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.

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.

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.

Construction of a representative cDNA library from prostatic intraepithelial neoplasia.

We report the construction of a plasmid-based cDNA library made from microdissected cells derived from prostatic intraepithelial neoplasia. Total RNA was extracted and converted to blunt-ended, double-stranded cDNA by oligo(dT)-mediated reverse transcription followed by linker addition. A linker-specific primer with UDG-compatible ends was used to amplify the cDNA and the resulting PCR product was subcloned. A total of 154 clones were sequenced and results indicated that 81.5% of the clones derived from either known genes, anonymous expressed sequence tags, or novel transcripts with very little redundancy of screened clones. These results demonstrate the feasibility of constructing complex representative cDNA libraries from specific microdissected cell populations that represent microscopic precursor stages of cancer progression. This method should facilitate identification of transcripts specifically expressed in cells of a distinct histological origin and tumorigenic stage.

Laser capture microdissection.

Laser capture microdissection (LCM) under direct microscopic visualization permits rapid one-step procurement of selected human cell populations from a section of complex, heterogeneous tissue. In this technique, a transparent thermoplastic film (ethylene vinyl acetate polymer) is applied to the surface of the tissue section on a standard glass histopathology slide; a carbon dioxide laser pulse then specifically activates the film above the cells of interest. Strong focal adhesion allows selective procurement of the targeted cells. Multiple examples of LCM transfer and tissue analysis, including polymerase chain reaction amplification of DNA and RNA, and enzyme recovery from transferred tissue are demonstrated.

Genetic analysis of synchronous mucinous tumors of the ovary and appendix.

The coexistence of mucinous ovarian and appendiceal tumors in association with pseudomyxoma peritonei (PP) is well established. However, it has not been determined whether they represent independent or metastatic neoplasms. The authors analyzed microsatellites on chromosome 17q 21.3-22 (nm23), 3p 25-26 (von Hippel Lindau disease [VHL] gene), and 5q 21-22 (D5S346 locus) in 12 synchronous ovarian and appendiceal mucinous lesions. Loss of heterozygosity (LOH) at the nm23 locus has been shown previously in ovarian carcinomas, and genetic alterations at both the 3p and 5q loci have been reported in colorectal carcinomas. The ovarian lesions consisted of nine mucinous tumors of low malignant potential and three invasive adenocarcinomas, and the appendiceal lesions consisted of eight carcinomas without invasion, two invasive carcinomas, and two mucosal hyperplasias. DNA was extracted from microdissected cells obtained from formalin-fixed, paraffin-embedded tissue sections and amplified by polymerase chain reaction. In three specimens, genetic alterations occurred at 17q 21.3-22 in only the ovarian tumors. One of these cases showed LOH on chromosome 5q 21-22 in only the appendiceal tumor. In three other specimens, LOH at the same locus was found in both tumors. Six specimens did not show LOH at any locus. These results suggest that a subset of synchronous mucinos ovarian and appendiceal lesions showing different LOH patterns in both sites most likely represent patients with two separate primary lesions. Another group of specimens with the same allelic loss in both tumors most likely represent patients with a single primary and metastatic spread. Thus, genetic analysis of these lesions may be useful in investigating the origin of histologically similar synchronous tumors.

Loss of heterozygosity on the short arm of chromosome 8 in male breast carcinomas.

Identification of loss of heterozygosity (LOH) at specific genetic loci in neoplastic cells suggests the presence of a tumor suppressor gene within the deleted region. LOH on chromosome 8p has been identified in colorectal, bladder, hepatocellular, and prostatic carcinomas. Little is currently known about the molecular events occurring during the development of male breast cancer. We studied LOH on chromosome 8p in 23 male breast carcinomas. Five polymorphic DNA markers were used: D8S136 and D8S137 on 8p12-21.3; and D8S254, D8S258, and D8S349 on 8p22. DNA was extracted from microdissected normal and tumor cells obtained from formalin-fixed, paraffin-embedded tissue sections and amplified by the PCR. LOH was identified in 19 of 23 cases (83%) with at least one marker. Seven cases showed LOH only at 8p22, six cases showed LOH only at 8p12-21.3, and six cases showed LOH at both 8p22 and 8p12-21.3. In five of these last six cases, at least one locus was retained between the two deleted regions; thus, the whole short arm of chromosome 8 was not lost in these tumors. Our results show that there are two discrete areas of deletion on chromosome 8p in male breast cancer, suggesting the presence of one or more tumor suppressor genes that may play a role in the development or progression of the disease.