Processing of cytological specimens.

Individual cells often need to be examined with antibodies apart from the surrounding tissue. They may be cells in fluid, cells encased in mucus from a swab, or cells directly extracted from a piece of tissue. Cells can be viewed on a glass slide as cell smears produced from a cell enriched source, introduced as a touch preparation from a piece of wet tissue, concentrated on a slide by the use of a cytocentrifuge, or applied directly to a slide from a solid medium such as a cotton swab. These cell preparations can then be optimally fixed in weakly or nondenaturing solutions such as acetone or those that are alcohol based. They can also be postfixed in formalin if desired. Incubation in buffers containing 0.25% Triton X-100 and 5% dimethylsulfoxide (DMSO) allow for easier antibody penetration. The endogenous peroxidase enzyme or oxidative compounds can be quenched in a mild hydrogen peroxide solution. The sections are then ready to test with antibody after an incubation in a normal serum solution blocks any available charged sites.

Preparation of frozen sections for analysis.

The analysis of frozen tissue by antibodies can be accomplished by the quick freezing of a small tissue sample in liquid nitrogen. Super-cooled isopentane can also be used to further the preservation process. Freezing preserves the available proteins in a near-native state for their identification by antibodies raised against naturally folded proteins. The tissues are sectioned onto charged glass slides where they can be optimally fixed in weakly or non-denaturing solutions such as acetone or those that are alcohol-based. Following mild pretreatment steps to allow for antibody use with low background, (the endogenous peroxidase enzyme or oxidative compounds quenched in a hydrogen peroxide solution and available charged sites blocked by incubation in a normal serum solution) the sections are ready for antigen detection.

Processing of tissue specimens.

In order to test tissue specimens with antibody, they first have to be preserved in fixative, embedded in paraffin, and sectioned very thinly onto glass microscope slides. Any piece of tissue, immediately after excision, must be placed into an adequate volume of fixative. Fixatives vary, but the standard one is 10% buffered formalin. After an optimum fixation time (for formalin, about 16 h), the sample must be embedded in paraffin and sectioned on a microtome. Paraffin-embedded sections placed on positively charged slides (either coated or commercially prepared) are then ready for various pretreatment steps. First, the paraffin must be replaced with water through a series of rehydration steps. Then, depending on the antigen to be tested, the section can be proteolytically digested with enzymes or heat-treated in low or high pH solutions. Following that, the endogenous peroxidase enzyme or oxidative compounds can be quenched in a hydrogen peroxide solution. The sections are then ready to be tested with antibody after an incubation in a normal serum solution blocks any available charged sites.

Processing of tissue culture cells.

Live cells are often studied, in vitro, bathed in nutrient growth media. It is sometimes necessary to study individual compounds produced by these cells and antibodies work well for this purpose. These cells must first be concentrated and fixed before testing. There are a couple of ways to study cells in culture using antibodies. One is to fix the cells in place as they adhere to a solid surface and then test them as though they were cells on a slide. Another is to retrieve them and pellet the cells, fixing them in a test tube and then embedding and sectioning them as though they were a solid tissue. Fixatives can be mild to moderate depending on the antigens to be studied. Sectioned cells can be tested following mild pretreatment steps. Cells fixed in the culture dish can be tested following mild pretreatment steps in buffers containing 0.25% Triton X-100 and 5% dimethylsulfoxide (DMSO) to allow for easier antibody penetration. The endogenous peroxidase enzyme or oxidative compounds can be quenched in a mild hydrogen peroxide solution. The sections are then ready to test with antibody after an incubation in a normal serum solution blocks any available charged sites.

The peroxidase-antiperoxidase (PAP) method and other all-immunologic detection methods.

Immunoenzyme procedures take on many forms, including, simply, antibody coupled to enzyme. These direct techniques require the labeling of all the primary antibodies and can produce more background. A more economical method uses a secondary antibody or one that has the primary antibody as its antigen. Labeling this secondary antibody with enzyme provides detection for many primary antibodies directed against different antigens of interest. A more sensitive approach involves the use of antibodies directed against enzyme connected to same-species primary antibodies by a secondary linking antibody. This "all immunologic" technique is more sensitive and can result in less background than the covalently labeled methods. Finally, an immune polymer consisting of several secondary antibodies along with many enzyme molecules embedded on one long chain carbon polymer, can be used. This can provide a faster, more universal detection procedure with adequate sensitivity and specificity.

