hMSH5: a human MutS homologue that forms a novel heterodimer with hMSH4 and is expressed during spermatogenesis.

MutS homologues have been identified in nearly all organisms examined to date. They play essential roles in maintaining mitotic genetic fidelity and meiotic segregation fidelity. MutS homologues appear to function as a molecular switch that signals genomic manipulation events. Here we describe the identification of the human homologue of the Saccharomyces cerevisiae MSH5, which is known to participate in meiotic segregation fidelity and crossing-over. The human MSH5 (hMSH5) was localized to chromosome 6p22-21 and appears to play a role in meiosis because expression is induced during spermatogenesis between the late primary spermatocytes and the elongated spermatid phase. hMSH5 interacts specifically with hMSH4, confirming the generality of functional heterodimeric interactions in the eukaryotic MutS homologue, which also includes hMSH2-hMSH3 and hMSH2-hMSH6.

Reduced Fhit expression in sporadic and BRCA2-linked breast carcinomas.

Evidence for alteration of the FHIT gene in a significant fraction of breast carcinomas has been reported, in apparent concordance with loss of heterozygosity (LOH) at chromosome region 3p14.2 in breast cancer and benign proliferative breast disease. A significantly higher frequency of LOH at the FHIT locus was reported for BRCA2-/- tumors, possibly due to misrepaired double-strand breaks at this common fragile region. To determine whether such genomic alterations lead to Fhit inactivation, we have assessed the level of Fhit expression by immunohistochemical detection in sporadic tumors and cancers occurring in BRCA2 999del5 carriers. To determine whether Fhit inactivation may have prognostic significance, we have also assessed expression of breast cancer markers and clinical features in sporadic tumors relative to Fhit expression. Of 40 consecutive sporadic breast carcinomas studied for tumor markers, 50% showed reduced Fhit expression. In these sporadic cancers, loss of Fhit expression was not correlated significantly with the presence or absence of other tumor markers. In a study of 58 sporadic and 34 BRCA2 999del5 Icelandic invasive cancers, there was a significant association of LOH at 3p14.2 with reduced expression of Fhit (P = 0.001); also the lower expression of Fhit and higher LOH at 3p14.2 in BRCA2 999del5 tumors relative to sporadic cancers was significant (P = 0.002). Thus, genetic alteration at the fragile site within the FHIT gene leads to loss of Fhit protein in a significant fraction of sporadic breast cancers and a much larger fraction of familial breast cancers with an inherited BRCA2 mutation, consistent with the idea that loss of BRCA2 function affects stability of the FHIT/FRA3B locus.

Steroid induction of therapy-resistant cytokeratin-5-positive cells in estrogen receptor-positive breast cancer through a BCL6-dependent mechanism.

Therapy resistance remains a major problem in estrogen receptor-α (ERα)-positive breast cancer. A subgroup of ERα-positive breast cancer is characterized by mosaic presence of a minor population of ERα-negative cancer cells expressing the basal cytokeratin-5 (CK5). These CK5-positive cells are therapy resistant and have increased tumor-initiating potential. Although a series of reports document induction of the CK5-positive cells by progestins, it is unknown if other 3-ketosteroids share this ability. We now report that glucocorticoids and mineralocorticoids effectively expand the CK5-positive cell population. CK5-positive cells induced by 3-ketosteroids lacked ERα and progesterone receptors, expressed stem cell marker, CD44, and displayed increased clonogenicity in soft agar and broad drug-resistance in vitro and in vivo. Upregulation of CK5-positive cells by 3-ketosteroids required induction of the transcriptional repressor BCL6 based on suppression of BCL6 by two independent BCL6 small hairpin RNAs or by prolactin. Prolactin also suppressed 3-ketosteroid induction of CK5+ cells in T47D xenografts in vivo. Survival analysis with recursive partitioning in node-negative ERα-positive breast cancer using quantitative CK5 and BCL6 mRNA or protein expression data identified patients at high or low risk for tumor recurrence in two independent patient cohorts. The data provide a mechanism by which common pathophysiological or pharmacologic elevations in glucocorticoids or other 3-ketosteroids may adversely affect patients with mixed ERα+/CK5+ breast cancer. The observations further suggest a cooperative diagnostic utility of CK5 and BCL6 expression levels and justify exploring efficacy of inhibitors of BCL6 and 3-ketosteroid receptors for a subset of ERα-positive breast cancers.

