Yan-Gao Man

Androgen Receptor Expression Along with Loss of bcl‐2, ER, and PR Expression in Benign and Malignant Apocrine Lesions of the Breast: Implications for Therapy

Abstract: Apocrine lesions of the breast are not uncommon. Limited studies have suggested that apocrine cells may be under the influence of an androgenic, rather than an estrogenic, regulatory system. Furthermore, a recent study has shown that metaplastic apocrine cells of the breast lack expression of bcl‐2, a B‐cell leukemia/lymphoma gene protein whose expression in the breast is believed to be regulated by estrogen. This study was undertaken to immunohistochemi‐cally assess the status of expression of estrogen receptor (ER), progesterone receptor (PR), bcl‐2, and androgen receptor (AR), in a series of mammary apocrine lesions ranging from simple metaplasia to metastatic carcinoma, and to determine whether there is any possible interrelationship or correlation between the expression of these hormone receptors and that of bcl‐2.

Morphofunctional Features of Intraductal Hyperplasia, Atypical Intraductal Hyperplasia, and Various Grades of Intraductal Carcinoma

Abstract: The spectrum of epithelial proliferations within the mammary duct system includes lobular, ductal, and papillary patterns of proliferation. Among those with a ductal growth pattern, the highest proportion qualifies as ordinary intraductal hyperplasia (IDH), a small proportion shows atypical intraductal hyperplasia (AIDH), and a steadily increasing percentage is composed of intraductal carcinomas (IDCA). AIDH and some variants of IDCA share many characteristics and their distinction can be quite troublesome, in some cases relying completely on an arbitrary quantitative criterion. IDH, however, is quite distinctive from both a morphologic and biologic standpoint and should not be confused with AIDH.

Evidence that Leydig cells in Sertoli-Leydig cell tumors have a reactive rather than a neoplastic profile.

Leydig cells are a variable and an inconstant feature of Sertoli‐Leydig cell tumors (SLCT). Controversy exists regarding their neoplastic versus reactive nature, and their molecular biologic profile is unknown.

An improved method for DNA extraction from paraffin sections.

The acquisition of comparable quality and quantity of DNA extracts is the prerequisite to the success of comparative genetic analyses. Although several DNA extracting protocols on paraffin sections have been introduced, the importance of deparaffinization, the procedure for obtaining an adequate hematoxylin staining, the significance of the ratio of the cell number to the enzyme volume, and a practical means for monitoring the digestion process have not been sufficiently addressed. These, however, are the most important factors accountable for a failure of DNA extraction. To minimize the impact of these factors, we have developed several unique strategies, including: (1) incubating sections at 80 degrees C for 30-60 minutes prior to xylene treatment, (2) checking each section to insure the complete removal of paraffin; (3) treating hematoxylin stained sections or cells with de-staining solutions; (4) using a micrometer inserted into the eyepiece of a microscope to estimate the number of cells collected and adjusting the enzyme volume according to the cell number; and (5) monitoring the digestion process with a magnifier. With these strategies, we have been able to consistently obtain comparable quality and quantity of DNA extracts which yielded uniform PCR products regardless of variations in tissue embedding and processing.

An antigen unmasking protocol that satisfies both immunohistochemistry and subsequent PCR amplification

Immunohistochemical elucidation of many proteins in formalin-fixed, paraffin-embedded tissues requires a prior antigen unmasking treatment, which often damages both the morphology and genetic materials, making subsequent assessments difficult or impossible. This study attempted to develop a method that satisfies both immunohistochemical and genetic analyses. Consecutive sections were made from formalin-fixed, paraffin-embedded breast and other tissues, and a set of four adjacent sections from each case were treated with (1) routine H & E staining; (2) our unmasking protocol; (3) microwave oven irradiation; (4) pressure cooker incubation. After immunohistochemical staining, the tissue in each section was scraped off, or the same cell clusters in four sections were separately microdissected for DNA extraction and PCR amplification. Compared to microwave and pressure cooker methods, our protocol showed the following advantages: (1) a better preservation of the morphology; (2) a substantial reduction of tissue detachments from slides; (3) effectiveness on all antibodies tested, including those requiring enzyme digestion or no prior unmasking; (4) higher PCR yields; (5) larger (higher molecular weight) amplified PCR products. Compared to the routine method on untreated tissues, our method consistently produced a comparable quality and quantity of PCR products. Our protocol, however, takes a longer time to yield results.