Overview of antigen detection through enzymatic activity.

The identification of antigenic substances with antibodies can only occur through the use of a reporter molecule. One way of doing this is through the use of enzymes. Enzymes act upon a substrate and that substrate, or a molecule affected by that substrate, in turn becomes detectable by a variety of methods. There are many enzymes available for this purpose. The most common is peroxidase. Another widely used enzyme is alkaline phosphatase. Each enzyme has a few chromogenic substrate solutions with which it can react to change a color visualized through the use of selected instruments, including the microscope. Antibodies can be labeled with an enzyme directly, or secondary antibodies can be labeled with the enzyme and employed in an indirect technique. Also, immunoglobulin labeled polymers labeled with enzyme can be used and the enzymes themselves can serve as antigens in immunoenzyme complex procedures. Finally, avidin or biotin can be labeled with enzyme, and used either singly or in complexes, and peroxidase mediated biotin amplification can be used to increase the sensitivity in some procedures.

The avidin-biotin complex (ABC) method and other avidin-biotin binding methods.

Immunoenzyme methods can be enhanced by the use of the high affinity molecules, avidin and biotin. The binding of avidin to biotin is almost irreversible. By labeling a detection enzyme such as horseradish peroxidase with biotin, and a secondary antibody (reactive against the antigen detecting primary antibody) with biotin as well, these two compounds can then be linked irreversibly with avidin. For this process, the biotinylated enzyme is complexed with avidin in solution and this avidin-biotin complex (ABC) is then introduced to the biotinylated secondary antibody, where it binds to primary antibody-antigen sites. Also, enzyme-labeled avidin molecules can be used to bind biotinylated secondary antibodies with greater resolution. Finally, biotinylated tyramide used in conjunction with peroxidase precipitates even greater amounts of biotin molecules for detection by enzyme-labeled avidin molecules.

Reversed Expression of the JAK/STAT Pathway Related Proteins Prolactin Receptor and STAT5a in Normal and Abnormal Breast Epithelial Cells.

The JAK/STAT pathway is important for cellular metabolism. One component, STAT5a, is activated in the breast upon prolactin to prolactin receptor (PRLR) binding facilitating the transcription of genes involved in lobule development. STAT5a was previously found to be expressed in most normal breast epithelial cells but not in many in situ or invasive carcinomas except secretory carcinomas which retain STAT5a expression. This report examines the JAK/STAT pathway in the breast through the detection of PRLR and STAT5a. Fifty breast tissues, including benign secretory change, microglandular adenosis, usual and atypical hyperplasia and in situ and invasive ductal carcinoma both usual and secretory, were obtained from the files of the Armed Forces Institute of Pathology. Sections were immunostained with antibodies to PRLR and STAT5a. PRLR was minimally detected on the surface of a few normal breast epithelial cells whereas STAT5a was greatly expressed in over 80% of normal cell nuclei. PRLR was also minimally detected in secretory carcinomas expressing STAT5a. However, the opposite pattern was seen in breast carcinomas lacking STAT5a expression. PRLR was abundantly expressed in these cells. This reversed expression may indicate a JAK/STAT pathway disturbance that could play a role in the initiation or maintenance of an abnormal breast phenotype.

Diagnostic utility of WT1 immunostaining in ovarian sertoli cell tumor.

