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.

Identical allelic loss on chromosome 11q13 in microdissected in situ and invasive human breast cancer.

Human breast carcinoma is thought to develop through progressive stages from atypical hyperplasias to in situ carcinoma and finally to invasive and metastatic cancer. In situ breast carcinoma consists of small, isolated neoplastic foci which cannot be selectively studied by conventional methods. In this study, we used tissue microdissection to examine the loss of heterozygosity (LOH) of chromosome 11q13 in both in situ and invasive lesions of the breast, as compared to normal breast epithelium from the same patients. Forty-one cases of sporadic breast cancer were analyzed. Tissue microdissection allows for procurement and PCR-based analysis of small lesions using either frozen or formalin-fixed, paraffin-embedded tissue sections. LOH on chromosome 11q13 was found in 24 of 36 (67%) of the informative invasive breast cancer cases using two polymorphic DNA markers specific for this region (INT2 and PYGM). Twenty-one of the cases which demonstrated LOH in the invasive tumor also contained in situ carcinoma in the same tissue section. Seventy-one % (15 of 21) of the microdissected in situ lesions showed LOH of chromosome 11q13. Every case (15 of 15) of in situ tumor with LOH showed loss of the same allele in the corresponding invasive tumor cells. The results of this study suggest that a tumor suppressor gene located on chromosome 11q13 may play an important role in the early stages of development of sporadic human breast cancer. This finding provides molecular genetic support for the hypothesis that invasive breast cancer arises from in situ lesions.

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.

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.

Histologic assessment of the age of recent brain infarcts in man.

In order to design a dating system based on the microscopic picture of brain infarcts of recent onset, we performed the histological examination of 31 infarcts covering the first 4 weeks of evolution in 30 autopsy cases. The date of the cerebral vascular accident was clinically established in every case. There were 13 men and 17 women with a mean age of 65 years. Hemorrhagic infarcts were found in 15 cases and anemic infarcts in 16 cases. Based on the histological features four periods were identified: the first period, from day 1 through day 4, was characterized by the predominance of eosinophilic neurons and necrotic oligodendrocytes; the second period, from day 5 through day 7, differed from the first by the appearance of macrophages and of newly formed blood vessels; the third period, from day 8 through day 14, showed neuronal ghosts, macrophages, astrocytic proliferation, gemistocytes, and absence of neutrophils; and in the fourth period, from day 15 through day 27, there were no eosinophilic neurons, and neither necrotic oligodendrocytes nor myelin in the central portion of the infarct were identified. By assessing the histological features and accurately correlating the findings with the corresponding clinical data, we have been able to describe four distinct microscopic patterns of the first month of evolution of brain infarcts. The present findings may be considered useful morphological clues to better characterize the early evolutional phase of brain infarcts in humans.

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.

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.

Meningothelial meningioma with "amianthoid" fibers. Case report with ultrastructural study.

The case of a meningothelial meningioma with 'amianthoid' fibers in a 48-year-old woman is presented. By light microscopy the tumor showed the typical features of meningothelial meningioma and rounded, deeply eosinophilic, and fibrillary areas, especially around and/or in the vicinity of blood vessels. These fibers are also called 'amianthoid' fibers. Ultrastructurally, these foci were made up of disorderly arranged and interwaving mature collagen fibrils with a variable width between 40 and 190 nm. No evidence of intracellular collagen synthesis by the tumor cells was found. The presence of 'amianthoid' fibers does not seem to carry any prognostic significance.

Intestinal microsporidiosis in a Chilean patient with acquired immunodeficiency syndrome (AIDS).

A 24-year-old male patient with AIDS diagnosed in 1989, and with several episodes of pneumocystosis, was admitted because of a chronic diarrheic syndrome and severe epigastric pain. Endoscopy showed a granular duodenal mucosa. Light microscopy showed a moderate villous atrophy with round-cell inflammatory infiltration of the chorion. Giemsa, Ziehl-Neelsen, and Gram stains showed microsporidial spores measuring between 1.5 and 2 microns in the supranuclear cytoplasm of some enterocytes. Electron microscopy showed sporoblasts and spores consistent with Enterocytozoon bieneusi, with an apparently non-tubular, rather electron-dense polar filament showing up to 7 coils and also a microtubular internal structure with annular disposition, a finding which has not been adequately emphasized in the pertinent literature, probably representing a contractile property of the polar filament, rather than a mere duct for the parasitic sporoplasm to be inoculated.

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.

Detection of heterozygosity loss in microdissected fine needle aspiration specimens of breast carcinoma.

