Theresa L. Pasha

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.

WT1 is differentially expressed in serous, endometrioid, clear cell, and mucinous carcinomas of the peritoneum, fallopian tube, ovary, and endometrium

Summary:The Wilms’ tumor gene WT1 plays complex roles in the development of the organs of the genitourinary tract and mesothelium, as well as Wilms’ tumors. Although its biologic role is still unclear, most serous carcinomas of the ovary and peritoneum, mesotheliomas, and Wilms’ tumor have been shown to express WT1. A recent study, however, found no WT1 expression in serous carcinomas of the endometrium, suggesting that WT1 could be useful in identifying the primary site of serous carcinomas. We examined the expression of WT1 and p53 by immunohistochemistry in 69 cases of endometrial carcinoma (35 endometrioid, 18 clear cell, 16 serous), 68 cases of ovarian carcinoma (28 serous, 11 endometrioid, 18 clear cell, and 11 mucinous), 14 fallopian tube carcinomas (12 serous, 2 endometrioid), and 20 primary peritoneal serous carcinomas. WT1 nuclear reactivity of any extent and intensity was considered positive. Immunohistochemical stains were evaluated semiquantitatively using a four-tiered scale. Among endometrial carcinomas, WT1 immunoreactivity was seen in 10 of 16 serous, but in none of 35 endometrioid or 18 clear cell carcinomas. Among ovarian tumors, WT1 expression was seen in 24 of 28 serous and 4 of 18 clear cell carcinomas, but in none of 11 endometrioid and 11 mucinous tumors. All 12 serous carcinomas but none of 2 endometrioid carcinomas of the fallopian tube were positive for WT1. WT1 expression was seen in 19 of 20 serous primary peritoneal carcinomas. The difference in WT1 expression was highly significant between serous and other types of tumors in all sites (p<0.0001, chi-square test), although the level of WT1 expression was significantly different among serous carcinomas arising at different sites (p<0.0001, Kruskal-Wallis test). A significant positive correlation was found between the level of p53 and WT1 expression in all carcinomas combined (r = 0.3935, p<0.0001, Spearman test), but when only serous carcinomas were analyzed, the correlation between p53 and WT1 expression levels did not reach statistical significance. Our results suggest that WT1 expression in epithelial tumors of the female genital tract may be related to cell differentiation and histologic subtypes of carcinomas, rather than to primary site of origin.

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.

Low D2-40 immunoreactivity correlates with lymphatic invasion and nodal metastasis in early-stage squamous cell carcinoma of the uterine cervix

Lymphatic invasion and nodal metastasis are predictors of shorter disease-free and overall survival in carcinoma of the uterine cervix. The monoclonal antibody D2-40, which reacts with the oncofetal membrane antigen M2A, is a new selective marker for lymphatic endothelium, and has been shown to be useful in identifying the presence of lymphatic invasion in various malignant neoplasms, including cervical carcinoma. However, the reactivity of the tumor cells with D2-40 has not yet been evaluated. In this study, we examined the pattern of D2-40 immunoreactivity in a series of 138 invasive squamous cell carcinomas of the uterine cervix. We correlated the presence and extent of D2-40 immunoreactivity in the tumor cells with various clinicopathologic features, the presence of lymphatic invasion, lymph node metastasis and outcome. Diffuse or focal D2-40 immunoreactivity was present in 17 (12%) and 81 (59%) tumors, respectively, while 40 (29%) tumors showed no immunoreactivity. Lymphatic invasion and nodal metastasis were present in 56 and 29% of tumors, respectively. Tumor emboli within lymphatic spaces and metastatic tumor foci in lymph nodes showed no immunoreactivity in 86 and 80% of the cases, respectively. Lymphatic invasion and nodal metastasis were significantly more common in tumors showing low D2-40 immunoreactivity (P<0.0001 and 0.022, respectively). D2-40 immunoreactivity showed no correlation with any other clinicopathologic features examined, including tumor size, grade and FIGO stage. In univariate analysis low D2-40 immunoreactivity was significantly associated with shorter recurrence-free, but not with overall survival. Our studies suggest that D2-40 immunostaining may serve as a marker for increased risk of lymphatic invasion and tumor recurrence in cervical biopsy material. Further study of the biological function of the M2A antigen may shed some light on the interaction of tumor cells with lymphatics.

