Cells with minimal expression of the JAK/STAT pathway related proteins STAT5a and the prolactin receptor: evidence of an alternate prolactin receptor isoform in breast disease.

Usually, wherever breast STAT5a is present, PRLR is reduced; without STAT5a PRLR becomes abundant. Five breast lesions essentially lacking both were tested immunohistochemically for PRLR isoforms. The intermediate isoform was essentially only detected in these lesions. In some breast lesions PRLR isoforms may be involved in JAK/STAT pathway disturbances.

Antibodies targeting p63 react specifically in the cytoplasm of breast epithelial cells exhibiting secretory differentiation.

AIMS: The nuclear detection of p63 in myoepithelial cells of the breast has been useful in identifying possibly invasive carcinomas. While examining myoepithelial cells for p63 a very strong cytoplasmic reaction product was noted in secretory cells. The aim was to determine whether this reaction is specific for p63 and indicative of all breast secretory cells. METHODS: Thirty breast specimens were tested immunohistochemically for p63 protein. These included seven with benign secretory changes, 10 secretory carcinomas (nine invasive), one microglandular adenosis, three lobular neoplasias, four invasive ductal carcinomas, three clear cell carcinomas, one squamous cell carcinoma and one mucinous carcinoma. RESULTS: Only cells exhibiting secretory changes or secretory carcinoma were cytoplasmically reactive for p63. The positive reaction was also present as an intraluminal secretory product. This reaction was not seen in cells undergoing apocrine differentiation or in other cells containing secretory vacuoles. CONCLUSIONS: Cells with secretory changes contain p63 protein or an antigenic equivalent. The detection of p63 protein continues to have considerable value for the identification of myoepithelial cells and thus the determination of invasion, but will also have value in the determination of secretory carcinomas of the breast and in understanding their development.

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.

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 degrees C. Results show that a decrease of the electrophoresis temperature from 51 degrees C (recommended by the User's Manual) to 45 degrees 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 degrees C) electrophoresis temperature permits multiple uses of a given gel with consistent results, consequently reducing the electrophoresis time and reagent costs.

Allelic losses at 3p and 11p are detected in both epithelial and stromal components of cervical small-cell neuroendocrine carcinoma.

Microdissected epithelial and stromal cells from 15 cervical small-cell carcinoma patients and 9 healthy control subjects were assessed for loss of heterozygosity with polymorphic DNA markers at chromosomes 3p and 11p. Among malignant lesions assessed with 7 markers at 3p, 21 allelic losses were detected from 193 informative samples. Of losses, 20 were in epithelial and 1 was in normal-appearing stromal cells. Among losses in epithelial cells, 16 were from 44 samples informative for 3 markers within 3p21.2-p14.2 (0.36 loss/sample), whereas only 4 were from 54 samples informative for 4 markers outside the region (0.09 loss/sample), suggesting a "hot spot" of genetic alterations within 3p21.2-p14.2. Among malignant lesions assessed with 2 markers within 11p14-p12, 15 losses were seen in 52 informative samples. Of losses, 10 were in epithelial and 5 were in normal-appearing stromal cells. Of 10 epithelial samples showing losses within 11p14-p12, 8 also displayed losses within 3p21.2-p14.2, suggesting a concurrent involvement of these loci in tumor development or progression. The five losses in stromal cells were in four cases that showed no loss in epithelial cells with same markers, suggesting that stromal cells might play initiative roles in tumor development.

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.

Genetic abnormalities in mammary ductal intraepithelial neoplasia-flat type ("clinging ductal carcinoma in situ"): a simulator of normal mammary epithelium.