WT1, the Wilms tumor gene product, can be expressed in various tumors from different anatomic sites, including some types of ovarian tumors. Regarding the latter, most studies have focused on surface epithelial-stromal tumors in which serous carcinomas are usually positive and endometrioid carcinomas are negative. Very few studies have specifically investigated this marker in ovarian sex cord-stromal tumors; however, limited data in the literature suggest that WT1 may be frequently expressed in sex cord-stromal tumors. As pure Sertoli cell tumor can be in the histologic differential diagnosis of endometrioid tumors (particularly borderline tumor and carcinoma) and carcinoid, immunostaining for WT1 might be of diagnostic value. Immunohistochemical staining for WT1 was performed in 108 ovarian tumors: pure Sertoli cell tumor (n=26), endometrioid borderline tumor (n=25), classic well-differentiated endometrioid carcinoma (n=23), sertoliform endometrioid carcinoma (n=12), and carcinoid (n=22). Additionally, inhibin and calretinin immunostaining were performed in all cases of Sertoli cell tumor for purposes of comparing expression with WT1. Extent of immunostaining was scored on a 0 to 4+ semiquantitative scale, and immunohistochemical composite scores based on a combination of extent and intensity of immunostaining were calculated in positive cases (possible range, 1 to 12). Nuclear expression of WT1 was present in 96% of Sertoli cell tumors, 16% of endometrioid borderline tumors, 13% of classic well-differentiated endometrioid carcinomas, 25% of sertoliform endometrioid carcinomas, and 0% of carcinoids. In Sertoli cell tumors, expression was diffuse (>50% of positive cells) in all positive cases. When positive in the non-Sertoli cell tumors, the extent of expression tended to be focal to patchy (50% or less positive cells). In Sertoli cell tumors, inhibin and calretinin were expressed in 96% and 54% of cases, respectively. The extent of expression of inhibin tended to be diffuse, similar to WT1; however, the extent of immunostaining for calretinin tended to be focal to patchy. The immunohistochemical composite scores for WT1, inhibin, and calretinin were 11.2, 7.6, and 4.8, respectively. Coordinate patterns for the extent of expression of WT1, inhibin, and calretinin in pure Sertoli cell tumor showed that all 3 markers were positive in 54% of cases; however, 42% were positive for WT1 and inhibin but negative for calretinin. In cases positive for both WT1 and inhibin, expression of both markers was diffuse in 84% of cases, but WT1 was diffuse while inhibin was focal to patchy in 16% of cases. We conclude that ovarian Sertoli cell tumor should be added to the growing list of WT1-positive tumors. This marker is useful for the distinction of Sertoli cell tumor from endometrioid tumors and carcinoid. The diagnostic utility of WT1 in Sertoli cell tumor is similar to inhibin but better than that of calretinin.

Immunohistochemical analysis of sox9 in ovarian Sertoli cell tumors and other tumors in the differential diagnosis.

The distinction of ovarian Sertoli cell tumor from other tumors in the histological differential diagnosis, particularly endometrioid carcinoma and carcinoid tumor, may be difficult. Many immunohistochemical markers have been studied for this differential diagnosis, but currently available markers are neither 100% sensitive nor specific. Sox9 is a transcription factor involved in Sertoli cell differentiation in the testis. The role that this molecule plays in the pathogenesis of ovarian Sertoli cell tumors and the potential use as an immunohistochemical marker for differential diagnosis have not been investigated. Immunohistochemical staining for Sox9 was performed in 152 ovarian tumors: pure Sertoli cell tumor (n = 36), endometrioid borderline tumor (n = 38), well-differentiated endometrioid carcinoma (n = 26), sertoliform endometrioid carcinoma (n = 13), and carcinoid tumor (n = 39). Nuclear expression was considered positive. Extent and intensity of staining were semiquantitatively scored. In addition, immunohistochemical composite scores in positive cases (ranging from 1 to 12) were calculated based on the extent score multiplied by the intensity score. Sox9 was expressed in 44% of Sertoli cell tumors, 55% of endometrioid borderline tumors, 65% of well-differentiated endometrioid carcinomas, 39% of sertoliform endometrioid carcinomas, and 10% of carcinoid tumors. The mean Sox9 immunohistochemical composite scores in positive cases were 6.3 for Sertoli cell tumor, 5.3 for endometrioid borderline tumor, 8.0 for well-differentiated endometrioid carcinoma, 2.8 for sertoliform endometrioid carcinoma, and 6.8 for carcinoid tumor. The differences in the mean Sox9 composite scores between Sertoli cell tumor and the other tumor categories were not statistically significant (p values ranged from 0.092 to 0.523). We conclude that Sox9 is variably expressed in ovarian Sertoli cell tumor and other tumors that are in the differential diagnosis and, thus, is not helpful for immunohistochemical distinction. Understanding the role of Sox9 in the pathogenesis of ovarian Sertoli cell tumor requires further study.

Comparative analysis of alternative and traditional immunohistochemical markers for the distinction of ovarian sertoli cell tumor from endometrioid tumors and carcinoid tumor: A study of 160 cases.

