Immunohistochemical studies of metastatic germ-cell tumors in retroperitoneal dissection specimens: a sensitive and specific panel.

Germ-cell tumors (GCTs) are the most common malignancies in adolescent and young men. These tumors are highly treatable, even at an advanced stage; therefore, accurate diagnosis is imperative. In this study, we evaluated immunohistochemical stains for SALL4, NANOG, glypican-3 (GPC3), D2-40, and CD30 with adequate control in retroperitoneal dissection specimens under the same laboratory conditions. The study groups included 31 cases of metastatic testicular GCTs with the following components: 11 seminomas, 14 embryonal carcinoma (ECs), 12 yolk sac tumor (YSTs), 8 teratomas, 10 cases of metastatic melanomas, 14 cases of malignant lymphomas, and 11 cases of metastatic, poorly differentiated carcinoma. SALL4 showed diffuse nuclear labeling for all seminomas, ECs, and YSTs. NANOG showed diffuse nuclear positivity in all seminomas and ECs. Metastatic carcinomas, melanomas, and malignant lymphomas were negative for these 2 markers. Gypican-3, D2-40, and CD30 showed sensitive staining for YSTs, seminomas, and ECs, respectively. In conclusion, SALL4 and NANOG are sensitive and specific markers for GCTs. GPC3, D2-40, and CD30 are sensitive but not specific for individual components of GCTs and may be useful in aiding in the differential diagnosis for the individual component of GCTs when the identity of GCT is established.

Sirtuin 1 (SIRT1): a potential immunohistochemical marker and therapeutic target in soft tissue neoplasms with myoid differentiation.

Sirtuin, silent mating-type information regulation 2 homolog Saccharomyces cerevisiae 1 (SIRT1), is a protein that has been implicated in multiple mammalian functions including cell aging, stress resistance, and differentiation. SIRT1 has also been shown to be involved in multiple tumors. In addition, new pharmacotherapies have recently been approved that target SIRT1. The purpose of this study was to use immunohistochemistry to characterize SIRT1 protein expression in human soft tissue neoplasms with the hopes of finding new diagnostic and therapeutic modalities. SIRT1 immunoreactivity was reviewed in a series of 164 soft tissue tumors including alveolar soft part sarcoma, angiomyolipoma, clear cell sarcoma, desmoid/fibromatosis, desmoplastic small round cell tumor, Ewing sarcoma, gastrointestinal stromal tumor, glomus tumor, leiomyoma, leiomyosarcoma, lipoma, liposarcoma, malignant peripheral nerve sheath tumor, nodular fasciitis, osteosarcoma, rhabdomyosarcoma, schwannoma, solitary fibrous tumor, synovial sarcoma, undifferentiated pleomorphic sarcoma, and Wilms tumor. In addition, numerous benign tissues were tested for SIRT1 reactivity. In nonneoplastic tissue, strong cytoplasmic SIRT1 reactivity was observed in all prostate stroma, smooth muscle, and striated muscle. A similar pattern of cytoplasmic SIRT1 expression was observed in soft tissue neoplasms with myoid differentiation, namely, angiomyolipoma (100%), glomus tumor (100%), leiomyoma (90%), leiomyosarcoma (76.5%), and rhabdomyosarcoma (87%). The other lesions examined were negative. Although the physiologic role of SIRT1 remains to be clarified in myoid tissues and neoplasms differentiating along these lines, this observation points to a potential role for this marker in diagnostic immunohistochemistry. Furthermore, the recent emergence of drugs capable of selectively inhibiting SIRT1 raises the possibility of a potential application for targeted therapy. Additional studies are necessary to further characterise the role of SIRT1 in myoid tissues and neoplasms.

Aberrant connexin 43 and 26 expression in cervical dysplasia.

OBJECTIVE: To determine whether connexin (Cx) expression is altered in cervical dysplasia. STUDY DESIGN: Cx proteins form gap junctions and are expressed in squamous epithelia including ectocervix. We used multispectral imaging to perform a quantitative immunohistochemical survey of Cx43 Cx26 in 37 archival human cervical specimens. RESULTS: Cx43 expression was very low in normal cervix (100%), but was increased in low-grade squamous intraepithelial lesions (LSILs) (64%), primarily in a parabasal distribution. High-grade squamous intraepithelial lesions (HSILs) showed weak full-thickness Cx43 staining (53%) or lacked Cx43 (47%). An aberrant increase in Cx43 expression was often (62%) present in histologically normal areas of specimens that elsewhere harbored dysplasia. Cx26 was highly expressed in the basal layer of normal ectocervix (100%). In LSIL, 57% showed a decrease in Cx26 and the rest showed no change relative to the normal pattern. In HSIL, Cx26 was expressed in the full thickness of the epithelium, at a high level in 80% of cases and a low level in the rest. CONCLUSION: Cx alteration is moderately consistent in cervical dysplasia, and for Cx43 can precede histologic changes. The resulting changes in Cx signaling may be important in the pathogenesis of cervical intraepithelial neoplasia.

