The effects of frozen tissue storage conditions on the integrity of RNA and protein.

Unfixed tissue specimens most frequently are stored for long term research uses at either -80° C or in vapor phase liquid nitrogen (VPLN). There is little information concerning the effects such long term storage on tissue RNA or protein available for extraction. Aliquots of 49 specimens were stored for 5-12 years at -80° C or in VPLN. Twelve additional paired specimens were stored for 1 year under identical conditions. RNA was isolated from all tissues and assessed for RNA yield, total RNA integrity and mRNA integrity. Protein stability was analyzed by surface-enhanced or matrix-assisted laser desorption ionization time of flight mass spectrometry (SELDI-TOF-MS, MALDI-TOF-MS) and nano-liquid chromatography electrospray ionization tandem mass spectrometry (nLC-ESI-MS/MS). RNA yield and total RNA integrity showed significantly better results for -80° C storage compared to VPLN storage; the transcripts that were preferentially degraded during VPLN storage were these involved in antigen presentation and processing. No consistent differences were found in the SELDI-TOF-MS, MALDI-TOF-MS or nLC-ESI-MS/MS analyses of specimens stored for more than 8 years at -80° C compared to those stored in VPLN. Long term storage of human research tissues at -80° C provides at least the same quality of RNA and protein as storage in VPLN.

Identification of a unique epigenetic sub-microenvironment in prostate cancer.

The glutathione S-transferase P1 (GSTP1) gene promoter is methylated in tumour cells in more than 90% of prostate carcinomas. Recently, GSTP1 promoter methylation was identified in tumour-associated stromal cells in addition to the tumour epithelium. To define the extent and location of stromal methylation, epigenetic mapping using pyrosequencing quantification of GSTP1 promoter methylation and an anatomical three-dimensional reconstruction of an entire human prostate specimen with cancer were performed. Normal epithelium and stroma, tumour epithelium, and tumour-associated stromal cells were laser capture-microdissected from multiple locations throughout the gland. As expected, the GSTP1 promoter in both normal epithelium and normal stromal cells distant from the tumour was not methylated and the tumour epithelium showed consistently high levels of promoter methylation throughout. However, tumour-associated stromal cells were found to be methylated only in a localized and distinct anatomical sub-field of the tumour, revealing the presence of an epigenetically unique microenvironment within the cancer. Morphologically, the sub-field consisted of typical, non-reactive stroma, representing a genomic alteration in cells that appeared otherwise histologically normal. Similar epigenetic anatomical mapping of a control prostate gland without cancer showed low background methylation levels in all cell types throughout the specimen. These data suggest that stromal cell methylation can occur in a distinct sub-region of prostate cancer and may have implications for understanding tumour biology and clinical intervention.

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.

[Serous peritoneal papillary tumor of low malignancy potential. Report of a case]. / Tumor papilar peritoneal seroso de bajo potencial maligno. Comunicación de un caso.

Papillary serous carcinoma with low malignant potential, similar to those described in the ovary, can also originate in the peritoneum. Characteristically they show peritoneal spread without involvement of the ovary and, if present, it corresponds to a superficial implant similar to those seen in the rest of the peritoneal cavity. Histologically they correspond to a low malignant potential tumor; they are slow growing and have good prognosis. They are common in young women, usually they present few symptoms and are frequently discovered during other surgical procedures. The treatment is surgical and it can be conservative in cases of women who want to preserve their fertility, without coadjuvant therapy. We report a 34 years old woman with a primary peritoneal serous carcinoma with low malignant potential and discuss its management.

Analysis of mRNA quality in freshly prepared and archival Papanicolaou samples.