The main neoplasms in the differential diagnosis for primary ovarian tumors with a tubule-rich pattern are pure Sertoli cell tumor, endometrioid tumors (including borderline tumor, well-differentiated carcinoma, and the sertoliform variant of endometrioid carcinoma), and carcinoid tumor. Because traditional immunohistochemical markers [pan-cytokeratin (pan-CK), low molecular weight cytokeratin (CK8/18), epithelial membrane antigen (EMA), inhibin, calretinin, CD99, chromogranin, and synaptophysin] can occasionally have diagnostic limitations, the goal of this study was to determine whether or not any alternative markers [cytokeratin 7 (CK7), estrogen receptor (ER), progesterone receptor (PR), CD10, and CD56] have better diagnostic utility when compared with traditional markers for this differential diagnosis. Immunohistochemical stains for alternative, as well as traditional, markers were performed on the following primary ovarian tumors: pure Sertoli cell tumor (n = 40), endometrioid borderline tumor (n = 38), sertoliform endometrioid carcinoma (n = 13), well-differentiated endometrioid carcinoma (n = 27), and carcinoid tumor (n = 42). Extent and intensity of immunostaining were semiquantitatively scored. In addition, immunohistochemical composite scores (ICSs) in positive cases were calculated on the basis of the combination of extent and intensity scores. Cytokeratin 7 (CK7) was positive in 97% of endometrioid tumors, 13% of Sertoli cell tumors, and 24% of carcinoid tumors. The differences in the mean ICSs for endometrioid tumors versus Sertoli cell tumor or carcinoid tumor were statistically significant (P values ranging from <0.001 to 0.018). ER and PR were positive in 87% and 86% of endometrioid tumors, 8% and 13% of Sertoli cell tumors, and 2% each of carcinoid tumors, respectively. The differences in the mean ICSs for endometrioid tumors versus Sertoli cell tumor were statistically significant (P values ranging from <0.001 to 0.012). Among the epithelial markers, EMA seemed to be the most discriminatory but only slightly better than CK7, ER, or PR. Pan-CK and CK8/18 were not helpful. CD10 showed overlapping patterns of expression in all categories of tumors. Among the sex cord markers, CD10 was markedly less useful than inhibin or calretinin; CD99 was not discriminatory. CD56 showed overlapping patterns of expression in all categories of tumors. Among the neuroendocrine markers, CD56 was less useful than chromogranin or synaptophysin. When traditional immunohistochemical markers are problematic for the differential diagnosis of ovarian Sertoli cell tumor versus endometrioid tumors versus carcinoid tumor, adding CK7, ER, and/or PR to a panel of markers can be helpful. Endometrioid tumors more frequently express CK7, ER, and PR and show a greater extent of immunostaining in contrast to Sertoli cell tumor and carcinoid tumor. Compared with traditional epithelial markers, CK7, ER, and PR are nearly as advantageous as EMA. Inhibin is the most discriminatory sex cord marker, and CD10 is not helpful in the differential diagnosis. Chromogranin and synaptophysin are excellent discriminatory markers for carcinoid tumor, and CD56 is neither sufficiently sensitive nor specific enough for this differential diagnosis to warrant its use in routine practice.

STAT 5a expression in the breast is maintained in secretory carcinoma, in contrast to other histologic types.

The 7 signal transducer and activator of transcription (STAT) molecules are responsible for the transcription of a variety of regulatory and differentiation proteins. STAT 5a is activated through a variety of mechanisms; in the breast, this is predominantly through binding of prolactin to its receptor. Previously, we showed that STAT 5a expression is decreased in atypical and malignant breast ductal epithelial cells. Interestingly, STAT 5a overexpression was observed in cells undergoing secretory change. In this study, secretory carcinomas were examined by immunohistochemistry for the presence of STAT 5a. In contrast to usual in situ or invasive ductal carcinoma, which lacked STAT 5a expression, all secretory carcinomas (11 invasive and 7 in situ, including 4 cases with both) expressed STAT 5a. No expression was seen in apocrine metaplasia or in other specialized breast carcinomas, such as mucinous or clear cell carcinoma. This retention of signal in the secretory carcinomas may be explained by the higher STAT 5a concentration present in cells undergoing secretory changes in general. Alternatively, STAT 5a expression may be related to the t(12;15)(p13;q25) chromosomal translocation, associated with certain pediatric tumors and recently demonstrated in many secretory carcinomas of the breast, which results in the expression of a tyrosine kinase through ETV6 and NTRK3 fusion. ETV6 also has been associated with the STAT 5a signaling pathway in another gene translocation and may be altering STAT 5a expression in secretory carcinomas. Breast cancer causes significant morbidity and mortality, and, regardless of the mechanism for retention of STAT 5a expression in this uncommon variant, the examination of STAT 5a will aid our understanding of normal and abnormal breast tissues.