TFE3 expression in tumors of the microphthalmia-associated transcription factor (MiTF) family.

The DNA-binding factor TFE3 is closely related to microphthalmia-associated transcription factor (MiTF) and is over-expressed in alveolar soft part sarcoma (ASPS) and select renal cell carcinomas. Reports of TFE3 expression in PEComa prompted investigation into TFE3 expression among other members of the putative MiTF group of neoplasms. The authors examined cases of PEComa (n = 6), conventional angiomyolipoma (AML; n = 22), metastatic melanoma (n = 16), and clear cell sarcoma (CCS; n = 9) for TFE3 expression. Nuclear immunostaining was observed in 74% (39/53) of cases, as follows: 5/6 PEComas, 18/22 AMLs, 10/16 metastatic melanomas, and 6/9 CCSs. However, with the exception of PEComas, compared with ASPS controls, TFE3 staining was significantly less intense in the tumors examined. These results illustrate that TFE3 immunoreactivity is detectable in other members of the MiTF family of neoplasms. For this reason, such neoplasms warrant consideration in the differential diagnosis with nuclear TFE3 immunoreactivity, particularly when staining is focal and less intense.

Analysis of HIF-1a and its regulator, PHD2, in retroperitoneal sarcomas: clinico-pathologic implications.

Hypoxia is known to play important role in cancer biology.  In sarcomas, hypoxia-induced protein biomarkers such as Hypoxia Inducible Factor-1a (HIF-1a), vascular endothelial growth factor (VEGF), and Erythropoietin (Epo) have been previously reported in only a few studies.  Moreover, the biologic significance and relationship to tumorigenesis of these hypoxia-induced biomarkers is not well understood in the context of sarcoma. The HIF negative regulator, Prolyl Hydroxylase Domain protein 2 (PHD2) has not been evaluated in sarcomas.  We examined the expression of PHD2, HIF-1a, and several other hypoxia induced biomarkers in a series of clinically characterized, retroperitoneal sarcomas with immunohistochemical methods.  Expression of these proteins was analyzed and correlated with clinical outcome.  Increased HIF-1a expression was associated with shorter overall and disease free survival.  PHD2 expression was detected in the majority of sarcoma cases, with increased expression correlating with high tumor grade but not with survival.  Though changes in PHD2 expression alone did not correlate with overall and disease free survival, reduced/absent PHD2 expression in the presence of HIF-1a expression was associated with shorter overall and disease-free survival than that of other HIF-1a/PHD2 expression profiles.  These observations suggest that regulation and expression of both PHD2 and HIF-1a are important to the biology of sarcomas, and that loss of PHD2 function has an additional adverse effect in the prognosis of sarcomas in tumors expressing HIF-1a.  The biologic and therapeutic implications of HIF-1a and PHD2 expression in retroperitoneal sarcomas warrant further investigation.

Thyroid transcription factor-1 expression in normal gynecologic tissues and its potential significance.

