver damage accompanying HCC. The specimens obtained by microdissection from paraffin-embedded tissues were analyzed using an assay based on the polymerase chain reaction for highly polymorphic nucleotide sequences of microsatellites in the RB gene. Out of 22 cases, 15 showed constitutional heterozygosity for the microsatellite markers. In 11 (73.3%) of these 15 informative cases, the primary HCC foci showed loss of heterozygosity (LOH). In 8 of these 11 doubly informative (informative and LOH-positive in primary HCC) cases, LOH was found in 20 (64.5%) of 31 microdissected non-tumorous foci. All of the non-tumorous foci showing RB loss were cirrhotic lesions but there were no foci of chronic hepatitis. The remaining 4 cases without LOH in HCC foci showed no LOH in non-tumorous lesions. In our study, LOH of the RB gene was frequently observed in liver cirrhosis surrounding tumor.

Aged↗
PubMed1985

Alteration in the capacities as well as in the zonal and cellular distributions of pyruvate kinase L and M2 in regenerating rat liver.

Pyruvate kinase L (PKL), the glucoregulatory isoenzyme of adult parenchymal cells, and M2 (PKM2), the isoenzyme of proliferating and non-parenchymal cells, were measured, using a specific anti-PKL antibody for differentiation, in total liver homogenates, in isolated parenchymal and non-parenchymal cells as well as in microdissected periportal and perivenous liver tissue from regenerating rat liver after two-thirds partial hepatectomy. Moreover, the zonal distribution of PKL was studied using immunohistochemical techniques. In total liver homogenates PKL activity per g liver decreased after partial hepatectomy, while PKM2 increased. Total PKL activity per 100 g body weight was restored to preoperational levels much more slowly than liver weight. During liver regeneration parenchymal cells acquired high PKM2 besides PKL activity. The isoenzyme outfit of non-parenchymal cells remained unchanged. Microdissection studies showed that PKL lost its normal perivenous to periportal gradient after partial hepatectomy and became evenly distributed within the liver acinus. PKM2 did not retain its even distribution, it became predominant in the periportal zone. Immunohistochemical staining revealed that after partial hepatectomy PKL was present in all parenchymal cells in an atypical non-zonal heterogeneous distribution. Normal specific activities as well as zonal and cellular distributions of both pyruvate kinase isoenzymes were restored 14-21 d after partial hepatectomy. During regeneration after 2/3 partial hepatectomy the liver loses its glucostat function as corroborated in this study by the decrease of the glycolytic capacity via the glucoregulatory PKL; this change of function is accompanied by a loss of PKL-zonation. This finding corroborates the view that zonation of carbohydrate-metabolizing enzymes is required only when the liver functions as a glucostat. The increase of PKM2 and the appearance of a zonal PKM2 heterogeneity are in line with the pattern of hepatocyte proliferation after partial hepatectomy.

Animals↗
PubMed2004

Microdissection techniques for molecular testing in surgical pathology.

OBJECTIVE: To describe the techniques for microdissection of paraffin-embedded and frozen tissue sections for the use in molecular applications. DATA SOURCES: Original research papers and review papers and the authors' personal experiences. DATA SYNTHESIS: Manual and laser-capture microdissection are described in detail, with specific protocols for sample preparation and instructions for performing the microdissection. A section addressing frequently asked questions is also included. CONCLUSIONS: Microdissection is a technique that is very useful both in the research setting and for clinical molecular testing in paraffin-embedded tissue samples. The available techniques range from simple and inexpensive (manual microdissection) to complex and expensive (laser-capture microdissection). All of the techniques, however, require the user to be familiar with microscopy and histology.

Genetic Techniques↗
PubMed2005

Differential expression and distribution of alternatively spliced transcripts of PDGF-A and of PDGF receptor-alpha in mouse reproductive tissues.

