SOX10 immunohistochemistry in sweat ductal/glandular neoplasms.

BACKGROUND: SOX10 is a newer Schwannian and melanocytic marker that has generated great interest for its relative sensitivity and specificity in the diagnosis of neural crest-derived tumors. Previous studies with SOX10 have shown positive immunohistochemical expression in cutaneous eccrine glands and negative expression in eccrine ducts, apocrine glands and hair follicles. Thus, we hypothesized that some sweat gland tumors of presumed eccrine origin would be positive for SOX10, whereas apocrine-derived sweat gland tumors would not. METHODS: A mouse monoclonal anti-SOX10 (clone BC34: Biocare Medical; Concord, California) immunohistochemical antibody was performed on various sweat gland tumors and basal cell carcinoma. RESULTS: SOX10 showed positivity in spiradenomas (13/13), cylindromas (9/10), hidradenoma papilliferum (10/10), syringocystadenoma papilliferum (8/10), apocrine adenomas (8/10), and negativity in poromas (0/12), syringomas (0/10), and basal cell carcinomas (0/13). There was mixed staining of hidradenomas (6/15). CONCLUSIONS: SOX10 immunohistochemistry may be of utility in distinguishing some of the varying adnexal tumors from each other, and from basal cell carcinoma (BCC), but given the staining of both apocrine and eccrine tumors, does not seem to provide information as to their origins as either eccrine or apocrine tumors.

Prostate cancer marker panel with single cell sensitivity in urine.

BACKGROUND: Over one million men undergo prostate biopsies annually in the United States, a majority of whom due to elevated serum PSA. More than half of the biopsies turn out to be negative for prostate cancer (CaP). The limitations of both the PSA test and the biopsy procedure have led to the development for more precise CaP detection assays in urine (e.g., PCA3, TMPRSS2-ERG) or blood (e.g., PHI, 4K). Here, we describe the development and evaluation of the Urine CaP Marker Panel (UCMP) assay for sensitive and reproducible detection of CaP cells in post-digital rectal examination (post-DRE) urine. METHODS: The cellular content of the post-DRE urine was captured on a translucent filter membrane, which is placed on Cytoclear slides for direct evaluation by microscopy and immuno-cytochemistry (ICC). Cells captured on the membrane were assayed for PSA and Prostein expression to identify prostate epithelial cells, and for ERG and AMACR to identify prostate tumor cells. Immunostained cells were analyzed for quantitative and qualitative features and correlated with biopsy positive and negative status for malignancy. RESULTS: The assay was optimized for single cell capture sensitivity and downstream evaluations by spiking a known number of cells from established CaP cell lines, LNCaP and VCaP, into pre-cleared control urine. The cells captured from the post-DRE urine of subjects, obtained prior to biopsy procedure, were co-stained for ERG, AMACR (CaP specific), and Prostein or PSA (prostate epithelium specific) rendering a whole cell based analysis and characterization. A feasibility cohort of 63 post-DRE urine specimens was assessed. Comparison of the UCMP results with blinded biopsy results showed an assay sensitivity of 64% (16 of 25) and a specificity of 68.8% (22 of 32) for CaP detection by biopsy. CONCLUSIONS: This pilot study assessing a minimally invasive CaP detection assay with single cell sensitivity cell-capture and characterization from the post-DRE urine holds promise for further development of this novel assay platform. Prostate 75: 969-975, 2015. © 2015 The Authors. The Prostate, published by Wiley Periodicals, Inc.

Expression of uroplakin II and GATA-3 in bladder cancer mimickers: caveats in the use of a limited panel to determine cell of origin in bladder lesions.

Antibodies targeting uroplakin II (UPII) are highly specific for urothelial cells and are frequently used to determine if a primary bladder lesion or a metastatic lesion originates from the urothelium. However, to date, no studies have tested the expression of UPII in histological mimickers of bladder cancer that are nonurothelial in origin. Given the potential risk of misdiagnosis, immunohistochemical markers are often used to better characterize these lesions. In the present study, we analyzed the immunohistochemical expression of UPII in a set of urothelial carcinoma mimickers that included conventional nephrogenic adenoma (n = 8), papillary nephrogenic adenoma (n = 6), endometriosis/endosalpingiosis (n = 5), inflammatory myofibroblastic tumor (n = 4), ectopic prostate tissue (n = 2), and malakoplakia (n = 2). We also examined the expression of GATA-3, another commonly used immunohistochemical marker in bladder cancer diagnosis, in the same lesions. Weak immunoreactivity for UPII was identified in 6 of 27 mimickers (22%), and GATA-3 was expressed in 16 of 27 mimickers (59%). Strong immunoreactivity for UPII appeared to be a specific marker for urothelial cell of origin, although weak staining was seen in a significant proportion of mimickers. GATA-3 immunostaining was present in a greater number and broader spectrum of mimickers; however, only one case of papillary nephrogenic adenoma showed dual positivity for UPII and GATA-3. These findings support the immunohistochemical panel-based approach in the diagnosis of bladder lesions, especially if nonurothelial bladder cancer mimickers are in the differential diagnosis. Additional larger studies would be of value to expand on these findings.