SUMMARY: Thyroid transcription factor-1 (TTF-1) is a 38-kd nuclear protein, and a member of the NKx2 family of homeodomain transcription factors. It is highly expressed in normal and neoplastic thyroid and lung tissues, and is considered a reliable marker for lung adenocarcinoma and thyroid carcinoma. Recently, expression of TTF-1 has also been reported in ovarian, endometrial, and endocervical epithelial neoplasms. Little is known about TTF-1 immunoreactivity in normal gynecologic tissues. In this study, TTF-1 expression in various non-neoplastic gynecologic tissues was investigated by standard immunohistochemistry. One hundred and eight samples of benign gynecologic tissues from adult patients who had no known history of neoplastic condition were collected. Twenty-eight endometria (12 proliferative, 11 secretory, and 5 inactive), 26 fallopian tubes, 28 cervixes (14 endocervical and 14 ectocervical), 14 myometria, and 12 ovaries were studied. In addition, 4 normal fallopian tubes and 2 ovaries from 5 pediatric patients (aged from 3 mo to 11-yr old) were evaluated. Variable TTF-1 nuclear reactivity was identified in 25 of 26 (96%) fallopian tubes (extent of positivity ranged from 2% to 60%, median 25%), 15 of 28 (54%) endometria (1% to 10%, median 5%), and 6 of 14 (43%) endocervical samples (<5%). TTF-1 was also identified in 2 of 4 (50%) pediatric fallopian tubes with 5% and 20% of the tubal epithelium being positive, respectively. No TTF-1 expression was detected in ovarian tissue (neither epithelial nor stromal tissue; neither adult nor pediatric samples), ectocervical squamous epithelium or myometrium, nor in stromal tissue in endometrium, tube, or cervix. TTF-1 reactivity was detected in both proliferative and secretory endometrium, but not in 5 inactive endometria. TTF-1 is frequently expressed in normal/non-neoplastic tubal, and less frequently in functional endometrial and endocervical epithelia, but not in ovarian surface epithelium. TTF-1 might have a functional and developmental role in normal fallopian tube and endometrium, as it is highly expressed in tubal epithelium of both adults and young children, and in functional endometrium but not in inactive endometrium. The high TTF-1 expression in tubal epithelium but not in normal ovarian surface epithelium suggests that some TTF-1-positive ovarian tumors might be related to the tubal epithelium.

TTF-1 expression in ovarian and uterine epithelial neoplasia and its potential significance, an immunohistochemical assessment with multiple monoclonal antibodies and different secondary detection systems.

Thyroid transcription factor-1 (TTF-1) is a 38-kd homeodomain containing DNA-binding protein, identified in thyroid and lung as a regulator of thyroid-specific genes and surfactant and Clara cell secretory protein gene expression. TTF-1 has been used as a reliable lineage marker for lung adenocarcinoma and thyroid carcinoma in surgical pathology. However, TTF-1 expression has been recently reported in carcinomas of other origins including female genital tract. We evaluated TTF-1 expression with 3 primary different antibodies (8G7G3/1, SPT24, and BGX-397A) and 2 secondary automated detection systems (Envision+/Dako autostainer versus Refine/Bond Max) in 104 ovarian and endometrial tumors on routine surgical specimens and 108 ovarian tumors on tissue microarray (TMA) specimens. SPT24 and Refine/Bond Max autostainer was the most sensitive system among the primary antibodies and secondary detection/autostainers tested. By using SPT24/Refine/Bond Max, TTF-1 reactivity could be detected in all major histologic subtypes of gynecologic tumors and up to 26% of all cases tested on routine surgical specimens and 6.4% on TMA. TTF-1 was most frequently detected in uterine malignant mixed Müllerian tumor (82%), more common in uterine tumors than ovarian tumors, and more common in surgical specimen than TMA. When present, tumor cells can be rarely positive or diffusely positive for TTF-1 reactivity. In addition to malignant tumors, TTF-1 was also detected in benign tumors and benign tubal and endometrial epithelia. TTF 1 immunostaining has the potential to misguide a pathologist to conclude an ovarian or endometrial tumor being a lung metastasis. However, the role of TTF-1 in female genital tract and its tumors is unknown.

Expression of glial cell line-derived neurotropic factor receptor alpha-1 in immature teratomas.

The immaturity of teratomas is usually manifested as immature neuroepithelium. The amount of immature neuroepithelium has been correlated with the survival of adult patients with ovarian immature teratoma. To date, no immunohistochemical marker has been found to facilitate the identification of immature teratoma. In this study, we evaluated the expression of glial cell line-derived neurotropic factor receptor alpha-1 (GFRalpha-1) for this purpose. We retrieved 38 cases of germ cell tumors: 26 cases contained immature teratoma, of which 24 had immature neuroepithelium and showed strong membrane staining for GFRalpha-1. No significant staining was seen in other components including embryonal carcinoma, seminoma, yolk sac tumor, choriocarcinoma, immature mesenchyme, and intratubular germ cell neoplasia. Immunohistochemical staining for GFRalpha-1 in immature neuroepithelium may facilitate its identification.

Cytoplasmic overexpression of WT-1 in gastrointestinal stromal tumor and other soft tissue tumors.