OBJECTIVE: To study the feasibility of utilizing mRNA recovered from cytologic Papanicolaou (Pap) specimens as a resource for gene expression studies of normal and diseased cells. STUDY DESIGN: To assess the effects of fixation on mRNA recovery and analysis, fresh Pap samples were processed by three separate methods: (1) routine cytologic fixation (2) 70% ethanol fixation, and (3) air drying without fixation. One-week-old, 1-month-old, 1-year-old and 10-year-old samples were studied to determine the quality of mRNA in archival samples. mRNA quality was analyzed by RT-PCR for the HPRT gene, and by complete transcript amplification. Both heterogeneous (whole slide scrapes) and microdissected cell populations were studied. RESULTS: Reverse transcriptase-polymerase chain reaction (RT-PCR) for the hypoxanthine guanine phosphoribosil transferase gene was positive in all fresh and archival samples and was not affected by fixative, processing methodology or microdissection. Complete transcript amplification followed by gel electrophoresis showed cDNA smears in all fresh samples with a maximum intensity between 1 and 2 kilobases (kb). Amplification of mRNA was not affected by fixation. Smaller cDNA smears were seen in archival specimens with a maximum intensity between 0.5 and 1.5 kb in both one-week-old and one-month-old samples. Smears of approximately 500 base pairs were observed in the 1-year-old and 10-year-old samples. Successful mRNA amplification was possible from microdissected cell populations. CONCLUSION: Messenger RNA recovery and analysis is possible from archival cytologic specimens, suggesting that they can serve as a useful template for RT-PCR analysis of individual genes as well as newly developing high-throughput gene expression methodologies, such as microarrays. Cytologic samples may be particularly useful for study of archival samples as well as diseases from which tissue samples amenable to mRNA-based studies are not available.

Allelic loss on chromosome arm 8p: analysis of sporadic epithelial ovarian tumors.

OBJECTIVE: Our objective was to determine the frequency of allelic loss at 8p21 in sporadic epithelial ovarian cancer. We recently described allelic loss at this locus in 7/9 ovarian cancers from patients with BRCA1 gene mutations. METHODS: We anonymously obtained and examined 40 unselected invasive epithelial ovarian cancers and 5 low-malignant-potential (LMP) ovarian tumors for loss of heterozygosity (LOH) at 8p12-22. Pure epithelial and stromal cell populations were procured selectively by laser capture microdissection and extracted DNA was amplified with polymorphic microsatellite markers spanning the region of interest. RESULTS: LOH was highest (50%) at marker D8S136 located at 8p21 with 15 of 30 informative cases exhibiting an allelic deletion. None of the LMP tumors evaluated showed LOH at 8p12-22. A trend toward more frequent LOH at 8p12-22 was identified with increasing disease aggressiveness from LMP to early stage invasive ovarian cancer to advanced stage invasive ovarian cancer (Lehman's test, P2 < 0.024). CONCLUSIONS: Fifty percent allelic loss at the distal portion of 8p21 has not been reported to date for sporadic epithelial ovarian carcinomas. The higher rate of loss in our cohort, in contrast to previous allelotyping studies, is due likely to analysis from homogenous cell populations. These results, in concert with our previous study of BRCA1 mutation-positive patients, suggest a tumor suppressor gene locus at 8p21 for epithelial ovarian cancer.

Long term organ culture of human prostate tissue in a NASA-designed rotating wall bioreactor.

PURPOSE: To maintain ex vivo integral prostatic tissue including intact stromal and ductal elements using the NASA-designed Rotating Wall Vessel (RWV) which maintains colocalized cells in an environment that promotes both three-dimensional cellular interactions together with the uniform mass transfer of nutrients and metabolic wastes. MATERIALS AND METHODS: Samples of normal prostate were obtained as a byproduct of transurethral prostatectomy or needle biopsy. Prostatic tissue dissected into small 1 x 1 mm. blocks was cultured in the Rotating Wall Vessel (RWV) Bioreactor for various time periods and analyzed using histological, immunochemical, and total cell RNA assays. RESULTS: We report the long term maintenance of benign explanted human prostate tissue grown in simple culture medium, under the simulated microgravity conditions afforded by the RWV bioreactor. Mesenchymal stromal elements including blood vessels and architecturally preserved tubuloglandular acini were maintained for a minimum of 28 days. Cytokeratins, vimentin and TGF-beta2 receptor and ligand were preserved through the entire culture period as revealed by immunocytochemistry. Prostatic acid phosphatase (PAP) was continuously expressed during the culture period, although somewhat decreased. Prostatic specific antigen (PSA) and its transcript were down regulated over time of culture. Prostatic carcinoma cells from the TSU cell line were able to invade RWV-cultured benign prostate tissue explants. CONCLUSIONS: The RWV bioreactor represents an additional new technology for culturing prostate tissue for further investigations concerning the basic physiology and pathobiology of this clinically important tissue.

Immuno-LCM: laser capture microdissection of immunostained frozen sections for mRNA analysis.