BACKGROUND: Mammary ductal intraepithelial neoplasia (DIN)-flat type ("clinging ductal carcinoma in situ [DCIS]") generally is a subtle epithelial alteration characterized by one or a few layer(s) of atypical cells replacing the native epithelium. The "low power" appearance of DIN-flat type can be misinterpreted easily as "normal" because of the frequent absence of multilayered proliferation and often subtle cytologic atypia. Because it presents as an often unrecognized lesion or in association with tubular carcinoma, to the authors' knowledge the clinical and biologic significance of this lesion has not been well established. METHODS: Using polymerase chain reaction, the authors examined DNA extracts from microdissected areas of 22 cases with extensive "clinging DCIS," including 13 cases associated with infiltrating ductal carcinoma as well as 5 cases associated with more conventional types of DCIS. Eight polymorphic DNA markers with a high rate of loss of heterozygosity (LOH) in classic types of DCIS were selected to identify possible genetic alterations on chromosomes 2p, 3p, 11q, 16q, and 17q. Two cases also were used for the assessment of clonality by means of X chromosome inactivation (methylation pattern of the human androgen receptor [HUMARA] gene). RESULTS: LOH was detected in 17 of 22 lesions (77%), and monoclonality was established in the 2 cases analyzed. The most common genetic alterations were at chromosomes 11q21-23.2, 16q23.1-24.2, and 3p14.2 with LOH in 50%, 45%, and 41%, respectively, of informative cases. The DIN-flat type showed the same genetic alterations (LOH) identified in adjacent in situ and infiltrating ductal carcinoma. In contrast to the DIN-flat type, the perfectly normal mammary epithelium was associated very infrequently (1 of 16 cases; 6%) with LOH. CONCLUSIONS: The DIN-flat type represents one of the earliest, morphologically recognizable, neoplastic alterations of the breast. Recognition of the DIN-flat type is important not only for the early detection of intraductal neoplasia but also to prevent misinterpretation and utilization of this lesion as a normal control in studies. This distinctive lesion could be crucial as an explanation for at least part of the > 20% reported incidence rate of breast carcinoma recurrence observed despite ostensibly "negative" margins of breast biopsies.

Concurrent and independent genetic alterations in the stromal and epithelial cells of mammary carcinoma: implications for tumorigenesis.

The high frequency of loss of heterozygosity (LOH) in epithelial cells of mammary ductal carcinoma in situ (DCIS) and IDC is a well known phenomenon, whereas the genetic abnormalities in the mammary stroma and its influence on the epithelial component have not been sufficiently studied. Using the PCR, we examined DNA extracts from microdissected stromal and epithelial tissues of 11 breast samples containing DCIS, including five cases associated with IDC. In each case, the mesenchymal tissue consisting of normal-appearing stroma at a distance from DCIS and IDC or stroma close to either DCIS or IDC was manually microdissected. Epithelial cells from morphologically clear-cut normal ducts and lobules, DCIS, and IDC were also microdissected. Twelve polymorphic DNA markers were tested to identify possible genetic alterations in the mesenchymal and epithelial cells on chromosomes 2p, 3p, 11q, 16q, and 17q. Samples from bilateral reduction mammoplasty from 10 women without any clinical, radiological, or pathological abnormalities were also selected as a control (reduction mammoplasty group). Whereas most cases (8/11, 73%) displayed at least one identical LOH in both epithelial and mesenchymal components, LOH at several loci was noted exclusively in stromal cells. The most frequent genetic alterations in the mesenchymal cells were at chromosomes 17q24, 16q23.1-24.2, 3p14.2, and 11q21-23.2, in 87.5, 62, 60, and 45% of informative cases, respectively. The LOH frequency in the stroma close to cancer ranged from 10 to 66.5% for DCIS and from 20 to 75% of informative cases for IDC. Furthermore, 10 of the 12 polymorphic markers revealed LOH in the stroma at a distance, ranging from 11 to 57% of informative cases. None of the control cases (women without any breast disease) revealed LOH either in the epithelial or in the stromal components. Our findings strongly support the concept of stromal-epithelial interaction in the development and progression of mammary neoplasia. Furthermore, this study suggests that genetic alterations in the stromal cells may precede genotypic changes in the epithelial cells. At least in some cases, the mammary stroma in DCIS or IDC apparently represents a neoplastic interactive component rather than a reactive response to the carcinoma. The frequent allelic loss (LOH) in the mammary stroma, identified in our study, may explain some of the fibroblastic abnormalities previously observed in patients with breast carcinoma or a variety of cancer-associated hereditary diseases. We conclude that the mammary stroma may play a key role in inducing neoplastic transformation of epithelial cells, recapitulating its role in normal mammary duct development.