STAT 5a expression in various lesions of the breast.

The seven signal transducer and activator of transcription (STAT) molecules are effectors of hormonal or cytokine stimulation through receptors. STAT 5a, isolated from prolactin-stimulated mammary cells, contributes to normal proliferation and is essential for mammary gland differentiation. Using a monoclonal antibody, we tested 100 formalin-fixed, paraffin-embedded breast tissues representing everything from simple hyperplasia to invasive carcinoma for the expression of STAT 5a in comparison to normal breast epithelial cells. Immunohistochemical analysis was performed following heat treatment in a pressure cooker. STAT 5a was found in endothelial cells, adipocytes, and leukocytes as well as in the cytoplasm and nucleus of normal epithelial cells, usual ductal hyperplasia, and benign lesions such as fibroadenoma. Myoepithelial cells and stromal fibroblasts failed to demonstrate any STAT 5a in addition to most atypical proliferations including in situ and invasive carcinomas. A few examples of lobular intraepithelial neoplasia and invasive carcinoma demonstrated some reactivity, albeit comparatively reduced. The absence of STAT 5a in the abnormal breast epithelial cells may indicate a defect contributory to the abnormal state.

Tubulolobular carcinoma of the breast: an analysis of 27 cases of a tumor with a hybrid morphology and immunoprofile.

Tubulolobular carcinoma (TLC) is a rare subtype of mammary carcinoma that has eluded precise classification, exhibiting features of both ductal and lobular differentiation. The clinicopathologic features of 27 cases of TLC were analyzed by both hematoxylin and eosin and immunohistochemical stains for E-cadherin and 34betaE12 (high molecular weight cytokeratin). Five cases of both pure tubular and classic lobular carcinoma were included as controls. Patients with TLC ranged in age from 43 to 79 years (median, 60 years). Tumor characteristics were as follows: size, 0.5 cm to 2.5 cm (median, 1.4 cm); bilaterality, 1 of 27 (4%); and multifocality, 5 of 27 (19%). Twenty-two of the 27 cases (81%) contained an in situ component: 8 (36%) lobular (LIN); 4 (18%) ductal (DIN); and 10 (46%) mixed. All 27 cases were intensely positive (3+) for E-cadherin, a feature of ductal differentiation, while 25 of 27 (93%) cases showed variable positivity for 34betaE12 (1 to 3+), a feature far more common in tumors with lobular differentiation. Clinical follow-up was available on 25 of 27 (93%) patients. Three of 24 (13%) patients developed axillary lymph node metastases and 1 of 25 (4%) patients developed a local recurrence over a follow-up period of 2 to 91 months (median, 39 months). In conclusion, TLCs are a distinct subtype of mammary carcinoma with overlapping morphologic features that are mirrored by a hybrid immunohistochemical profile. The uniform 3+ expression of E-cadherin in TLC supports the ductal differentiation of these tumors, despite a dominant lobular growth pattern. The prognosis of these tumors appears to be excellent, especially in those cases that are unilateral and less than 2 cm in size.

Morphologically similar epithelial and stromal cells in primary bilateral breast tumors display different genetic profiles: implications for treatment.