The Wilms tumor 1 (WT-1) is a zinc finger transcription factor essential for the development of the kidneys and gonads. Alterations in the WT-1 gene were observed in several tumor types. Depending on the tumor types, WT-1 might function as a tumor suppressor or as a survival factor. WT-1 immunoreactivity in gastrointestinal stromal tumor (GIST) was currently not known. We, therefore, investigated the expression of WT-1 in GIST in comparison to other soft tissue tumors by immunohistochemistry and Western blot analysis. We found that all 28 cases (100%) of GIST are positive for WT-1, diffusely (>75%, 3+) in 13 (46.4%) cases, moderately (26% to 75%, 2+) in 13 (46.4%) cases, and focally (5% to 25%, 1+) in 2 (7.2%) cases. The staining intensity is usually strong. The staining pattern is predominantly cytoplasmic with rare scattered nuclear staining. Similar but less extensive cytoplasmic WT-1 immunoreactivity was detected in 16 of 25 (64%) uterine leiomyosarcoma and 14 of 24 (58.3%) soft tissue leiomyosarcoma. Rare scattered nuclear staining was also seen in uterine leiomyosarcoma and soft tissue leiomyosarcoma, which showed positive cytoplasmic WT-1 reactivity. Only 1 of the 10 solitary fibrous tumors showed weak cytoplasmic WT-1 positivity (10%). No WT-1 staining was detected in 6 cases of fibromatosis. The significance of cytoplasmic expression of WT-1 in GIST and some smooth muscle tumors is unclear and warrant further investigation. The potential roles of WT-1 in the diagnosis and treatment of GIST were discussed.

The selective expression of CD43 in adenoid cystic carcinoma.

CD43, a sialoglycoprotein expressed on hematopoietic cells, has only rarely been reported in nonhematopoietic tumors, mostly of colon. We describe CD43 expression by immunohistochemistry and mRNA in situ hybridization in adenoid cystic carcinomas (ACCs). CD43 immunoreactivity with 2 different antibodies was detected mainly in ACC but also 1 membranous type basal cell adenocarcinoma and 1 colonic adenocarcinoma. Nine of 20 (45%) ACC (8/17 salivary/mucoserous and 1/3 mammary) showed immunoreactivity with clone MT1 whereas 4 (23.5%) salivary ACC also showed immunoreactivity with clone DF-T1. The few high-grade, angioinvasive ACC in our study did not seem to express CD43. CD43 mRNA was detected by in situ hybridization in 2 of the 3 ACC that were CD43 positive on immunohistochemistry but not on any CD43 negative cases tested. Hence, CD43 seems to be selectively expressed in a subset of ACC and its significance in salivary tumors is discussed.

Immunoprofile of endocervical and endometrial stromal cells and its potential application in localization of tumor involvement.

To evaluate and compare the immunophenotype of endocervical and endometrial stromal cells and to asses its potential application in tumor localization. Paraffin sections of benign endocervix (n = 24), benign endometrium (n = 33), endocervical adenocarcinoma (n = 9), endometrial carcinoma (n = 13), and endometrial hyperplasia (n = 16) were stained with antibodies to CD10, Wilms Tumor-1, CD34, smooth muscle actin, and factor XIIIa by immunohistochemistry. In 16 cases, lower uterine segment was also available. Immunoreactivity of stromal cells was recorded as positive (>/=50% staining), focally positive (>/=5%-<50%) or negative (<5%). Endocervical stromal cells (ECSC) in either benign or malignant cervical epithelial lesions were predominantly CD34/CD10 (CD34 dominant immunophenotype). Endometrial stromal cells (EMSCs) in either benign or malignant epithelial lesions were primarily CD34/CD10 (CD10 dominant immunophenotype). Expression of Wilms Tumor-1 was decreased in EMSC of the EMCA when compared to their counterpart in endometrial hyperplasia. There was no differential expression of smooth muscle actin and factor XIIIa identified between ECSC and EMSC. The immunophenotypes of the ECSC and EMSC overlapped in the lower uterine segment. The functional status of the endometrium had no effect on the immunoprofile. The pattern of CD34 and CD10 immunostaining in stromal cells might be helpful in determining tumor involvement in uterine and cervical sites.

Cancer/testis (CT) antigens are expressed in fetal ovary.