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

BACKGROUND: 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. METHODS: Six SLCT and one pure Leydig cell tumor were studied. Mitotic counts and immunohistochemistry for Ki-67 were performed in all cases. Leydig cells, neoplastic tissues, and normal nonneoplastic tissues were microdissected. DNA extracts of these samples were assessed for loss of heterozygosity (LOH) by polymerase chain reaction amplification with ten polymorphic DNA markers that have shown high rates of LOH in a variety of human tumors. Three SLCT and the Leydig cell tumor were assessed for clonality by examining the DNA methylation pattern at a polymorphic site on the androgen receptor gene. RESULTS: Leydig cells in SLCT had a low mitotic count (0-1/50 high-power fields [HPF]) compared with the neoplastic stroma (median, 40/50 HPF). Ki-67 was positive in < 2% of Leydig cells in all SLCT, compared with a median of 7% in the neoplastic stroma. Clonality analysis confirmed the monoclonality of the neoplastic cells in the Leydig cell tumor. However, the Leydig cells from three SLCT were polyclonal, whereas the monoclonal nature of the neoplastic Sertoli tubules was confirmed in one of these cases and that of mucinous heterologous elements in another case. The Leydig cell tumor showed LOH at four of the eight loci evaluated. Leydig cells from five SLCT were evaluated: one showed LOH at one locus, two showed LOH at two loci, and the remaining two showed no LOH. CONCLUSIONS: The demonstration that Leydig cells from SLCT are polyclonal strongly suggests that they are nonneoplastic in nature. This is supported by a low proliferation fraction and a lower fraction of LOH compared with the truly neoplastic Leydig cells.

A clinicopathologic and immunohistochemical study of 22 intraductal papillary mucinous neoplasms of the pancreas, with a review of the literature.

Intraductal papillary-mucinous neoplasms (IPMNs) of the pancreas are rare lesions. We undertook this study to analyze these tumors by focusing on the diagnostic criteria and correlating the histologic features with clinical prognosis. Twenty-two cases of IPMN were retrieved from the Endocrine Tumor Registry of the Armed Forces Institute of Pathology. Blocks or unstained slides were available for histochemical and immunohistochemical studies (including proliferative markers and cell cycle regulators) and K-ras oncogene mutations in 15 cases. Patient follow-up was obtained in all of the cases. IPMN occurs in both genders with a slight male predominance, with a mean age at presentation of 64.4 years (range, 48-85 yr). The patients presented with abdominal pain. The neoplasms were radiologically and grossly cystic, usually (18 cases of 22) located in the head of the pancreas. Histologically, the tumors consisted of intraductal papillary proliferations protruding into and expanding the pancreatic ducts. Invasion into the surrounding pancreatic parenchyma was detected in 15 cases. Chronic pancreatitis was present in all of the cases. p27 immunoreactivity always exceeded the immunoreactivity of cyclin E. K-ras oncogene mutations were detected in two cases. Patients were treated with a complete surgical resection (n = 7) or a Whipple procedure (n = 13). Only 2 of 22 patients died of disease (3 died immediately postoperatively and 3 died of unrelated causes), whereas the remaining 14 patients were alive at last follow-up, without evidence of disease, an average of 58.2 months after initial presentation. IPMNs are rare, distinctive neoplasms, with complex intraductal papillae, that can be easily separated from in situ ductal adenocarcinoma and mucinous cystic neoplasms. The high ratio of p27 protein to cyclin E supports the excellent prognosis of these neoplasms, despite the presence of invasion and K-ras oncogene mutation.