The morphologic features of primary bilateral breast carcinoma have been well elucidated, but it is not known whether tumors at two sides share a common genetic profile and undergo the same clinical course. To address this issue, morphologically comparable epithelial and stromal cells in 18 paired primary bilateral breast tumors were microdissected and subjected to comparisons for the frequency and pattern of loss of heterozygosity (LOH) and microsatellite instability (MI), as well as the profiles of comparative genomic hybridization. Of 18 paired bilateral epithelial samples assessed with 10 DNA markers at five chromosomes, 78 altered loci were found; of these, 23 (29.5%) displayed concurrent and 55 (70.5%) showed independent LOH, MI, or both. Of 18 paired bilateral stromal samples assessed with the same markers, 70 altered loci were seen; of these, 9 (12.9%) displayed concurrent and 61 (87.1%) showed independent LOH, MI, or both. Collectively, all the markers and 30 (83.3%) of 36 paired bilateral epithelial and stromal cells displayed significantly more (P < 0.01) independent than concurrent LOH, MI, or both. In contrast, the epithelial cells of a pulmonary small cell carcinoma metastasized to both breasts displayed concurrent LOH at each of the four altered loci. Of seven selected cases for comparative genomic hybridization, six (86%) displayed chromosomal changes, but none showed an identical pattern and frequency of changes in both breasts. The significantly higher rate of independent genetic alterations in morphologically comparable cells of paired bilateral primary breast tumors supports the notion that the development and clinical course of tumors in two sides differ substantially; consequently, different interventions might be needed for the optimal management of bilateral breast tumors.

The T cell receptor gamma chain alternate reading frame protein (TARP), a prostate-specific protein localized in mitochondria.

We previously showed that mRNA encoding TARP (T cell receptor gamma chain alternate reading frame protein) is exclusively expressed in the prostate in males and is up-regulated by androgen in LNCaP cells, an androgen-sensitive prostate cancer cell line. We have now developed an anti-TARP monoclonal antibody named TP1, and show that TARP protein is up-regulated by androgen in both LNCaP and MDA-PCa-2b cells. We used TP1 to determine the subcellular localization of TARP by Western blotting following subcellular fractionation and immunocytochemistry. Both methods showed that TARP is localized in the mitochondria of LNCaP cells, MDA-PCa-2b cells, and PC-3 cells transfected with a TARP-expressing plasmid. We also transfected a plasmid encoding TARP fused to green fluorescent protein into LNCaP, MDA-Pca-2b, and PC-3 cells and confirmed its specific mitochondrial localization in living cells. Fractionation of mitochondria shows that TARP is located in the outer mitochondrial membrane. Immunohistochemistry using a human prostate cancer sample showed that TP1 reacted in a dot-like cytoplasmic pattern consistent with the presence of TARP in mitochondria. These data demonstrate that TARP is the first prostate-specific protein localizing in mitochondria and indicate that TARP, an androgen-regulated protein, may act on mitochondria to carry out its biological functions.

Assessment of lesions coexisting with various grades of ductal intraepithelial neoplasia of the breast.

Ductal intraepithelial neoplasia (DIN) is descriptive of in situ breast lesions from usual ductal hyperplasia (UDH) to advanced ductal carcinoma in situ (DCIS). A total of 2628 cases of DIN diagnosed at the Armed Forces Institute of Pathology were separated based on their grade. These were assessed for the presence of invasive carcinoma (ductal or lobular) and lobular intraepithelial neoplasia (LIN) grades 1-3. The frequency of invasive cancer (ductal and lobular) appearing with DIN increased with increasing DIN grade from 2% in low-risk DIN (UDH) to 37% in DIN 2-3 (DCIS grades 2-3). The frequency of these invasive carcinomas, which were either lobular or displayed lobular features, however, decreased with increasing grade of DIN with a peak of 28% in DIN 1-flat type, (flat epithelial atypia) to a low of 2% in DIN 3. Likewise, the frequency of LIN appearing with DIN decreased as the grade of DIN increased, with a peak of 26% in DIN 1-flat type to a low of 9% in DIN 3. Lower-grade LIN 1 comprised 14% of the LIN in low-risk DIN cases, but only 4% of the LIN seen in DIN 3 cases. Conversely, higher-grade LIN 3 comprised only 6% of the LIN seen in low-risk DIN cases, while accounting for 15% of the LIN in DIN 3 cases. The frequency of invasive carcinoma in DIN 1 ranged from 4% in quantitatively limited DIN 1 less than or equal to 2 mm (atypical ductal hyperplasia) to 27% among the more abundant DIN 1 greater than 2 mm (DCIS grade 1). The frequency of LIN associated with DIN 1 less than or equal to 2 mm was 13.4%, and the frequency of LIN associated with DIN 1 greater than 2 mm was 16.6% when there was no DIN 1-flat type present. However, the frequency of the LIN seen in combination with DIN 1-flat type was reduced by 50% as the quantity of DIN exceeded 2 mm. Based on this retrospective analysis of DIN, we noted that: (1) invasive carcinoma is most frequently associated with the higher grades of DIN; (2) the grade of LIN parallels the grade of coexisting DIN; (3) a relationship exists between DIN 1-flat type and the occurrence of LIN and (4) this relationship in association with DIN less than or equal to 2 mm is not maintained in DIN greater than 2 mm.