Platelet-derived growth factor-A (PDGF-A) affects cellular activities such as proliferation, differentiation, and development by way of paracrine or autocrine interaction with PDGF-A receptor alpha (PDGFR-alpha). Two forms of alternatively spliced PDGF-A mRNA, a long and a short isoform, have been found in several mammalian species. Expression of PDGF-A and its cognate receptor PDGFR-alpha has been well studied in various tissues. However, these investigations did not distinguish between the individual isoforms of PDGF-A. In the present investigation, we identified the differential cellular expression patterns of the two isoforms of PDGF-A and of PDGFR-alpha in mouse reproductive tissues by using laser capture microdissection coupled with reverse transcriptase polymerase chain reaction. The long PDGF-A mRNA isoform was primarily detected in the epithelium, while the short isoform was ubiquitously distributed in epithelium, stroma, and muscle cells, although it was still more prominent in epithelium. PDGFR-alpha was mainly detected in stromal and muscle cells. Also, it was found in the epididymal epithelium, mucosal folds of the seminal vesicle, and ovarian granulosa cells. Thus, the complete PDGF-A/PDGFR-alpha signaling system is present in murine reproductive tissues, but the distribution of the long and short isoforms of PDFG-A differs.

Alternative Splicing↗
PubMed2005

Enzymatic digestion plus mechanical searching improves testicular sperm retrieval in non-obstructive azoospermia cases.

BACKGROUND: In non-obstructive azoospermic patients (NOA) besides the mechanical treatment, vital spermatozoa from the tissue obtained from testes by biopsy can be enzymatically prepared. OBJECTIVE: To increase the sperm recovery success of testicular sperm extraction (TESE), suitable for ICSI. STUDY DESIGN: Prospective, clinical study. In 177 consecutive men who presented with clinical and laboratory data indicating NOA, tissue samples were obtained by microdissection TESE method. Initially, mature spermatozoa were searched for by mechanical extraction technique shredding the biopsy fractions. In cases with no spermatozoa was observed after maximum 30 min of initial searching under the inverted microscope, the procedure was then followed by enzymatic digestion using DNAse and collagenase type IV. In cases of at least a mature spermatozoon could be obtained properly, ICSI was performed. RESULTS: Of 177 cases with NOA, conventional mincing method extended with enzymatic treatment yielded successful sperm recovery for ICSI in 102 (57%). Overall in vitro tissue-processing time for patients with sperm recovery failure after 30 min of mechanical searching, was between 80 and 105 min (mean 96+/-9). Cleavage, embryo transfer and clinical pregnancy rates in mechanical plus enzymatic TESE patients were not significantly different from those of only mechanically TESE performed patients (p>0.05). CONCLUSION: Combination of conventional TESE and enzymatic digestion is an effective method to recover spermatozoa suitable for ICSI. The benefit of the mincing combined with enzyme to sperm retrieval for NOA is firstly to shorten the mechanical searching time, thus minimizing further cellular damage as well as exposure to external conditions, and secondly to reduce the number of cases with sperm recovery failures.

Adult↗
PubMed2000

Proteomic analysis of laser capture microdissected human prostate cancer and in vitro prostate cell lines.

Specific populations of normal and malignant epithelium from three radical prostatectomy tissue specimens were procured by laser capture microdissection (LCM) and analyzed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Six proteins that were only seen in malignant cells and two proteins that were only seen in benign epithelium were reproducibly observed in two of two cases examined. Furthermore, these proteins were not observed in the 2-D PAGE profiles from the patient-matched microdissected stromal cell populations, but were seen in the protein profiles from the undissected whole cryostat sections. One of these proteins was determined to be prostate-specific antigen (PSA) by Western blot analysis, and intriguingly the remaining protein candidates were found to be at least as abundant as the PSA protein. Comparison of 2-D PAGE profiles of microdissected cell with matched in vitro cell lines from the same patient, and metastatic prostate cancer cell lines (LnCaP and PC3) showed striking differences between prostate cells in vivo and in vitro with less than 20% shared proteins. The data demonstrate that 2-D PAGE analysis of LCM-derived cells can reliably detect alterations in protein expression associated with prostate cancer, and that these differentially expressed proteins are produced in high enough levels which could allow for their clinical utility as new targets for therapeutic intervention, serum markers, and/or imaging markers.