Intratumoral recombinant GM-CSF-encoding virus as gene therapy in patients with cutaneous melanoma.

Seven immunocompetent, revaccinated patients with surgically incurable cutaneous melanoma underwent treatment of dermal and/or subcutaneous metastases with twice-weekly intratumoral injections of escalating doses (10(4)-2 x 10(7) plaque-forming units (PFU)/lesion; 10(4)-8 x 10(7) PFU/session) of a vaccinia/GM-CSF recombinant virus for 6 weeks. Patients with stable or responding disease were maintained on treatment until tumor resolution or progression. Systemic toxicity was infrequent, dose-related, and limited to mild flu-like symptoms that resolved within 24 hours. Local inflammation, at times with pustule formation, was consistently seen with doses of > or =10(7) PFU/lesion. Chronically treated lesions showed a dense infiltration, with CD4+ and CD8+ lymphocytes, histiocytes, and eosinophils. All seven patients developed an antivaccinia humoral immune response 14-21 days following revaccination. Despite the presence of these antivaccinia antibodies, the reporter gene was expressed, as judged by the development of anti-beta-galactosidase antibodies in all patients. Passenger cytokine gene function was evidenced by the presence of virally encoded GM-CSF mRNA at injection sites both early (weeks 1 and 5) and late (week 31) in the course of treatment. Eosinophilia at treatment sites indicated that physiologically significant levels of functional cytokine were generated. However, there were no changes in the total number of peripheral white blood cells or in the numbers or percentages of polymorphonuclear leukocytes, monocytes, or eosinophils. GM-CSF was not detected in the sera. The two patients with the largest tumor burdens failed to respond even at treatment sites. Three patients had mixed responses, with regression of treated and untreated dermal metastases and progression of disease elsewhere. One patient had a partial response, with regression of injected and uninjected regional dermal metastases. Residual melanoma was excised, rendering the patient disease free. One patient with only dermal metastases confined to the scalp achieved a complete remission. Sequential administration of escalating doses of a GM-CSF recombinant vaccinia virus is safe, effective at maintaining passenger gene function, and can induce tumor regression.

Gastrointestinal stromal tumors/smooth muscle tumors (GISTs) primary in the omentum and mesentery: clinicopathologic and immunohistochemical study of 26 cases.

Gastrointestinal stromal tumor or smooth muscle tumor (GIST) is the designation for a major subset of gastrointestinal mesenchymal tumors that histologically, immunohistochemically, and genetically differ from typical leiomyomas, leiomyosarcomas, and schwannomas. Because GISTs, like the interstitial cells of Cajal, the gastrointestinal pacemaker cells, express CD117 (c-kit protein), the origin of GISTs from the interstitial cells of Cajal has been recently proposed. Comparison of GISTs primary in the omentum and mesentery to GISTs primary in the tubular gastrointestinal tract is of particular diagnostic and histogenetic interest in view of the possible similarity of these tumors with the GIST group. In this study, we analyzed 14 omental and 12 mesenteric primary mesenchymal tumors representing smooth muscle tumors or GISTs. These tumors were phenotypically compared with gastric and small intestinal GISTs, leiomyomas of the esophagus, and leiomyosarcomas of the retroperitoneum. Most (13 of 14) omental and mesenteric (10 of 12) tumors showed histologic features similar to GISTs with elongated spindle cells or epithelioid cells with high cellularity; most of these tumors showed low mitotic activity. Omental and mesenteric GISTs were typically positive for CD117 and less consistently for CD34. They often showed alpha-smooth muscle actin reactivity but were virtually negative for desmin and S-100 protein. One omental and two mesenteric tumors showed features of leiomyosarcoma with ovoid, less elongated nuclei, cytoplasmic eosinophilia; all these tumors had significant mitotic activity. These tumors were positive for alpha-smooth muscle actin and two of them for desmin, but all were negative for CD34 and CD117, similar to retroperitoneal leiomyosarcomas. Tumor-related mortality occurred in the group of mesenteric GISTs, but not in the group of omental GISTs. In contrast, all three patients with a true leiomyosarcoma of the omentum or mesentery had documented liver metastases or died of tumor. In summary, we show that tumors phenotypically identical with GISTs occur as primary tumors in the omentum and mesentery. The occurrence of CD117-positive tumors outside the gastrointestinal tract militates against an origin of these tumors exclusively from the interstitial cells of Cajal.