Diagnostic value of differential expression of CK19, galectin-3, HBME-1, ERK, RET, and p16 in benign and malignant follicular-derived lesions of the thyroid: An immunohistochemical tissue microarray analysis

BackgroundSeveral immunohistochemical markers have been used to aid in the diagnosis of follicular-derived lesions of the thyroid (FDLT). In this study we analyze the diagnostic efficacy of an immunopanel of antibodies to cytokeratin-19 (CK19), galectin-3 (GAL-3), HBME-1, anti-MAP kinase (ERK), ret-oncoprotein (RET), and p16 using a tissue microarray consisting of both benign and malignant FDLT.DesignThe study cohort consisted of 90 cases of FDLT (53 benign, 37 malignant) embedded in a microarray and immunostained with antibodies to CK19, Gal-3, HMBE-1, ERK, RET, and p16. Staining was scored as positive when >25% of the lesional cells showed positive immunostaining.ResultsHMBE-1 was expressed in 70% of malignant and 10% of benign FDLT (p value: <0.0001). (CK19 and GAL-3 were positive in 70% and 73% of malignant lesions, respectively, and 34% of benign FDLT (p value 0.0005 and 0.0015, respectively). ERK was positive in 4% of the benign and 32% of the malignant cases (p value 0.0002). p16 was expressed in 2% and 46% of the benign and malignant lesions, respectively (p value 0.0001). RET positivity was identified in 15% of the benign lesions and 27% of the malignant cases (p value 0.0016).ConclusionsHBME-1, ERK, and p16 were more specific for malignancy, whereas CK19 and GAL-3 stained benign lesions with a higher frequency and were not specific for malignant FDLT. RET-oncoprotein showed poor sensitivity and specificity.

Histopathologic comparison of nephrogenic fibrosing dermopathy and scleromyxedema

Background:  Nephrogenic fibrosing dermopathy (NFD) clinically presents as indurated plaques and papules in patients with renal dysfunction. The differential diagnosis generally includes scleromyxedema (SMX), an idiopathic systemic disorder with cutaneous manifestations, in which patients also develop indurated papules and plaques. The two entities can be extremely difficult to distinguish microscopically. Histopathologic differences with immunophenotypic comparison, to our knowledge, have not been thoroughly studied. We compared these two entities with an emphasis on immunohistochemistry.

Undesirable Cytokeratin Immunoreactivity of Native Nonepithelial Cells in Sentinel Lymph Nodes From Patients With Breast Carcinoma

CONTEXT Use of cytokeratin immunohistochemistry on histologically negative sentinel lymph nodes (SLNs) in patients with breast carcinoma has been shown to be efficient in detecting false-negative nodes. Several recent studies have shown that micrometastases detected by immunohistochemistry constitute an independent predictor of disease-free period and overall survival for breast cancer patients. It has been demonstrated that the fibroblastic type of reticulum cells in lymph nodes also express cytokeratin, and unawareness of such cytokeratin reactivity in SLNs could result in difficulty in the interpretation of the results of immunohistochemistry. OBJECTIVES To study the incidence of undesirable cytokeratin reactivity in reticulum cells and other native nonepithelial cells of SLNs and to compare the immunoreactivity of 3 commonly used cytokeratin antibodies (AE1/AE3, pancytokeratin [pan-CK], and CAM5.2). DESIGN Immunohistochemistry with pan-CK, AE1/AE3, and CAM5.2 antibodies was performed on paraffin sections of SLNs from patients with breast cancer. Correlation of undesirable cytokeratin reactivity with size and metastatic status of the SLNs was also analyzed. PATIENT MATERIAL Paraffin sections of 84 SLNs from 38 consecutive patients with breast cancer in our tertiary-care, teaching hospital. RESULTS Cytokeratin reactivity was found in reticulum cells and plasma cells in 29 (35%) and 9 (10%) of the 84 SLNs, respectively, with pan-CK and CAM5.2 but not with AE1/AE3 (P <.001). The presence of cytokeratin-positive reticulum cells did not correlate with the size and metastatic involvement of the SLNs. CONCLUSIONS The incidence of undesirable keratin reactivity in SLNs from breast cancer patients could be limited by using an AE1/AE3 antibody cocktail. The AE1/AE3 antibody cocktail is a sensitive epithelial marker and appears to be more specific in recognizing epithelial cells in SLNs.