Blotting, Western↗
PubMed1983

Energy dispersive x-ray analysis of inner ear fluids and tissues during the ontogeny of cochlear function.

Energy dispersive x-ray analysis (EDXA) was used to characterize freeze-dried, microdissected samples of inner ear fluids and tissues from neonatal gerbils throughout the period of cochlear functional development, between 10 and 18 days after birth. EDXA spectra from endolymph residue demonstrated that the adult ionic composition of this fluid is established before functional onset, and before the appearance of the endocochlear potential. However, stria vascularis spectra showed substantial increases in relative phosphorus and sodium content during the period in which auditory thresholds improve by approximately 100 dB, in which the endocochlear potential appears and increases to its adult value, and in which the level of metabolism of the stria increases dramatically. The increase in relative phosphorus content of stria in particular occurred with a time course which was very similar to, though slightly earlier than, that of the developmental increase in the endocochlear potential. It is concluded that increases in the relative phosphorus content of stria may represent increases in inorganic phosphate associated with strial metabolism. Increases in relative sodium content may reflect the action of an ion transport system which directs this element into strial cells during generation of the endocochlear potential.

Aging↗
PubMed1984

The substantia innominata and adjacent regions in the human brain: histochemical and biochemical observations.

The substantia innominata was investigated in the adult human brain with particular reference to the cholinergic nature of the nucleus of Meynert. Based on histochemical observations of acetylcholinesterase activity and biochemical estimations of choline acetyltransferase the relations of the Meynert nucleus to adjacent structures in the substantia innominata region were identified precisely. A new dissection procedure is described which permits combined histochemical and histological examination of anatomically complex regions of the human brain, such as the substantia innominata, to be carried out in conjunction with accurate tissue dissection for neurochemical analysis. Using this technique, various acetylcholinesterase-positive and choline acetyltransferase-containing structures are apparent in coronal sections removed from the rostrocaudal length of the substantia innominata. These include, in addition to the prominently stained, putative cholinergic neurons, acetylcholinesterase-positive tracts which contain putative cholinergic projections to the neocortex, and 'islands' of acetylcholinesterase-positive neuropil which presumably reflect a collateral or intrinsic cholinergic innervation in the area. This anatomical complexity of cholinergic structures in the substantia innominata suggests that neurochemical analysis should be conducted on microdissected as opposed to macrodissected tissue samples. Neuropathologically, the present report provides a further basis for optimising quantification of putative cholinergic perikarya. Continued systematic analysis of the nucleus of Meynert at the morphological and biochemical level should thus establish the role of this nucleus in normal brain function and in disease.

Acetylcholinesterase↗
PubMed2000

Laser capture microdissection: methodical aspects and applications with emphasis on immuno-laser capture microdissection.

Laser capture microdissection (LCM) is an easy, extremely fast and versatile method for the isolation of morphologically defined cell populations from complex primary tissues for molecular analyses. However, the optical resolution is limited due to the use of dried sections without coverslip necessary for tissue capture, and routine stains such as hematoxylin and eosin are sometimes insufficient for precise microdissection, especially in tissues with diffuse intermingling of different cell types and lack of easily discernible architectural features. Therefore, several groups have adapted immunohistochemical staining techniques for LCM. In addition to providing high contrast targets for microdissection, immunostaining allows selection of cells not only according to morphological, but also phenotypical and functional criteria. In order to allow reliable tissue transfer on one hand and preserve the integrity of the target of analysis such as DNA, RNA and proteins on the other hand, immunostaining protocols have to be modified for the purposes of LCM. The following review gives an overview of immuno-LCM and describes some applications, e.g. in the field of hematopathology.