Cancer/testis (CT) antigens are named after their expression pattern as they are typically present in various types of tumors and in the germ cells of normal adult testis. Adult ovarian tissue is usually reported to be CT antigen negative. Based on the differences in female versus male gonadal development, the ovarian counterpart of the most predominant CT antigen positive testicular germ cells are not prevalent in the adult ovary. Hence, we analyzed the protein expression of several CT antigens in fetal ovary by immunohistochemistry with various monoclonal antibodies (mAbs) previously generated by our group. The mAbs used were: MA454 (MAGE-A1), M3H67 (MAGE-A3), 57B (MAGE-A4), CT7-33 (CT7/MAGE-C1), and ES121 (NY-ESO-1). All mAbs showed some immunopositivity in fetal ovarian germ cells. The most intense staining was seen with mAbs M3H67, 57B, and CT7-33 during weeks 16-23 of gestation. The most prevalent cells stained were oogonia, with only focal staining of oocytes of the primordial follicle. We conclude that CT antigens are regularly expressed in fetal ovarian germ cells and might play an important role in male and female germ cell biology.

D2-40 functions as an effective chondroid marker distinguishing true chondroid tumors from chordoma.

Chordomas and low-grade chondrosarcomas of the central nervous system share many histological features, generating, at times, considerable diagnostic difficulty and, not infrequently, requiring immunohistochemical analysis for appropriate classification. While both chordomas and chondrosarcomas stain positively for S100, only chordomas typically express epithelial antigens like cytokeratins and epithelial membrane antigen. Positive or negative staining with these latter two markers currently represents the only immunohistochemical technique that effectively distinguishes chordomas from chondrosarcomas. A marker that is reliably positive in chondrosarcomas and negative in chordomas has, to date, not been reported. D2-40 is a monoclonal antibody initially developed against M2A, a fetal testis-related antigen now known as podoplanin (aggrus), which has been found to stain a diverse collection of both benign and malignant tissues. In this study, we systematically investigated D2-40 immunoreactivity in a series of 22 chordomas, 20 chondrosarcomas, and 12 enchondromas, in conjunction with cytokeratin and S100 immunostaining. We found that D2-40 robustly and reliably immunostains low-grade chondroid neoplasms (100% of enchondromas and 94% of grades I and II chondrosarcomas), but not chordomas. By contrast, we observed generally strong and diffuse cytokeratin positivity in all cases of chordoma, but not in cases of enchondroma or low-grade chondrosarcoma. Thus, we show that D2-40 behaves as a chondroid marker differentiating true chondroid neoplasms from chordoma. We also demonstrate D2-40 immunoreactivity in two cases of chordoid meningioma and, in doing so, tentatively provide a means to distinguish this tumor from chordoma.

Comparison of p63 and p73 expression in benign and malignant salivary gland lesions.

BACKGROUND: The p63 and p73 genes are members of the p53 family and play an important role in stem cell identity and cellular differentiation and are expressed in basal and myoepithelial cells. In this study, we examined the expression of p63 and p73 in 50 various benign salivary gland lesions and 45 malignant salivary gland tumors. METHODS: The 95 salivary gland tumors were selected from the archives of the Department of Pathology and Laboratory Medicine at the Hospital of the University of Pennsylvania. Sectioned formalin-fixed paraffin-embedded tissue cut at 3 mum was immunostained with antibodies that recognize all isozymes of p63 and p73 and evaluated with respect to percentage of positive cells and localization. RESULTS: In benign lesions, p63 and p73 nuclear reactivity was seen in 46 (92%) of 50 and 47 (94%) of 50 cases, respectively. In malignant tumors, p63 and p73 were seen in 34 (76%) of 45 and 40 (89%) of 45 cases, respectively. A significant difference between p63 and p73 positivity was only seen in adenoid cystic carcinomas (p = .006). Also, p73 was found in tumors with minimal basal/myoepithelial differentiation. CONCLUSIONS: Hence, p63 and p73 expression is retained in both benign and malignant salivary gland tumors with basaloid or myoepithelial differentiation. Hence, p63 seems to be a more specific marker of myoepithelial differentiation than p73.

Utility of D2-40, a novel mesothelial marker, in the diagnosis of malignant mesothelioma.