Immunohistochemical localization of p21(WAF1/CIP1) in normal, hyperplastic, and neoplastic uterine tissues.

p21WAF1/CIP1 is a nuclear protein that binds to cyclin-dependent kinase complexes (CDKs) and inhibits the activity of multiple kinases. These CDKs are involved in the regulation of cell cycle progression at several checkpoints. In this study, the authors have analyzed by immunohistochemistry the expression of p21WAF1/CIP1 in normal uterine tissues, 12 endometrial hyperplasias, 17 endocervical adenocarcinomas, and 31 endometrial adenocarcinomas. In addition, a group of 10 leiomyomas and 10 uterine leiomyosarcomas were also stained. To evaluate cell proliferation, the monoclonal antibody Ki-67 was used in all of the available cases. Terminally differentiated epithelial endocervical and endometrial cells showed variable expression of p21WAF1/CIP1, whereas the endometrial hyperplasias, and endocervical and endometrial adenocarcinomas showed decreased expression or were negative. All of the cases of cervical squamous dysplasia were positive. Normal smooth muscle cells and 50% of leiomyomas were negative, whereas all leiomyosarcomas showed expression of p21WAF1/CIP1. These results indicate that p21WAF1/CIP1 contributes to differentiation in normal endometrial and endocervical glands. The decreased expression of p21WAF1/CIP1 in endometrial hyperplasias and carcinomas may be important in the process of neoplastic transformation. The role of certain CDK inhibitors, such as p21WAF1/CIP1, is different in epithelial and mesenchymal tumorigenesis in the uterus.

Colorectal carcinomas with high microsatellite instability: defining a distinct immunologic and molecular entity with respect to prognostic markers.

Molecular analysis of hereditary nonpolyposis colorectal carcinomas (HNPCC) has identified DNA mismatch repair deficiencies with resulting microsatellite instability (MSI) as a pathway of carcinogenesis that appears to be relevant for prognosis, treatment, and possibly prevention. In this study, expression of cell cycle proteins and other known prognostic markers is correlated with the microsatellite status of colorectal cancers (CRC). One hundred consecutive cases from the CRC Registry at Thomas Jefferson University were analyzed for MSI. Immunohistochemistry was performed for the mismatch repair proteins hMLH1 and hMSH2, tumor suppressor p53, apoptosis inhibitor bcl-2, cell cycle proteins p21(WAF1/CIP1), and p27 and the proliferation markers Ki-67 and topoisomerase II. High MSI (MSI-H) is significantly correlated with loss of either hMLH1 or hMSH2, presence of bcl-2, and absence of p53. p21(WAF1/CIP1) is positive in all tumors with MSI-H. Previous findings of a lower proliferation rate were confirmed with a topoisomerase II stain. Microsatellite stable (MSS) tumors generally express both MSH2 and MLH1. Other highly significant differences are positive p53 in 56% of MSS cases and negative bcl-2 in 98% of MSS cases. p27 expression is found in approximately 50% of all CRCs irrespective of the microsatellite status. MSI-H tumors follow the mutator pathway, with loss of expression of one mismatch repair protein, wild-type p53, lower proliferation, and positivity for p21(WAF1/CIP1). MSS tumors follow the suppressor pathway, characterized by p53 overexpression, higher proliferation, and absence of bcl-2 expression; p21(WAF1/CIP1) expression can be variable. These data provide a molecular basis for the clinical observation that patients with HNPCC appear to have a more favorable prognosis. HUM PATHOL 31:1506-1514.