Immunohistochemistry to identify Leishmania parasites in fixed tissues.

The definitive diagnosis of leishmaniasis currently depends on the identification of characteristic amastigote morphology in tissue, or isolation of promastigotes by culture. Histopathological identification can be difficult, and is variably sensitive; culture is considered "the gold standard", but is not uniformly diagnostic or available. In this study, we compared light microscopic immunohistochemistry (IHC) using a monoclonal anti-Leishmania antibody (G2D10) to standard hematoxylin and eosin (H&E) stain in the diagnosis of Leishmania on skin. Sixty-one archived specimens from patients suspected of being infected with Leishmania were used; 41 of these had leishmaniasis confirmed by culture. Although not statistically significant, both sensitivity and specificity were higher for IHC compared to H&E: 51% (95% CI: 35-67%) compared to 42% (CI: 26-58%; 2p=0.29) for sensitivity, and 100% (CI: 83-100%) compared to 85% (CI: 62-97%, 2p=0.25) for specificity, respectively. Furthermore, because organisms could be diagnosed by IHC at low power (x20-40), this assay was more rapid than H&E, in which parasite morphology could best be identified at oil immersion power. The G2D10 antibody has broad Leishmania species recognition, and offers promise as a simple, rapid diagnostic screen for leishmaniasis. Further study is underway to better characterize this antibody.

Osteocalcin and osteonectin immunoreactivity in extraskeletal osteosarcoma: a study of 28 cases.

Extraskeletal osteosarcoma (EOSA), a rare malignant soft tissue tumor, is by definition unattached to the skeleton and composed of malignant cells of osteoblastic phenotype which produce osseous matrix (ie, neoplastic bone). Because of its location, it can mimic other soft tissue tumors, and its matrix can be mistaken for hyalinized collagen. Antiosteocalcin (OC) and antiosteonectin (ON), antibodies against two abundant human bone proteins, are explored in the diagnosis of EOSA. Twenty-eight cases coded as EOSA (n=24) or probable EOSA (n = 4) were identified from the Soft Tissue Registry of the Armed Forces Institute of Pathology (Washington DC). All cases had paraffin blocks available for immunohistochemistry. OC and ON (Biodesign International, Kennebunk, ME, clones OC1 and OST1) immunostaining for tumor cells and matrix was graded on a four-tiered grading system: 1 = focal (< 50%) weak staining; 2 = focal strong staining; 3 = diffuse (> or = 50%) weak staining; and 4 = diffuse strong staining. Patient ages ranged from 9 to 80 years, with a mean age of 57 years. There were 9 female patients and 19 male patients. The tumor sizes ranged from 1.5 to 15 centimeters, with a mean size of 5.8 centimeters. Locations included the lower extremity (n=14), trunk (n=9), upper extremity (n=4), and head and neck (n=1). Subtypes included 12 osteoblastic, 4 fibroblastic, 2 chondroblastic, 2 well differentiated, 1 telangiectatic, 1 small cell, and 6 giant cell rich EOSAs; the latter resembled giant cell rich malignant fibrous histiocytomas with neoplastic bone formation. All tumors had both neoplastic cells and bony tumor matrix to evaluate. OC was 82% sensitive for EOSA neoplastic cells (1 to 4+), with immunostaining of neoplastic cells away from bone in 91% of cases, and 75% for bony tumor matrix (2 to 4+). ON was 93% sensitive for EOSA neoplastic cells (2 to 4+), yet only 39% for bony tumor matrix (1 to 4+). In 100% giant cell rich EOSA, neoplastic cells were positive for OC and ON (2 to 4+). OC showed 100% specificity for osteoblasts as it was nonreactive in all nonbone cells. ON was not specific for osteoblasts but consistently immunostained other cell types in our EOSA tumors: fibroblasts (100%), pericytes (96%), endothelial cells (92%), chondrocytes (5/5), basal layer of skin epithelium (1/4), nerves (2/2), and osteoclastic giant cells (64%). ON also stained several other cell types in normal and neoplastic tissues in our battery of preliminary stainings; OC was negative in all nonosteoblastic tissues and tumors. Both OC and ON were specific for osteoid matrix as they were nonreactive in both collagen and cartilage matrix. OC is a sensitive and specific marker for bone cells and would be helpful in identifying EOSA, even in the absence of neoplastic bone on small biopsies. ON and OC (more sensitive) will both distinguish malignant bone from collagen and cartilage matrix, essential to the diagnosis of EOSA.