OBJECTIVE: To use the polymerase chain reaction (PCR) to detect loss of heterozygosity (LOH) in microdissected cells form cytologic smears obtained by fine needle aspiration (FNA) from 20 cases of invasive breast carcinoma. STUDY DESIGN: In each case, histologic sections of the primary tumor were also available. Tumor and nontumor cells were dissected from both the cytologic smear and tissue section in all cases except in three smears that showed only tumor cells. RESULTS: LOH was identified in 10 of 19 informative cases using two polymorphic DNA markers at chromosome 11q13 (INT-2, PYGM). The same results were obtained in both the cytologic and histologic specimens, including three cases that had hypocellular cytologic smears. CONCLUSION: FNA of breast lesions provides adequate samples for direct microdissection of the cytologic smear to detect LOH using PCR amplification.

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.

[Diagnosis of human papillomavirus infections in cervical cytology in the absence of classical signs]. / Diagnóstico de infección por virus papiloma humano en citología cervical con ausencia de signos clásicos.

"Nonclassic" cytologic signs (NCS) of infection by human papillomavirus (HPV) were analyzed in two groups of patients: A: (condylomata) 81 women with both condylomata confirmed by biopsy and a cervical smear not showing the "classic" signs of HPV infection, and B: (controls) 50 cervical smears diagnosed as negative. In group A, 96% showed nuclear hyperchromatism, 78% showed perinuclear halos, 77% showed clear cytoplasm, and 74% showed mild koilocytosis. In 89% of the condylomata cases at least four NCS were found. In the control group only three cases showed nuclear hyperchromatism; the most frequent NCS in this group was perinuclear halo (50%). The frequency in which NCS of condylomata are found in smears without the classic signs is high. Nevertheless, 11% of these showed less than four NCS; in all of them, nuclear hyperchromatism was prominent. This is the most useful NCS in the diagnosis of HPV infection.

[Cutaneous angiitis: direct immunofluorescence and morphopathology in 23 cases]. / Angeítis cutáneas: inmunofluorescencia directa y morfopatología de 23 casos.

We reviewed the findings in 23 patients with histologically diagnosed cutaneous angiitis, between 1981 and 1985. There were 8 males and 15 females, with a mean age of 43 years. Four histological patterns were identified: leukocytoclastic angeitis in 9 (31%), lymphocytic angeitis in 8 (35%), capillaritis in 2 (9%) and minimal alterative angeitis in 4 (17%). Serum complement levels were normal in 56% and low in the remainder. Fourteen patients (61%) showed positive direct immunofluorescence for complement fractions and different immunoglobulins in skin lesions. All cases with lymphocytic angeitis yielded a positive reaction as compared to only 66% in leucocytoclastic angeitis. No correlation was found between serum complement levels and histologic pattern of angeitis. The present results suggest that direct immunofluorescence in diseased skin is a more sensitive procedure for identifying active cutaneous angeitis.

[Epithelioid smooth muscle tumor of the uterus, of unknown malignant potential]. / Tumor muscular liso epitelioideo del útero, de potencial maligno incierto.

A uterine smooth muscle tumor belonging to the category of unknown malignant potential in a 20-year-old woman is reported. The major axis of the tumor was 5 cm, and the tumor was soft and homogeneous. Histologically, it was composed of cellular groups, arranged in an epithelioid pattern, with hyaline bands dissecting the groups, moderate nuclear hyperchromasia and pleomorphism, and mitotic index of 2 per 10 high power fields; the tumor had infiltrating margins and tumoral vascular invasions were found. Immunohistochemically, the tumor was positive for vimentin, and negative for keratins, desmin, actin, and S 100 protein. Epithelioid smooth muscle tumors of the uterus should be classified as tumors of unknown malignant potential under the following conditions: if they are larger than 5 cm, if they show at least moderate cellular pleomorphism, if their mitotic index is at least 1 per 10 high power fields, or if they show cellular necrosis. The present immunohistochemical findings suggest more a fibroblastic than a smooth muscle cell origin of this neoplasm.

[Miniature squamous cells in gynecological smears: high association with epithelial lesions]. / Celulas escamosas en miniatura (CEM) en frotis ginecológicos: alta asociacion con lesiones epiteliales.

The frequence of abnormal smears in gynecological cytologies containing miniature squamous cells (MSC) was studied. Every smear containing MSC was collected from 5.000 consecutive smears. Two hundred thirty three cases (4.66%) containing MSC collected. The general frequence of abnormal smears was 2.04%, while this frequence was 24.89% in cases with MSC (p < 10(-8). 114 cases with MSC had an atrophic pattern, 10.5% of them being abnormal smears; 119 cases with MSC had an estrogenic/progestative pattern, 38.7% of them being abnormal smears (p < 0.05). 53.2% of smears containing MSC and keratohyaline granules had a cytological diagnosis of epithelial lesion or were atypic, while only 10.4% of cases with MSC and without keratohyaline granules had these diagnosis (p < 10(-6)). The frequence of abnormal smears was significantly higher in cases with MSC than the general frequence of abnormal smears, especially in cases also having a non-atrophic pattern or containing keratohyaline granules.