Although immunohistochemistry has proven to be valuable in the differentiation of epithelioid mesothelioma from pulmonary or metastatic adenocarcinoma, no single antibody has demonstrated absolute sensitivity or specificity in making this distinction. Using immunohistochemical analysis with D2-40, a recently available monoclonal antibody that has been used as a lymphatic endothelial marker, we examined 53 cases of mesothelioma, 28 cases of reactive pleura, 30 cases of pulmonary adenocarcinoma, 35 cases of renal cell carcinoma, 26 cases of ovarian serous carcinoma, 16 cases of invasive breast carcinoma, 11 cases of prostatic adenocarcinoma, and seven cases of urothelial carcinoma. In addition, immunohistochemistry using calretinin, cytokeratin 5/6, and WT1 was performed on all cases of mesothelioma, pulmonary adenocarcinoma, ovarian serous carcinoma, and renal cell carcinoma. Predominantly, membranous D2-40 immunoreactivity was present in 51 of 53 (96%) mesotheliomas, 27 of 28 (96%) cases of reactive pleura, and 17 of 26 (65%) ovarian serous carcinomas; membranous staining was not seen in any other tumors examined. Compared to other immunohistochemical markers of mesothelioma, D2-40 was as sensitive as calretinin and more sensitive than cytokeratin 5/6 and WT1. We conclude that D2-40 immunoreactivity is sensitive for cells of mesothelial origin, and may be useful in the differential diagnosis of epithelioid malignant mesothelioma vs adenocarcinoma.

PDGF-A, PDGF-Rbeta, TGFbeta3 and bone morphogenic protein-4 in desmoplastic small round cell tumors with EWS-WT1 gene fusion product and their role in stromal desmoplasia: an immunohistochemical study.

Histologically, desmoplastic small round cell tumor is composed of the characteristic neoplastic small round cells with divergent differentiation, and distinct desmoplastic stroma. Genetically, the tumor shows a characteristic 11;22 translocation, involving the EWS gene on chromosome 22 and the WT1gene on chromosome 11 to produce an EWS-WT1 fusion gene which generates a chimeric protein functioning as a novel transcription factor that activates expression of target genes such as PDGF-A. Expression of PDGF-A, a potent growth factor for fibroblasts, has been detected in desmoplastic small round cell tumors and has been linked to the characteristic desmoplasia in these tumors. Bone morphogenic proteins, which are members of the TGFbeta superfamily play a complex role in regulating cell growth and differentiation and bone formation but have not been evaluated in desmoplastic small round cell tumors. In all, 24 desmoplastic small round cell tumors with EWS-WT1 fusion product confirmed by RT-PCR analysis were evaluated for expression of PDGF-A, PDGF-Rbeta, TGFbeta3 and bone morphogenic protein-4 by standard immunohistochemical methods with antigen retrieval on paraffin sections. Immunoreactivity was evaluated semiquantitively. Tumor-associated desmoplasia was quantified using a three-tier scale on hematoxylin- and eosin-stained sections. Desmoplastic small round cell tumors showed variable immunoreactivity with TGFbeta3 (21/24), BMP4 (14/21), PDGF-A (19/24) and PDGF-Rbeta (16/22). Less frequently, the stromal cells showed reactivity with TGFbeta3, PDGF-Rbeta and PDGF-A. Tumor-associated desmoplasia was prominent in eight, intermediate in seven and weak in nine cases. There was no correlation between tumor-associated desmoplasia and the markers tested except PDGF-A. In contrast to a previous study, our study showed that the level of PDGF-A expression inversely correlated with tumor-associated desmoplasia. Other targets of the EWS-WT1 transcription factor other than PDGF-A may be directly responsible for the prominent tumor-associated desmoplasia seen in desmoplastic small round cell tumor.

Peroxisome proliferator-activated receptor gamma expression in follicular-patterned thyroid lesions. Caveats for the use of immunohistochemical studies.

To determine its usefulness as a specific diagnostic marker for follicular carcinomas (FCs) vs other follicular-patterned thyroid lesions and possible application to fine-needle aspiration specimens, we immunohistochemically studied peroxisome proliferator-activated receptor gamma (PPAR gamma) expression in histologic sections (FC, 13 cases; follicular adenoma [FA], 11; follicular variant of papillary carcinoma [FVPC], 9) and surrounding thyroid tissue by using a PPAR gamma monoclonal antibody. Positivity (detected by nuclear staining) was scored as absent, weak, moderate, or strong. When only moderate or strong nuclear staining was considered positive, 9 FCs (69%), 3 FAs (27%), and 2 FVPCs (22%) demonstrated positive nuclear immunoreactivity. The sensitivity and specificity of immunohistochemical detection of PPAR gamma expression in FCs were 69% and 75%, respectively. In nonlesional surrounding tissue, moderate to strong positive staining was seen in focal areas of chronic lymphocytic thyroiditis in 6 of 33 cases (FC, 2; FA, 3; FVPC, 1); diffuse moderate staining was detected in surrounding tissue in the absence of lymphocytic thyroiditis in 4 cases (12%; FC, 3; FA, 1). Staining in the follicular-patterned lesions and surrounding nonlesional thyroid was specific with peptide blocking experiments. PPAR gamma expression is not a specific marker for FCs, and its detection in nonlesional thyroid tissue suggests limited usefulness as a diagnostic marker for follicular-patterned lesions in general.