Contributions of leukocytes to tumor invasion and metastasis: The “piggy-back” hypothesis

The epithelium, which is the origin of over 85% of human malignant tumors, is physically separated from the stroma by the basement membrane. In breast, prostate, and major salivary glands, the epithelium is further segregated form the stroma by a layer of myoepithelial or basal cells. The epithelium is normally devoid of lymphatic ducts and blood vessels, and thus, totally relies on the stroma for its essential needs. Due to these structural relationships, the disruption of these structures is a prerequisite for tumor invasion or metastasis, which is believed to be a multistage process, progressing sequentially from normal to

Leukocyte-mediated cell dissemination and metastasis: findings from multiple types of human tumors.

Our previous studies revealed that leukocyte infiltration could trigger human breast and prostate tumor invasion through focal disruptions of the tumor capsule, which selectively favors monoclonal proliferation of tumor progenitors or a biologically more aggressive cell clone overlying the focal disruptions. Our current study, involving multiple types of human tumors, further shows that leukocyte infiltration could also trigger tumor metastasis through the following pathways: [1] more leukocytes migrate to focally disrupted tumor capsules, which forms leukocyte aggregates surrounding newly formed tumor cell clusters, [2] the physical movement of leukocytes into proliferating tumor cells disrupts the intercellular junctions and cell‐surface adhesion molecules, causing the disassociation of tumor cells from the tumor core, [3] leukocytes are conjoined with some of these tumor cells through plasma membrane fusion, creating tumor cell–leukocyte chimeras (TLCs), and [4] the leukocyte of TLCs impart migratory capacity to associated tumor cell partners, physically dragging them to different tissue sites. Our findings suggest a novel pathway for tumor cell dissemination from the primary sites and the subsequent journey to new sites. Our findings also provide a unique explanation for the cellular mechanism of leukocytes on tumor invasion and metastasis. If confirmed, our hypothesis and technical approach may significantly facilitate early detection and intervention of tumor invasion and metastasis. J. Cell. Biochem. 112: 1154–1167, 2011.

Five Useful Approaches for Generating More Valid Gel Images of Loss of Heterozygosity and Clonality Analysis With an Automated 377 Dna Sequencer

The recently introduced fluorescence-based gene scan system for assessment of loss of heterozygosity (LOH) and clonality with an automated DNA sequencer has several advantages over the traditional method. However, the production of gel images with this system is subjected to more technical challenges, including the interference of autofluorescence, weaker and less consistent signals that result from the restricted well size and difficulties in sample loading. To minimize the impact of these technical difficulties, several unique strategies were used, including the following: elimination of fabrics or paper towels in the cleaning of gel plates and containers; use of a modified loading buffer; use of more concentrated gels; use of an innovative apparatus to clean gel wells before and after the prerun; and covering the black printer cartridge with a sheet of scotch tape. With these strategies, the authors have been able to consistently obtain gel images that can be presented as either densitometric graphs or as band patterns for direct visual assessment.

Multiple use of slab gels in sequencing apparatus for separation of polymerase chain reaction products.

Attempting to assess whether a decrease of the electrophoresis temperature could prevent or reduce the extent of gel well deformations, and whether the utilization of native polyacrylamide gels (without urea) could speed up the separation of polymerase chain reaction (PCR)‐amplified products with an automated 377 DNA sequencer, denatured PCR products were subjected to electrophoresis in 6% native gels under 45°C. Results show that a decrease of the electrophoresis temperature from 51°C (recommended by the Users Manual) to 45°C substantially facilitates the preservation of gel wells, and that all PCR products tested migrate significantly faster in native than in denatured (with urea) gels of the same concentration. The combination of a 6% native gel and a lower (45°C) electrophoresis temperature permits multiple uses of a given gel with consistent results, consequently reducing the electrophoresis time and reagent costs.

Tissue microarrays: construction and uses.

Tissue microarrays (TMAs) are produced by taking small punches from a series of paraffin-embedded (donor) tissue blocks and transferring these tissue cores into a positionally encoded array in a recipient paraffin block. Though TMAs are not used for clinical diagnosis, they have several advantages over using conventional whole histological sections for research. Tissue from multiple patients or blocks can be examined on the same slide, and only a very small amount of reagent is required to stain or label an entire array. Multiple sections (100-300) can be cut from a single array block, allowing for hundreds of analyses per microarray. These advantages allow the use of TMAs in high-throughput procedures, such as screening antibodies for diagnostics and validating prognostic markers that are impractical using conventional whole tissue sections. TMAs can be used for immunohistochemistry, immunofluorescence, in situ hybridization, and conventional histochemical staining. Finally, several tissue cores may be taken without -consuming the tissue block, allowing the donor block to be returned to its archive for any additional studies.