Overexpression of p21WAF1/CIP1 and MDM2 characterizes serous borderline ovarian tumors.

Ovarian epithelial tumors are classically divided into benign, malignant, and borderline or of low malignant potential. It is controversial whether this last group of tumors should be considered benign or malignant. Expression of cell cycle markers has recently been linked to tumor behavior and response to treatment. It has been shown that one of the pathways through which the p53 gene controls the cell cycle is by transactivating p21WAF1/CIP1, a cyclin-dependent kinase (cdk) inhibitor. By inhibiting cdks, p21WAF1/CIP1 blocks the G-1 to S-phase transition in the cell cycle. p53 can be regulated by MDM2 (murine double minute-2) through direct inactivation or promotion of its cytoplasmic degradation. In an attempt to investigate the cell cycle checkpoint mechanisms of these tumors, we studied the expression of p53, Ki-67, MDM2, and p21WAF1/CIP1 by immunohistochemistry. We analyzed the expression of these proteins in 19 cystadenomas (8 serous and 11 mucinous), 40 borderline tumors (31 serous and 9 mucinous), and 18 serous carcinomas of the ovary. p21WAF1/CIP1 was expressed in 7 of 19 (37%) benign cystadenomas, 32 of 40 (80%) borderline tumors (93.5% of serous and 33% of mucinous), and in 9 of 18 (50%) serous carcinomas. Ki-67 was only weakly expressed in 8 of 19 (42%) benign cystadenomas, all borderline tumors showed Ki-67 staining in less than 50% of the cells, and 55% of serous carcinomas stained in more than 50% of tumor cells. p53 was absent in all but 1 of the cystadenomas, was expressed in 9 of 40 (22.5%) borderline tumors (25.8% of serous and 11% of mucinous), and in 10 of 18 (55%) carcinomas. All 11 implants of serous borderline tumors expressed p21WAF1/CIP1. Most serous borderline tumors expressed higher levels of MDM2 compared with the benign cystadenomas and carcinomas. Four of the serous borderline implants (40%) expressed MDM2. Coexpression of p21WAF1/CIP1 and MDM2 characterizes serous borderline tumors of the ovary and their implants, which suggests that these cell cycle control proteins are important in these tumors and may be related to tumor progression. Low expression of p53 protein in serous borderline tumors might be in part mediated by MDM2. This suggests that the p53 pathway is intact in most of these tumors, in contrast with carcinomas, in which high expression of p53 has been related to mutations of this gene.

Extramammary Paget disease is characterized by the consistent lack of estrogen and progesterone receptors but frequently expresses androgen receptor.

Extramammary Paget disease (EPD) is an uncommon cutaneous malignant neoplasm that arises in areas rich in apocrine glands (perineum, vulva, and axilla). Apocrine gland origin or apocrine differentiation of cells of EPD has been suggested. Estrongen, progesterone, and androgen hormone receptors have been reported to exhibit a characteristic pattern of expression in mammary apocrine type carcinomas; however, their expression in EPD has not been elucidated fully. By using immunohistochemical methods, we studied the expression of steroid receptors in EPD on formalin-fixed paraffin-embedded tissue samples from 28 patients with EPD without associated visceral malignant neoplasms or adnexal carcinoma. Androgen receptor (AR) was identified in 15 of 28 cases. The proportion of AR-positive cells varied from 1% to more than 75%; 8 cases expressed AR in more than 10% of cells. Strong AR expression also was seen in the invasive carcinoma arising from 1 case of EPD. All cases lacked immunohistochemically detectable estrogen and progesterone receptors. The immunophenotype characteristic of apocrine carcinomas (AR-positive, estrogen receptor-negative, progesterone receptor-negative) was seen in a substantial proportion of EPD cases. Results suggest that AR expression is a factor in pathogenesis of EPD. This may be important for the therapy of recurrent or invasive disease.

Keratin subsets in papillary and follicular thyroid lesions. A paraffin section analysis with diagnostic implications.