Microdissection of routinely stained or unstained frozen sections has been used successfully to obtain purified cell populations for the analysis of cell-specific gene expression patterns in primary tissues with a complex mixture of cell types. However, the precision and usefulness of microdissection is frequently limited by the difficulty to identify different cell types and structures by morphology alone. We therefore developed a rapid immunostaining procedure for frozen sections followed by laser capture microdissection (LCM) and RNA extraction, which allows targeted mRNA analysis of immunophenotypically defined cell populations. After fixation, frozen sections are immunostained under RNAse-free conditions using a rapid three-step streptavidin-biotin technique, dehydrated and immediately subjected to LCM. RNA is extracted from captured tissue, DNAse I treated, and reverse transcribed. Acetone-, methanol-, or ethanol/acetone-fixed sections give excellent immunostaining after 12 to 25 minutes total processing time. Specificity, precision, and speed of microdissection is markedly increased due to improved identification of desired (or undesired) cell types. The mRNA recovered from immunostained tissue is of high quality. Single-step PCR is able to amplify fragments of more than 600 bp from both housekeeping genes such as beta-actin as well as cell-specific messages such as CD4 or CD19, using cDNA derived from less than 500 immunostained, microdissected cells. Immuno-LCM allows specific mRNA analysis of cell populations isolated according to their immunophenotype or expression of function-related antigens and significantly expands our ability to investigate gene expression in heterogeneous tissues.

[Frozen-section biopsy in ovarian neoplasm diagnosis: diagnostic correlation according to diameter and weight in tumors of epithelial origin]. / Biopsia rápida por congelación en el diagnóstico de tumores de ovario: correlación diagnóstica según diámetro y peso en tumores de origen epitelial.

BACKGROUND: Adequate management and treatment of ovarian carcinoma requires a complete surgical staging supported by frozen-section examination. To achieve this goal it is necessary a high level of accuracy. AIM: To evaluate the accuracy of frozen-sections in ovarian carcinoma considering the influence of tumor diameter and weight. PATIENTS AND METHODS: Retrospective study of frozen-sections performed in patients with ovarian tumors who underwent surgery. Frozen- and permanent-sections were divided into three categories (benign, borderline and malignant) and stratified by diameter (< 10 cm, 10 to 20 cm, > 20 cm) and weight (< 700 g, 700 a 1400 g, > 1400 g). The diagnostic correlation, sensitivity, specificity, predictive values and accuracy of each frozen-section diagnosis were determined. RESULTS: Eight hundred forty two ovarian tumors that underwent frozen-sections between January 1988 and October 1998 were studied. Final diagnosis was 86.7% benign, 2.7% low malignant potential (LMP) and 10.6% malignant. The diagnosis correlation between frozen- and permanent-sections was 98.2%. Misdiagnosis was in epithelial ovarian tumors, particularly in LMP tumors. Sensitivity, specificity, positive- and negative-predictive values and accuracy of the four hundred eighty nine epithelial tumor were 92.6%, 99.2%, 96.7%, 98.2% and 97.9%, respectively. Diagnostic correlation was higher in epithelial ovarian tumors with diameter < 10 cm (98.2% v/s 93.8%) and weight < 700 g (96.9% v/s 88.9%). CONCLUSIONS: Diagnostic correlation with permanent-section examination, sensitivity, specificity and predictive values of frozen-sections are high in ovarian tumors. Accurate diagnosis at frozen sections of epithelial ovarian tumors with diameter > 10 cm or weight > 700 g (particularly in LMP tumors) is difficult because of the extensive sampling required. Frozen-sections diagnoses are important to determine the type and extent of surgery performed at the initial operation.

[Inactivation of tumor suppressor genes in uterine cervix carcinogenesis]. / Inactivación de genes supresores de tumores en la carcinogénesis del cuello uterino.