Cytokeratin immunoreactivity in lobular intraepithelial neoplasia.

Eighteen commercially available antibodies reactive against different cytokeratin proteins were tested on classic examples of lobular intraepithelial neoplasia (LIN) and of ductal intraepithelial neoplasia (DIN) of the breast. About 90% of higher-grade DIN (AIDH and DCIS) show no or substantially diminished reaction with clone 34betaE12 (specified as reactive against keratins 1, 5, 10, and 14 as determined by the manufacturer), while the cells of LIN were found to express the antigen reactive with this antibody. To determine which of these four keratins are present in the cells of LIN, antibodies reactive against these individual four keratins were tested. None of the four antibodies to keratins 1, 5, 10, or 14 reacted with the cells of LIN. To investigate this further, 13 additional monoclonal antibodies to various other keratin proteins were tested on the cells of LIN. Those that successfully reacted with the cells of LIN were further tested on the cells of DIN. All of the individual antibodies reactive with the cells of LIN were also reactive with the cells of DIN to a degree, with clone RCK108 (reactive against keratin 19) coming the closest to demonstrating the reactivity seen with 34betaE12. We conclude that the reactivity seen in the cells of LIN with 34betaE12 is due to either (a) a crossreaction with keratin 19 that is slightly less prominent than the reaction of the individual clone RCK108, (b) a crossreaction with a keratin protein that was not tested (3, 11, 12), (c) a crossreaction with a protein closely resembling keratin in formalin-fixed, paraffin-embedded tissue, or (d) the detection of a mutated or truncated form of keratin 1, 5, 10, or 14 that cannot be detected by the individual monoclonal antibody.

Cell clusters overlying focally disrupted mammary myoepithelial cell layers and adjacent cells within the same duct display different immunohistochemical and genetic features: implications for tumor progression and invasion.

INTRODUCTION: Our previous studies detected focal disruptions in myoepithelial cell layers of several ducts with carcinoma in situ. The cell cluster overlying each of the myoepithelial disruptions showed a marked reduction in or a total loss of immunoreactivity for the estrogen receptor (ER). This is in contrast to the adjacent cells within the same duct, which were strongly immunoreactive for the ER. The current study attempts to confirm and expand previous observations on a larger scale. METHODS: Paraffin sections from 220 patients with ER-positive intraductal breast tumors were double immunostained with the same protocol previously used. Cross-sections of ducts lined by > or = 40 epithelial cells were examined for myoepithelial cell layer disruptions and for ER expression. In five selected cases, ER-negative cells overlying the disrupted myoepithelial cell layer and adjacent ER-positive cells within the same duct were separately microdissected and assessed for loss of heterozygosity and microsatellite instability. RESULTS: Of the 220 cases with 5698 duct cross-sections examined, 94 showed disrupted myoepithelial cell layers with 405 focal disruptions. Of the 94 cases, 79 (84%) contained only ER-negative cell clusters, nine (9.6%) contained both ER-negative and ER-positive cell clusters, and six (6.4%) contained only ER-positive cell clusters overlying disrupted myoepithelial cell layers. Of the 405 disruptions, 350 (86.4%) were overlain by ER-negative cell clusters and 55 (13.6%) were overlain by ER-positive cell clusters (P < 0.01). Microdissected ER-negative and ER-positive cells within the same duct from all five selected cases displayed a different frequency or pattern of loss of heterozygosity and/or microsatellite instability at 10 of the 15 DNA markers. CONCLUSIONS: Cells overlying focally disrupted myoepithelial layers and their adjacent counterparts within the same duct displayed different immunohistochemical and molecular features. These features potentially represent an early sign of the formation of a biologically more aggressive cell clone and the myoepithelial cell layer breakdown possibly associated with tumor progression or invasion.