Previous studies indicate that keratins 7, 8 and 18 are present in all thyroid papillary and follicular lesions, but the distribution of other keratins has been incompletely characterized. The profile of individual keratin (K) polypeptides was evaluated immunohistochemically in over 200 non-neoplastic and neoplastic thyroid papillary and follicular lesions. Monoclonal antibodies to K19, K17, K16, K5/6 and K10 were applied in paraffin sections of formaldehyde-fixed tissue. K19 was present variably, often only focally in goitres, and was present only sporadically in papillary hyperplasia. However, K19 was strongly and uniformly expressed in virtually all papillary carcinomas, indicating differential diagnostic usefulness in differentiating papillary hyperplasia and papillary carcinoma. About half of the follicular carcinomas (defined as tumours strictly excluding the follicular variant of papillary carcinoma) were also strongly K19-positive, suggesting that K19 patterns are not reliable in differentiating papillary and follicular carcinoma. K17 and K5/6 were present in cysts and squamous metaplasia of goitres, and focally in papillary but only exceptionally in follicular carcinoma in areas of squamous differentiation and tumour cells in desmoplastic stroma. K16 in turn was present only focally in well-developed squamous metaplasia in goitres but was not found in differentiated thyroid carcinomas. K10, a high-molecular-weight keratin typical of epidermal differentiation, was identified neither in non-neoplastic nor in neoplastic differentiated thyroid lesions, including squamous metaplasia. These results indicate that papillary carcinomas differ from other differentiated thyroid tumours in their varying, usually focal, expression of stratified epithelial keratins that are partly but not exclusively related to squamous differentiation in such lesions. However, papillary carcinomas do not express truly epidermally restricted keratins; their previously described reactivity with polyclonal "epidermal keratin" antibodies most probably results from the reactivity of such antibodies with K19.

Cell-type- and tumour-type-related patterns of bcl-2 reactivity in mesenchymal cells and soft tissue tumours.

Bcl-2 is one of the many proteins that regulate programmed cell death and is overexpressed in B-cell lymphomas. The expression of bcl-2 in mesenchymal cells and soft tissue tumours was the subject of this study. Normal mesenchymal tissue and representative cases of soft tissue tumours of different types (n>200) were investigated immunohistochemically for bcl-2 expression. Although bcl-2 expression was normally relatively restricted to some smooth muscle cells and neural cells, bcl-2 immunoreactivity was widespread in different types of soft tissue neoplasms, both benign and malignant. Consistently positive tumours included solitary fibrous tumour, haemangiopericytoma, schwannoma and synovial sarcoma. The few soft tissue tumours that were consistently negative for bcl-2 included nodular fasciitis and desmoid tumour. Leiomyomas and leiomyosarcomas were heterogeneous; all uterine leiomyomas were bcl-2 positive, but all oesophageal leiomyomas were negative, paralleling the reactivity observed in the smooth muscle at those sites. Gastrointestinal stromal tumours showed bcl-2 reactivity; this was less consistent in malignant tumours. Along the malignancy gradient, there was no consistent trend in the bcl-2 reactivity. Dermatofibrosarcomas showed increase of bcl-2 expression with fibrosarcomatous transformation, whereas smooth muscle sarcomas and malignant peripheral nerve sheath sarcomas were less consistently positive than the corresponding benign neoplasms. We conclude that bcl-2 expression is widespread in soft tissue tumours, but shows constitutional expression patterns that are often parallel to the normal tissue counterparts. Compared with benign soft tissue tumours, bcl-2 expression is often reduced in sarcomas, but it cannot be used as a prognostic marker without correlation of the data to its phenotypic expression patterns.

Immunohistochemical localization of prostate-specific antigen in ductal epithelium of male breast. Potential diagnostic pitfall in patients with gynecomastia.