Use of monoclonal antibody against human inhibin as a marker for sex cord-stromal tumors of the ovary.

Inhibin is a glycoprotein hormone produced by normal ovarian granulosa cells and testicular sertoli cells. In the ovary, it inhibits the secretion of follicle-stimulating hormone. Patients with granulosa cell tumors (GCT) have elevated serum levels of inhibin and this finding has been used to detect recurrent tumor. This study attempts to determine whether inhibin antibody (IAB) can preferentially mark GCT and Sertoli-cell or Sertoli-Leydig cell tumors (SCT) in paraffin-embedded tissues and facilitate distinction of GCT from small cell carcinoma of hypercalcemic type (SCC), SCT from Sertoliform endometrioid carcinoma (SEC), and primitive gonadal-stromal tumors from a variety of poorly differentiated neoplasms. Applying microwave-enhanced immunohistochemistry, a total of 126 paraffin-embedded and microwave-enhanced archival ovarian tumors and tissues were studied by using monoclonal IAB. The tumors included 32 adult GCT, 7 juvenile GCT, 4 metastatic GCT, 8 SCT, 7 SCC, 6 primitive gonadal stromal tumors (PGST), 5 fibrothecomas, 6 lipid cell tumors (LCT), 5 extrauterine endometrial stromal sarcomas (ESS), 5 hemangiopericytomas (HPC), 1 metastatic malignant melanoma, 1 metastatic malignant lymphoma, and 27 epithelial tumors including 8 SEC, 5 mucinous tumors, and 4 Brenner tumors. Seven pregnancy luteomas (nodular theca lutein hyperplasia of pregnancy), 3 corpora lutea and 2 ovarian follicles were also studied. The intensity of immunostaining was scored from one to three and the percentage of the immunoreactive tumor cells was determined and expressed in 10% increments. Among 32 adult GCT, 31 (97%) tumors reacted positively with IAB. The percent of positive cells ranged from 30% to 100% (average 80%). Similarly, all four metastatic GCT, 7 juvenile GCT and 4 of 5 fibrothecomas were immunoreactive with monoclonal IAB. Seven of 8 (88%) SCT, 5 of 6 (83%) PGST, all 6 LCT, 7 pregnancy luteomas, 3 corpora lutea and the 2 ovarian follicles were also positive with IAB. The most intense positivity was observed in luteinized stromal cells regardless of tumor type. No immunoreactivity was observed in any of the 7 SCC, 5 ESS, 5 HPC, 1 metastatic malignant melanoma, 1 metastatic malignant lymphoma and the epithelial component of all 27 epithelial tumors including 8 SEC. Among the mucinous tumors of the ovary, however, 3 tumors with luteinized stromal cells showed immunoreactivity in these cells, but no positivity was seen in the mucinous epithelium. We conclude that IAB is an excellent marker for sex cord differentiation in ovarian tumors. It can be used effectively in the diagnosis of GCT and its distinction from epithelial neoplasms particularly SCC. The IAB may also be helpful in differentiating LCT from epithelial malignancies. However, it cannot be used to distinguish GCT from SCT.