The importance of inactivation of tumor suppressor genes in the development/progression of carcinomas of the uterine cervix is reviewed. It is well known that HPV-related oncogenes are strongly linked to cervical cancer. However, fewer studies have explored the occurrence of inactivation of tumor suppressor genes in this neoplasia. Genetic deletions affecting tumor suppressor genes are the most common mechanism of inactivation of these genes. Studies using conventional molecular techniques such as restriction fragment length polymorphism (RFLP) and Southern Blot showed low frequency of deletions in cervical carcinomas. Detection of deletions by using RFLP and Southern Blot presents several disadvantages, the most important being the difficulty in analyzing pure tumor cells. More sensitive approaches include tissue microdissection and PCR analysis of micro-satellites. Using these approaches, it has been shown that genetic deletions are, in fact, frequent events in cervical cancers, being detected in up to 95% of the cases. Multiple genetic loci are involved, including chromosomes 3p, 5p, 6p and 11q. Deletions are detected even in precursor lesions (cervical intraepithelial neoplasia, CIN). Some deletions have been correlated with prognostic parameters, such as stage, depth of invasion, and vascular space involvement. It is concluded that cervical carcinogenesis, like in other tumors, is a multistep process, characterized by the accumulation of events including activation of oncogenes, as well as inactivation of tumor suppressor genes.

Histopathology and molecular biology of ovarian epithelial tumors.

Carcinogenesis in the ovary presents special features related to that organ. First, the preinvasive or even invasive lesions are difficult to detect, which explains why most cases are diagnosed at an advanced stage. Second, the group of tumors of low malignant potential (borderline tumors) are still a controversial category of ovarian lesions. Finally, familial ovarian tumors represent an interesting hereditary model of carcinogenesis at the molecular level. Flow cytometry and immunohistochemistry for proliferative markers or oncogenes provide important prognostic information in patients with ovarian tumors. Molecular data, such as loss of heterozygosity at specific genetic loci, also have been correlated with prognosis. Clonality studies in patients with multiple ovarian/pelvian lesions analyzing chromosome X inactivation patterns and genetic deletions or mutations have contributed to the understanding of the origin of these lesions. New technologies to study gene expression patterns, such as cDNA library construction and DNA microarray technologies, are being applied to study histologic phases of tumor progression, such as normal, preinvasive, and tumor tissues. It is hoped that these studies will contribute important information not only for a better understanding of the process of carcinogenesis, but also for assessing the biology and behavior of individual tumors, determining patient prognosis, and eventually influencing therapy.

cDNA sequencing and analysis of POV1 (PB39): a novel gene up-regulated in prostate cancer.

We recently identified a novel gene (PB39) (HGMW-approved symbol POV1) whose expression is up-regulated in human prostate cancer using tissue microdissection-based differential display analysis. In the present study we report the full-length sequencing of PB39 cDNA, genomic localization of the PB39 gene, and genomic sequence of the mouse homologue. The full-length human cDNA is 2317 nucleotides in length and contains an open reading frame of 559 amino acids which does not show homology with any reported human genes. The N-terminus contains charged amino acids and a helical loop pattern suggestive of an srp leader sequence for a secreted protein. Fluorescence in situ hybridization using PB39 cDNA as probe mapped the gene to chromosome 11p11.1-p11.2. Comparison of PB39 cDNA sequence with murine sequence available in the public database identified a region of previously sequenced mouse genomic DNA showing 67% amino acid sequence homology with human PB39. Based on alignment and comparison to the human cDNA the mouse genomic sequence suggests there are at least 14 exons in the mouse gene spread over approximately 100 kb of genomic sequence. Further analysis of PB39 expression in human tissues shows the presence of a unique splice variant mRNA that appears to be primarily associated with fetal tissues and tumors. Interestingly, the unique splice variant appears in prostatic intraepithelial neoplasia, a microscopic precursor lesion of prostate cancer. The current data support the hypothesis that PB39 plays a role in the development of human prostate cancer and will be useful in the analysis of the gene product in further human and murine studies.

General mechanisms of metastasis.

In the present article, the steps involved in the process of tumor metastasis are discussed. Several events are required for malignant cells to leave the primary tumor and proliferate at a distant site: vessel formation (angiogenesis), cell attachment, invasion (matrix degradation, cell motility), and cell proliferation. Molecular mechanisms underlying each of these steps are described. Based on blocking these processes, new anti-metastasis therapies are being developed.

Identification of a novel transcript up-regulated in a clinically aggressive prostate carcinoma.