Enlargement of the male breast is frequently encountered in the course of adjuvant antiandrogen therapy for advanced prostate carcinoma. The clinical differential diagnosis in this setting includes hormonal imbalance-induced gynecomastia, primary breast carcinoma, and metastasis of prostatic carcinoma. Biopsy of the lesion with the identification of prostate-specific antigen (PSA) plays an important role in establishing the correct diagnosis. Recent studies showed that female mammary epithelium may be a significant source of PSA, but its expression in male breasts has not been sufficiently studied. We found that normal and hyperplastic duct epithelium in gynecomastia exhibited focal, strong (+3) PSA immunoreactivity in 5 of 18 cases (28%). In contrast, no PSA reactivity was found in eight cases of male breast carcinoma. No reactivity was seen with antiprostatic acid phosphatase (PsAP) antibody, in either benign or malignant epithelium. Frequent expression of PSA in gynecomastia may, in an appropriate clinical setting, cause confusion with metastatic prostatic carcinoma. The lack of immunoreactivity for PsAP in male breast epithelium indicates its usefulness in the differential diagnosis.

A comparative analysis of Ki-67, p53, and p21WAF1CIP1 expression in tamoxifen associated endometrial carcinomas.

OBJECTIVE: To determine clinicopathologic parameters, expression of proliferation markers, and immunohistochemical oncogene expression in endometrial cancers in patients with a history of breast cancer with and without tamoxifen use. METHODS: Thirty endometrial carcinoma specimens were examined from patients with a previous history of breast cancer. Patients who had taken tamoxifen (15) were compared to non-users (15). Immunohistochemical staining was performed for p53, Ki-67, and p21WAF1/CIP1, overexpression was defined as greater than 10% positivity. RESULTS: Patient populations were statistically similar. P53 was overexpressed in 73% of tamoxifen users compared to 53% of non-users. Ki-67 was overexpressed in over 90% of user and non-user specimens. p21WAF1/CIP1 was overexpressed in 33% of users and 47% of non-users. Tamoxifen users had shorter time to diagnosis of endometrial cancer than non-users. CONCLUSIONS: In this small study, tamoxifen associated tumors expressed p53 more frequently than non-users, while the opposite was observed with p21WAF1/CIP1. This suggests that p53 mutations might play a role in development of tamoxifen associated tumors.

Expression of p21WAF1/CIP1 in soft tissue sarcomas: a comparative immunohistochemical study with p53 and Ki-67.

The p53 gene controls the cell cycle by transactivating p21WAF1/CIP1, a cyclin dependent kinase (cdk) inhibitor. By inhibiting cdks, p21WAF1/CIP1 regulates the cell cycle by blocking the G1 to S phase transition. In this study, we analyzed the immunohistochemical expression of p21WAF1/CIP1 in 66 soft tissue sarcomas and its relationship to p53 and the cell cycle proliferation antigen Ki-67. Expression of p21WAF1/CIP1, was detected in 76% of the tumors and p53 in 26%. All malignant schwannomas, synovial sarcomas, leiomyosarcomas and gastrointestinal stromal tumors expressed p21WAF1/CIP1. The majority of angiosarcomas, dermatofibrosarcomas, and fibrosarcomas showed low expression or were negative for p21WAF1/CIP1. Ewing's sarcomas, liposarcomas, and malignant fibrous histiocytomas were heterogeneous in their expression of p21WAF1/CIP1. Combining p53 and p21WAF1/CIP1 staining, the following four patterns were observed: 23% of the tumors showed the p53+/p21+ pattern; 53% showed the p53-/p21+ pattern; 3% showed the p53+/p21- pattern and 21% were negative for both p53 and p21WAF1/CIP1. There was no correlation between Ki-67 and p21WAF1/CIP1 or p53 staining. Our results show that soft tissue sarcomas, independent of their histologic subtype, frequently express p21WAF1/CIP1 which is probably important in their tumorigenesis. Additionally, p21WAF1/CIP1 may play a role in determining the efficacy of various cell cycle-directed therapies in soft tissue sarcomas.

Detection of growth fraction in tumors by Ki67 monoclonal antibody in cytologic smears: a prospective study of 40 cases.

