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.

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.

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.

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.

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.

Eleven pure apocrine carcinomas (8 breast, 2 vulvar, 1 axillary) were evaluated for the presence of androgen and estrogen receptor mRNA. These immunohistochemically androgen receptor positive and estrogen receptor negative cases were microdissected and analyzed by the reverse transcription polymerase chain reaction for the presence of receptor message. Nine of these 11 estrogen receptor (ER) negative lesions were found to contain the ER mRNA through the first intron splice region. In 4 of the 11 cases, the androgen receptor (AR) mRNA could not be shown even though the protein was detected immunohistochemically. In the other seven, AR mRNA was identified. This indicates that the mechanism for production of the estrogen receptor is intact and functional in most cases through the first transcriptional splice region. Therefore, the immunohistochemical absence of the estrogen receptor in apocrine cells cannot be explained by an abnormal message at these common sites and should be sought beyond these points.

The terminal duct-lobular unit is the origin of 2 distinct variants of intraepithelial neoplasia traditionally separated into ductal and lobular types based on a combination of cytologic and architectural features. In general, distinction of the fully developed or classic lobular intraepithelial neoplasia (LIN) from various grades of ductal intraepithelial neoplasia (DIN) is not a problem. An increasing number of lesions that appear to have intermediate, overlapping ductal and lobular features are being sent to us for consultation because of the distinctly different clinical implication of the 2 diagnoses. We have separated and designated these as MIN (mammary intraepithelial neoplasia, not otherwise specified), whereas others have categorized them into either a definitive ductal or lobular subtype. The recent findings that LIN lacks immunoreaction for E-cadherin coupled with significantly diminished to absent expression of the high molecular weight (HMW) cytokeratins in more than 90% of grade 1b or higher DIN prompted us to evaluate intraepithelial neoplasias for a possibly more precise immunohistochemical categorization. One hundred and ten examples of intraepithelial neoplasias, consisting of 40 classic LIN, 20 unequivocal DIN 1c to DIN 3 (ductal carcinoma in situ), and 50 MIN, were acquired from the files of the Armed Forces Institute of Pathology. These specimens were tested with an antibody to E-cadherin and with antibody 34ssE12 reactive against HMW cytokeratins 1, 5, 10 and 14. All samples of LIN showed complete absence of reactivity with anti-E-cadherin, whereas all cases of DIN displayed a positive immunoreaction. In contrast, the DIN lesions displayed little or no reactivity with 34ssE12, whereas the lobular lesions showed cytoplasmic reactivity, often in a distinct perinuclear pattern. Twenty-three of the morphologically indeterminate cases could be classified as either ductal or lobular based on the immunoprofile, and 27 demonstrated an immunoprofile that differed from either typical DIN or classic LIN. Among the 27 MIN, 11 were negative for both markers (negative hybrids), whereas 16 were positive for both markers (positive hybrids). These 2 antibodies in combination are extremely useful in distinguishing lobular and ductal lesions and clarifying the nature of some of the morphologically intermediate cases. Also, they have confirmed the presence of a group of intraepithelial lesions (MIN) with not only overlapping morphologic features, but also immunoprofiles distinctly different from either DIN or LIN. These MIN lesions may reflect either a transient stage in the development of DIN and LIN (the immediate post-stem cell stage) or a plastic group in transition from one type to the other. This group needs further evaluation for better understanding of its significance, pattern of progression, and behavior.

BACKGROUND Central neurocytomas are composed of mature neuronal elements, frequently arranged in rosettes similar to those present in pineocytomas. This suggests the possibility of similar patterns of differentiation, including photoreceptor differentiation. The authors analyzed the immunoreactivity of central neurocytomas for retinal S-antigen, neuronal, glial, and neuroendocrine markers. METHODS Thirty-three central neurocytomas were analyzed with reference to their clinicopathologic characteristics, immunoreactivity, and the possibility that anaplastic histologic features correlated with aggressive clinical behavior. RESULTS There were 18 male and 15 female patients. The median age at diagnosis was 30 years (range, 3–69 years). All of the tumors with specified location were related to the ventricles. Thirty-two tumors were diagnosed at surgery and 1 at autopsy. Histologic features included mineralization (20 of 33), foci of necrosis (4 of 33), chronic inflammation (4 of 33), ganglion cell differentiation (1 of 33), and lipomatous differentiation (1 of 33). None of the lesions had significant nuclear pleomorphism, mitotic activity, or vascular endothelial proliferation. Immunohistochemistry included expression of synaptophysin (33 of 33), neuron specific enolase (31 of 33), S-100 protein (25 of 33), retinal S-antigen (14 of 24), somatostatin (8 of 27), glial fibrillary acidic protein (4 of 33), neurofilament protein (3 of 22), and leucine enkephalin (1 of 27). At follow-up, 15 of 23 patients were alive an average of 8.1 years (range, 0.91–35.9 years) after surgery. CONCLUSIONS Central neurocytomas behave as slowly growing neoplasms that remain confined within one or several supratentorial ventricles and are associated with long survival after surgical excision. Malignant forms with aggressive clinical behavior were not found. The neoplastic cells can express photoreceptor differentiation possibly relating central neurocytomas to pineocytomas. Adipocyte differentiation may be present, and the possibility of a relation between the central neurocytoma and cerebellar liponeurocytoma should be entertained. Cancer 2001;91:136–43. Published 2001 American Cancer Society.

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 3p 21.2–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.

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.

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.