OBJECTIVES: To identify differentially expressed genes in tumor cells of patients with prostate cancer by means of tissue microdissection and targeted differential display. METHODS: RNA was recovered from pure populations of microdissected normal epithelium and invasive tumor from frozen tissue sections of a radical prostatectomy specimen. Reverse transcription-polymerase chain reaction (PCR) using arbitrary and zinc finger PCR primers was performed. RESULTS: A 130-base pair product was identified that appeared selectively in the tumor sample. DNA sequence analysis revealed it to be a clone from the expressed sequence tag database (GenBank accession R00504). Microdissection of normal epithelium and the corresponding invasive tumor was subsequently performed on a test panel of 10 prostate carcinoma specimens. Comparison of R00504 levels in normal epithelium and invasive carcinoma, using beta-actin as an internal control, showed the transcript to be substantially overexpressed in 5 of 10 carcinomas. Northern blotting revealed R00504 to be a 2.6-kilobase gene. CONCLUSIONS: A novel transcript up-regulated in an aggressive prostate carcinoma was identified using degenerate zinc finger primers in microdissected tissue samples. The approach used in this study may be helpful in quantitative comparison of known genes and identification of novel genes in microdissected human tissue samples.

Molecular genetic events in the development and progression of ovarian cancer in humans.

Ovarian tumor development is characterized by specific clinical and pathological features that provide an interesting model of carcinogenesis: first, the pre-invasive and even invasive lesions are difficult to detect; second, a group of cases with a known familial predilection constitute an important heredltary model of carcinogenesis; and third, the category of morphologically borderline ovarian tumors (tumors of low malignant potential) poses several unanswered questions such as: what histologic criteria should be used for their diagnosis; what is their natural history; and what is their molecular relationship to invasive tumors? Recently, molecular studies have contributed to a better understanding of the biology of these tumors, their behavior in vivo, and their response to therapy. This article summarizes the most recent molecular advances.

Molecular analysis of synchronous uterine and ovarian endometrioid tumors.

The presence of simultaneous carcinomas involving both the ovary and uterine corpus presents a diagnostic challenge, particularly if the tumors have a similar histology. The classification of these lesions as either two separate primary tumors, or as a single primary tumor with a metastasis has significant implications with respect to patient prognosis and recommendations for therapy. Although several morphologic criteria have been proposed as guidelines for the classification of these lesions, certain cases remain difficult to confidently classify. The application of current molecular biology techniques to pathological specimens can provide genetic information than can be helpful in establishing the relationship between synchronous neoplasms. Specifically, the use of tissue microdissection and polymerase chain reaction (PCR) amplification of DNA can be helpful. In this study we used polymorphic DNA markers on chromosomes 17q21.3-22 and 11q13 to study loss of heterozygosity (LOH) in 13 patients who presented with endometrioid tumors in both the uterus and ovary. Ten of the 13 cases showed LOH in one or both tumors. In eight of the 13 cases the detected LOH either chromosome 17q21.3-22 or 11q13 occurred selectively in only one of the two tumor sites. The results of this study suggest that the eight cases with LOH selective for one tumor site represent patients with two separate primary tumors. Molecular analysis may be useful in determining the relationship of synchronous uterine and ovarian endometrioid neoplasms.

Analysis of loss of heterozygosity on chromosome 11q13 in atypical ductal hyperplasia and in situ carcinoma of the breast.

Identical allelic loss in invasive and adjacent in situ ductal breast carcinoma (DCIS) on chromosome 11q13 has been previously reported, providing molecular evidence for the progression of DCIS to invasive tumor. In this study we analyzed loss of heterozygosity (LOH) on 11q13 (PYGM, INT-2) in atypical ductal hyperplasia (ADH) and various histological types of in situ carcinomas of the breast in patients without invasive cancer. Twenty-four cases of in situ carcinoma and twelve cases of ADH were studied. Tissue microdissection of normal, hyperplastic, and tumor cells from fixed, paraffin-embedded sections was performed, and DNA was extracted for polymerase chain reaction. In situ tumors included both high- and low-grade DCIS. LOH was identified in six of twenty-two (27.3%) in situ tumors and in one of eleven (9%) ADH cases. Within in situ carcinomas, LOH was identified in six of seventeen (35%) high-grade DCIS but in none of six low-grade DCIS. The present results show that LOH at 11q13 occurs in an appreciable proportion of high-grade DCIS, although the rate is substantially less than in patients with concomitant DCIS and invasive tumor. LOH was identified less frequently in low-grade in situ tumors and ADH, suggesting that a putative tumor suppressor gene(s) located on chromosome 11q13 may be involved in the transition from early preneoplastic lesions to invasive breast cancer.