The monoclonal antibody (MAb) Ki67 detects a nuclear antigen in cycling tumor cells. Quantitation of proliferating cells is helpful in predicting the recurrence and metastatic potential of tumors as previously reported. We conducted a prospective study on 40 benign and malignant tumors by performing Ki67 immunocytochemical stains on cytologic smears and their corresponding frozen tissues. Quantitation of Ki67 positive cells was done by counting 300 cells in 5-7 high-power fields in cytologic smears and tissues. Only nuclear or nucleolar immunostaining was considered positive for MAb Ki67. The number of Ki67 positive tumor cells in cytologic smears correlated well with Ki67 positive cells from corresponding tissues. On the average, cytologic smears showed 1.9% higher Ki67 positivity in malignant tumors as compared to their corresponding frozen tissues (P < 0.001). The Ki67 positivity in malignant tumors was found to be significantly higher when compared with benign tumors (P < 0.001). We conclude that cytologic smears can be used for the determination of growth potential in tumors by MAb Ki67. Additionally, cytologic preparations can be used during intraoperative consultations when adequate tissue is not available for the above mentioned study.

Immunocytochemical evaluation of HBME-1, CA 19-9, and CD-15 (Leu-M1) in fine-needle aspirates of thyroid nodules.

Overlapping morphologic patterns that may be observed in goiter, follicular adenoma, and papillary carcinoma can limit the cytologic evaluation of the thyroid gland. In an attempt to develop a useful adjunctive test, the immunocytochemical reactivity of HBME-1, carcinoma antigen 19-9 (CA 19-9), and CD-15 (Leu-M1) was tested on 59 cell block preparations from fine-needle aspirations of the thyroid gland. HBME-1 monoclonal antibody was reactive in all 21 papillary carcinomas, in 4 of 18 adenomas, and in 5 of 20 goiters. CA 19-9 was identified in 13 of 21 carcinomas, 1 goiter, but none of the adenomas. CD-15 was present in 15 of 21 carcinomas, 1 goiter, and 1 adenoma. We conclude that HBME-1 is a sensitive marker of papillary thyroid carcinoma. CD-15 and CA 19-9 are less sensitive but more specific. This panel can be useful to help classify morphologically equivocal lesions. As with all immunocytochemical testing, caution must be exercised in the interpretation of results, and correlation made with morphologic and clinical data.

p53 immunohistochemistry for distinguishing reactive mesothelium from low grade ovarian carcinoma.

OBJECTIVE: To determine the utility of immunohistochemical staining for p53 in cell block material for distinguishing reactive mesothelium from borderline or low grade ovarian carcinoma. STUDY DESIGN: Paraffin-embedded cell blocks from paracentesis and pelvic wash fluid of 44 cases of ovarian carcinoma and 20 cases containing only reactive mesothelium were immunostained for p53 using monoclonal antibody DO-7. Tumor grades ranged from borderline to high grade and were serous papillary (33), clear cell (3), mucinous (2), endometrioid (2), mixed serous papillary/clear cell (3) and undifferentiated (1). The three authors independently evaluated the staining, including estimation of the percentage and intensity of positive nuclear staining. RESULTS: A separation of positive from negative cases was seen when staining intensity was considered the critical parameter; moderate to strong staining was considered truly positive. Seventy-three percent (8/11) of borderline tumors, 80% (8/10) of low grade tumors and 65% (15/23) of intermediate to high grade tumors showed moderate to strong positivity. Percentage of staining was a less-reliable parameter as 25% of negative cases were positive by this assessment. CONCLUSION: p53 Immunohistochemistry, using monoclonal antibody DO-7 combined with standard morphologic evaluation, may be useful in distinguishing benign reactive mesothelium from borderline or low grade ovarian carcinoma.

Cyclin E expression and early cervical neoplasia in ThinPrep specimens. A feasibility study.