Cell Separation↗
PubMed1988

Epidermal growth factor in the mouse kidney: developmental changes and intranephron localizations.

The developmental changes in epidermal growth factor (EGF) have been studied in tissue homogenates, kidney slices, and microdissected nephron segments of the mouse. Immunoreactive EGF concentration per milligram of protein increased in the kidney by about 20-fold from 1 week to 3 weeks of life, reaching the highest levels between 5 and 7 weeks, and decreasing by 10 weeks of life. The time course of the changes was different from those of submaxillary and urinary EGF. Above 7 weeks of age, kidney EGF was higher in female than in male mice. Among various zones of the kidney (outer cortex, inner cortex, outer medulla I, II and papilla), the outer medulla I and II contained the highest quantities of EGF per gram of wet tissue. The highest EGF content per millimeter length was observed in the medullary and cortical thick ascending limbs of Henle's loop. The amounts exceeded by about 4.5-fold those found in glomeruli and in proximal convoluted and proximal straight tubules, and by about 3-fold those present in distal and collecting tubules. Unilateral nephrectomy resulted in no significant changes in EGF levels in the contralateral kidney. The results suggest that the ontogeny of kidney EGF is different from that of the EGF found in the submaxillary gland, and that there is nephron heterogeneity in EGF content.

Animals↗
PubMed2005

Exfoliated buccal and microdissected lung cell expression of antioxidant enzymes.

INTRODUCTION: An exfoliated buccal cell biomarker assay for antioxidant gene transcript levels was used to measure inter-tissue concordance with lung, and inter-subject variability in a lung cancer case-control study. METHODS: First, qualitative RNA-specific RT-PCR was used to compare expression in exfoliated buccal cells with that in laser microdissected lung tissue remote from the tumor from 14 individuals providing both specimens. RESULTS: There was complete [100% for quinone oxidoreductase 1 (NQO1), glutathione peroxidase (GPX), and superoxide dismutase 1 (SOD1)], or predominant [85.7% for catalase (CAT)] inter-tissue concordance for qualitative expression. Second, quantitative real-time RT-PCR for antioxidant enzyme transcript levels was performed in exfoliated buccal samples from these same 14 individuals, as well as 28 additional individuals providing buccal cells only, for a total of 42 buccal specimens (19 current smokers and 23 ex- or never-smokers), of whom 26 (61.39%) had a new diagnosis of lung cancer. DISCUSSION: Wide inter-individual expression differences for each gene transcript (>10(1)-10(4)-fold) were observed in the exfoliated buccal cells, unrelated to smoking and case-control status. In multivariate analyses, family history of tobacco-related malignancy correlated inversely with buccal NQO1 and CAT mRNA levels (p=0.003, p<0.001, respectively). This antioxidant expression trait may relate to family risk of cancer, but is notably unrelated to oxidant challenges inherent in cigarette smoke.

Adult↗
PubMed2003

Molecular genetic alterations in the laser-capture-microdissected stroma adjacent to bladder carcinoma.