Overexpression of p16INK4A in liquid-based specimens (SurePath) as marker of cervical dysplasia and neoplasia.

Human papillomavirus (HPV) is recognized as a causal agent for cervical carcinomas. Assimilation of HPV oncogenes E6 and E7 into the host DNA promotes upregulation of cyclin dependent kinase inhibitor (CDKI) p16(INK4A), detectable by monoclonal antibody in the developing cervical cancer cells. The aim of this study was to 1) develop a protocol for p16(INK4A) immunocytochemical staining on SurePath preparations, and 2) determine its utility as an HPV marker on a spectrum of cervical reactive and neoplastic lesions. Seventy-two specimens consisting of 28 nonneoplastic/nondysplastic cases (NN), one reactive glandular cells (RGC), 27 low-grade squamous intraepithelial lesions (LSIL), 10 high-grade squamous intraepithelial lesions (HSIL), one squamous cell carcinoma (SCCA), four atypical glandular cells (AGUS), and two adenocarcinomas (ADCA) were reprepped by SurePath and antibody to p16(INK4A) applied at 1:100 dilution using the Dako Envision + System on the Dako Autostainer. Expression of p16(INK4A) within the nucleus principally and cytoplasm of at least 10-15 cells was considered positive. All initial Papanicolaou-stained discrepant cases (p16(INK4A) positivity of NN and RGC cases and lack of reactivity in LSIL, HSIL, and AGUS) were reviewed. Nine of ten (90%) HSIL, one (100%) SCCA, 21/27 (78%) LSIL, and some reactive and inflammatory specimens demonstrated the presence of p16(INK4A). Reevaluation of discrepant cases revealed that several were underinterpreted (four NN were LSIL, one RGC was AGUS) or overinterpreted (one LSIL was NN). Following reassessment, false-positive staining was present in only 1/25 (1.4%) NN. Six of 30 (20%) LSIL lacked p16(INK4A) positivity. One of 10 (10%) HSIL had no staining. Two of four AGUS did not react with p16(INK4A) antibody. Both SCCA (1) and ADCA (2) had positive expression. This study confirms the intimate relationship between p16(INK4A) and HPV cytopathic effect. The p16(INK4A) immunocytochemical stain can be applied to liquid-based cervical preparations. This technique offers a more objective approach to deciphering "gray areas" of gynecologic cytopathology.

Immunohistochemical expression of galectin-3 in benign and malignant thyroid lesions.

CONTEXT: The expression of galectin-3, a human lectin, has been shown to be highly associated with malignant behavior of thyroid lesions. DESIGN: We studied the immunohistochemical expression pattern of galectin-3 in a variety of follicular-derived thyroid lesions (13 benign and 62 malignant), including Hürthle cell and follicular carcinoma, papillary carcinomas and variants, and anaplastic and poorly differentiated carcinomas. RESULTS: Immunoreactivity was strongest in papillary thyroid carcinomas, whereas staining was less intense in Hürthle cell and anaplastic carcinomas, and even weaker in the follicular variant of papillary thyroid carcinoma. Staining was absent or weak in the 3 follicular thyroid carcinomas and was negative in both insular carcinomas. In several tumors, staining was stronger at the advancing invasive edge of the lesion than in the central portion of the tumor. Galectin-3 was also expressed focally and weakly in reactive follicular epithelium and entrapped follicles in chronic lymphocytic thyroiditis. A variety of thyroid lesions showed prominent endogenous, biotin-like activity, which could cause flaws in interpretation if a biotin-detection system were used. CONCLUSION: We conclude that galectin-3 immunostaining, when used in biotin-free detection systems, may be useful as an adjunct to distinguish benign from malignant thyroid lesions.