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.

Podoplanin is a highly sensitive and specific marker to distinguish primary skin adnexal carcinomas from adenocarcinomas metastatic to skin

Distinction of primary skin adnexal carcinomas from cutaneous metastasis of adenocarcinomas is challenging. In this study, we evaluated podoplanin immunoreactivity in a series of primary skin adnexal tumors and adenocarcinomas metastatic to skin using a D2-40 antibody. The initial test series were composed of a total of 93 cases including 32 primary skin adnexal carcinomas, 46 benign primary adnexal tumors, and 15 cutaneous metastatic adenocarcinomas. We found that variable D2-40 reactivity was seen in all of the primary cutaneous carcinomas including sebaceous carcinomas (10/10), squamous cell carcinomas (10/10), porocarcinomas (4/4), trichilemmal carcinomas (4/4), skin adnexal carcinomas not otherwise specified (4/4), and in the majority of benign skin adnexal tumors. In contrast, no podoplanin immunoreactivity was seen in any of the 15 (0/15) cutaneous metastases. To confirm the initial findings and to further explore the utility of podoplanin reactivity in the distinction of these tumors, we also examined a test set of 35 unknown cases, including 21 adenocarcinomas metastatic to skin and 14 primary adnexal tumors, in a blinded fashion. In this test set of cases, podoplanin was negative in 22 cases and positive in 13 cases. Of the 22 podoplanin negative cases, 20 were proven to be metastatic adenocarcinoma. Of the 13 D2-40 positive cases, 12 were proven to be primary adnexal tumors. Our results suggest that podoplanin can be a useful tool to distinguish primary skin adnexal carcinomas form adenocarcinomas metastatic to skin with high sensitivity (94.5%) and specificity (97.2%).

Expression of Melan-A and Ki-67 in desmoplastic melanoma and desmoplastic nevi

Background: Desmoplastic melanoma (DMM) is an uncommon melanoma variant with a distinct morphology, including a prominent spindle cell component with fibrosis, as well as a distinct immunohistochemical profile. Histologically, the spindle cell component of DMM can be confused with sclerotic/desmoplastic nevi, nonpigmented blue nevi, scar, and neural tumors. The histological distinction between sclerotic/desmoplastic/blue nevi and DMM using standard light microscopic techniques can be exceedingly subtle. Therefore, we investigated whether immunohistochemical staining for Melan-A and Ki-67 expression can be used to discriminate these lesions, distinguishing between epithelioid and spindle cell compartments of the lesions. Design: Fifty cases of DMM and 13 cases of sclerotic/desmoplastic/blue nevi were identified. Standard immunohistochemical techniques were used with antibodies towards HMB-45, Melan-A (A103), and Ki-67; 43 of 50 DMM cases were available for staining with Melan-A, 42 of 50 for HMB-45, and 31 of 50 cases were stained with Ki-67. All 13 nevi were stained for Melan-A and 8 for Ki-67. Immunoreactivity to Ki-67 antibody was scored as 0 to 5%, 6 to 10%, 11 to 30%, or greater than 30% positive tumor cells. Results: Only 3 of 43 and 3 of 42 of spindle cell compartments of DMMs were positive for Melan-A and HMB-45, respectively. Focal staining of epithelioid cells in the junctional component or superficial dermis was observed in 33% (14/43). In contrast, 100% of the 13 nevi were strongly positive for Melan-A (P < 0.001). Seventeen melanomas (55%) were 0 to 5% positive for Ki-67, five (16%) fell into the 6 to 10% category, three (10%) were between 11 and 30%, and six (19%) were at least focally greater than 30% positive. All 8 nevi (100%) had less than 5% positive cells for Ki-67 (P = 0.02), with only 2 cases having more than 2% positive cells. Conclusion: The sclerotic/desmoplastic and hypopigmented blue nevi were uniformly positive for Melan-A, while the vast majority of DMM were negative in their spindle cell compartments. Melan-A is very useful in distinguishing between DMM and sclerotic nevi. Ki-67 appears to be an inconsistent marker for DMM. However, a high labeling index (over 5%) may be used as a clue in diagnosing DMM.