BACKGROUND: Urothelial carcinoma commonly manifested loss of heterozygosity (LOH) at different regions of chromosomes 17p, 3p, and 9q. Recent studies suggested that bladder stromal cells may be implicated in the growth and progression of urothelial carcinoma. To better understand the genetic alterations in the stromal cells in patients with bladder carcinoma, the authors evaluated the prevalence of allelic loss at three microsatellite polymorphic markers on chromosomes 17p13 (TP53), 3p25-26 (D3S3050), and 9q32-33 (D9S177). In addition, the pattern of X-chromosome inactivation of the stromal cells was evaluated by analyzing the DNA methylation pattern at a polymorphic site on the androgen receptor gene. METHODS: The authors studied 18 female patients who underwent either transurethral resection (n = 2) or radical cystectomy (n = 16) for high-grade muscle-invasive urothelial carcinoma of the urinary bladder. Genomic DNA samples from the stromal cells immediately adjacent to the tumor and the tumor itself were prepared from formalin-fixed, paraffin-processed tissues using laser-assisted microdissection and LOH was determined. RESULTS: The stromal cells showed a high frequency of LOH on chromosomes 17p13 (29%), 3p25-26 (61%), and 9q32-33 (47%) with no clear concordance with the adjacent tumor cells. Fourteen specimens (78%) showed LOH in the stroma in at least 1 of 3 markers examined. Nonrandom X-chromosome inactivation was frequent in the stromal cells (50% of informative specimens). CONCLUSIONS: The current study revealed that some of the genetic changes that commonly occur with invasive urothelial carcinoma were frequently found in the adjacent stroma and suggested that the stroma of urothelial carcinoma may play an important role in bladder carcinogenesis.

Carcinoma, Transitional Cell↗
PubMed2006

Mutation detection in tumor suppressor genes using archival tissue specimens.

Tuberous sclerosis complex (TSC) is a neurocutaneous syndrome characterized by seizures, mental retardation, and benign tumors of many organs, including the brain, kidneys, skin, retina, and heart. TSC is caused by mutations in the TSC1 and TSC2 tumor suppressor genes. The genes follow the two-hit model for tumorigenesis, with germline mutations inactivating one allele and somatic mutations inactivating the remaining wild-type allele. Allelic loss (also called loss of heterozygosity [LOH]) in the 9q34 and 16p13 regions has been found in many tumor types from TSC patients. Cardiac rhabdomyomas are frequently found in infants with TSC. Because rhabdomyomas often spontaneously regress, access to fresh tissue is limited. In this chapter, we present methodology for detection of genetic inactivation of TSC1 and TSC2 in paraffin-embedded archival tissues. The template DNA is obtained either by direct scraping of tissue or after laser capture microdissection. LOH analysis is performed after polymerase chain reaction amplification of microsatellite markers in the 9q34 and 16p13 regions and denaturing polyacrylamide gel electrophoresis. Mutation detection is performed using single-strand conformation polymorphisms on mutation detection enhancement gels. Finally, variant bands are amplified and analyzed by direct sequencing.

DNA↗
PubMed2000

Clonality analysis of defined cell populations in paraffin-embedded tissue sections by RT-PCR amplification of X-linked G6PD gene.

This paper establishes a method of clonality analysis using the reverse transcription-polymerase chain reaction (RT-PCR) to amplify X-linked G6PD transcripts on defined cell populations microdissected from archival, paraffin-embedded tissue sections. Four known monoclonal low-grade B-cell lymphomas from females who were heterozygous (informative) at the 1131 exonic polymorphic locus of the G6PD gene were used to validate the method. Lymphoma and reactive lesions in each case were separated by microdissection. In order to preserve the intact RNA species in the lesion, sections were digested on the slides before microdissection. A one-step RT-PCR was performed with a single pair of primers, one of which contained a mismatched base adjacent to the polymorphic site, to generate a PvuI cutting site. Successful amplification and allele identification by PvuI digestion were achieved from all RNA samples studied. Three of four samples from non-neoplastic reactive lesions showed two bands with equal intensity, representing transcription of the two alleles of the G6PD gene, while the corresponding tumour samples demonstrated a biased intensity in one allele, indicating monoclonality. To assess the method further, the clonal nature of in situ and invasive breast cancers was examined, along with adjacent normal breast tissue and hyperplastic lesions from three informative females from our archives. Apart from the clusters of normal terminal duct-lobular units, all lesions were monoclonal. This result is in agreement with data derived from other X-linked gene studies and loss of heterozygosity (LOH) analyses of pre-invasive breast disease. The results suggest that the clonality analysis method presented here is simple and reliable, and is therefore potentially applicable in a wide range of pathological conditions.

Breast↗