OBJECTIVE: To investigate cyclin E expression as a possible marker for early cervical neoplasia using ThinPrep gynecologic specimens from premenopausal women. STUDY DESIGN: Archived ThinPrep liquid-based cervical/endocervical specimens (Cytyc Corporation, Boxborough, Massachusetts, U.S.A.) diagnosed as human papillomavirus infection (HPV) (20), atypical squamous cells of undetermined significance (ASCUS) (48) and within normal limits (WNL)/benign cellular changes (BCC) (21) were resampled in duplicate, fixed in 95% ethanol, subjected to immunocytochemical staining with the cyclin E antibody (clone 13A3, Novocastra Laboratories Ltd., Newcastle upon Tyne, U.K.) and HPV antibody (clone K1H8, Dako Corporation, Carpinteria, California, U.S.A.) and the expression scored by two pathologists and correlated with the cytologic diagnosis. A case was scored as positive if it contained > 10 abnormal squamous cells with nuclear immunocytochemical staining. RESULTS: The cylin E antibody assay was positive in 20 (100%) cases cytologically diagnosed as HPV. These cases were also anti-HPV antibody positive. Four cases (19%) cytologically diagnosed as WNL/BCC were cyclin E positive. Of these, two were anti-HPV antibody positive. Thirty-four (73%) cases cytologically diagnosed as ASCUS were positive for the cyclin E assay and for anti-HPV antibody staining. CONCLUSION: Cyclin E expression correlates strongly with morphologic features of HPV in ThinPrep specimens and may serve as a surrogate marker for HPV infection and early cervical preneoplastic lesions.

Prolactin suppresses a progestin-induced CK5-positive cell population in luminal breast cancer through inhibition of progestin-driven BCL6 expression.

Prolactin controls the development and function of milk-producing breast epithelia but also supports growth and differentiation of breast cancer, especially luminal subtypes. A principal signaling mediator of prolactin, Stat5, promotes cellular differentiation of breast cancer cells in vitro, and loss of active Stat5 in tumors is associated with antiestrogen therapy failure in patients. In luminal breast cancer, progesterone induces a cytokeratin-5 (CK5)-positive basal cell-like population. This population possesses characteristics of tumor stem cells including quiescence, therapy resistance and tumor-initiating capacity. Here we report that prolactin counteracts induction of the CK5-positive population by the synthetic progestin (Pg) R5020 in luminal breast cancer cells both in vitro and in vivo. CK5-positive cells were chemoresistant as determined by fourfold reduced rate of apoptosis following docetaxel exposure. Pg-induction of CK5 was preceded by marked upregulation of BCL6, an oncogene and transcriptional repressor critical for the maintenance of leukemia-initiating cells. Knockdown of BCL6 prevented induction of CK5-positive cell population by Pg. Prolactin suppressed Pg-induced BCL6 through Jak2-Stat5 but not Erk- or Akt-dependent pathways. In premenopausal but not postmenopausal patients with hormone receptor-positive breast cancer, tumor protein levels of CK5 correlated positively with BCL6, and high BCL6 or CK5 protein levels were associated with unfavorable clinical outcome. Suppression of Pg-induction of CK5-positive cells represents a novel prodifferentiation effect of prolactin in breast cancer. The present progress may have direct implications for breast cancer progression and therapy as loss of prolactin receptor-Stat5 signaling occurs frequently and BCL6 inhibitors currently being evaluated for lymphomas may have value for breast cancer.

Immunohistochemical staining for proliferating cell nuclear antigen, BCL2, and Ki-67 in vulvar tissues.

Dysplastic cells of the uterine cervix commonly react with antibodies to Ki-67 and proliferating cell nuclear antigen (PCNA), but less is known regarding the reactivity of vulvar lesions. Paraffin-embedded slides from seven normal vulvar tissues, eight cases of lichen sclerosus, and 10 cases of vulvar intraepithelial neoplasia (VIN), many with associated condylomatous changes, were immunostained with antibodies to PCNA, Ki-67, and BCL2 protein, a protooncogene product normally expressed only in basal cells of squamous epithelium. Ki-67 and PCNA nuclear staining was largely restricted to basal and parabasal cells in normal tissues and lichen sclerosus. Focal midepithelial staining with PCNA and Ki-67 was seen in one case of lichen sclerosus; a vulvar biopsy of this patient 1 year later was negative for VIN. Both antibodies stained dysplastic cells at higher epithelial levels in VIN, but Ki-67 was more consistently reactive and showed a sharper distinction from adjacent histologically uninvolved epithelium compared to PCNA. Condylomatous changes were variably stained. The pattern of BCL2 staining was identical in normal vulva, lichen sclerosus, and VIN, but BCL2 decorated occasional mitotic figures in VIN. Overall, Ki-67 was a better marker of vulvar dysplasia than PCNA.