Serous Carcinoma Mimicking Primary Urothelial Carcinoma on Clinical Evaluation and Pathology: A Potential Diagnostic Pitfall.

CONTEXT: - Serous carcinoma of the gynecologic tract often involves the external bladder wall and can occasionally mimic primary urothelial carcinoma of the bladder. OBJECTIVE: - To define the spectrum of morphologic and immunohistochemical features that characterize serous carcinoma involving the bladder wall and its distinction from urothelial carcinoma. DESIGN: - We reviewed all cases of serous carcinoma secondarily involving the bladder wall from the University of California San Diego and Polytechnic Institute for histopathologic and immunohistochemical features. RESULTS: - We identified 20 cases of Müllerian high-grade serous carcinoma involving the bladder wall. Five cases were clinical mimics of urothelial carcinoma, including 2 cases that presented as a large, transmural, primary bladder mass without precedent gynecologic history in women younger than 60 years, and 3 cases presumed to be new bladder carcinoma occurring distant to a serous carcinoma diagnosis. A subset of cases were morphologic mimics of urothelial carcinoma, which showed nested growth patterns (2 of 20; 10%), squamouslike foci (2 of 20; 10%), spindled/sarcomatoid growth (2 of 20; 10%), basaloid morphology (3 of 20; 15%), and syncytial growth patterns (1 of 20; 5%). Immunohistochemical stains in 17 cases showed immunoreactivity for CK7 (17 of 17; 100%), WT1 (17 of 17; 100%), uroplakin (UP) II (1 of 17; 6%), p63 (2 of 17; 12%), GATA3 (2 of 17; 12%), and PAX8 (17 of 17; 100%). CONCLUSIONS: - A subset of serous carcinomas involving the bladder wall can mimic urothelial carcinoma. Awareness of this mimicker and use of an immunohistochemical panel that includes CK7, WT1, UPII, PAX8, p63, and GATA3 can be helpful in confirming the diagnosis.

Correlation Between Programmed Death Receptor-1 Expression in Tumor-Infiltrating Lymphocytes and Programmed Death Ligand-1 Expression in Non-Small Cell Lung Carcinoma.

CONTEXT.­: The interaction between programmed death ligand-1 (PD-L1) and programmed death receptor-1 (PD-1) on activated T cells sends an inhibitory signal that dampens the immune response. Tumors can express PD-L1 and evade the immune system. In advanced non-small cell lung carcinoma, expression of PD-1 in tumor-infiltrating lymphocytes (TILs) correlates with PD-L1 expression in tumor cells (TCs). However, this relationship has not been thoroughly explored in early disease. OBJECTIVE.­: To investigate the correlation of PD-1 and PD-L1 in non-small cell lung carcinoma tumor samples, with emphasis on stage I disease. DESIGN.­: Whole tissue sections from non-small cell lung carcinoma tumors were retrospectively evaluated by immunohistochemistry for PD-1 and PD-L1 expression. The scoring was based on the percentage of cells positive for PD-1 in TILs and PD-L1 in TCs and tumor-infiltrating immune cells (ICs). RESULTS.­: Expression of PD-1 in TILs was observed in 147 of 161 non-small cell lung carcinoma cases (91%). The majority of cases negative for PD-1 also lacked PD-L1 in TCs. The 68 cases with highest PD-1 expression in TILs included 33 (49%) with expression of PD-L1 in TCs and ICs. Strong correlations were observed in patients with elevated PD-1 expression in TILs and PD-L1 in TCs ( P = .01) and ICs ( P = .003). Expression of PD-1 also correlated with increased PD-L1 in TCs and ICs when the 2 were grouped together ( P < .001). Finally, stage I patients with negative PD-1 and PD-L1 expression showed trends toward increased disease-specific survival. CONCLUSIONS.­: Expression of PD-1 in TILs correlates with PD-L1 expression in both TCs and ICs. Furthermore, negative expression of PD-1 and PD-L1 suggest trends toward disease-specific survival, even in early disease stages.

CDH17 Is a More Sensitive Marker for Gastric Adenocarcinoma Than CK20 and CDX2.

CONTEXT: -CDH17, which is expressed in the intestinal epithelium, is a novel oncogene involved in tumor invasion and metastasis. A panel consisting of cytokeratin (CK) 7, CD20, and CDX2 antibodies is typically used to diagnose gastrointestinal adenocarcinomas. However, studies have shown that CDH17 is a highly specific marker for gastrointestinal adenocarcinoma and may be important in clinical diagnosis. OBJECTIVE: -To evaluate the sensitivity and specificity of CDH17, CK20, and CDX2 antibodies in neoplastic tissues, with emphasis on colon, stomach, and esophageal gastrointestinal lineage. DESIGN: -Immunohistochemistry was performed with CDH17, CK20, and CDX2 antibodies on formalin-fixed, paraffin-embedded tissue microarrays from normal (n = 26) and neoplastic (n = 884) tissues. RESULTS: -CDH17 immunostaining was positive in 97.3% (145 of 149) of colon adenocarcinomas, whereas CK20 and CDX2 stained positively in 88.6% (132 of 149) and 93.3% (139 of 149), respectively. In metastatic colon cancers, CDH17, CK20, and CDX2 positive staining was observed in 90.6% (29 of 32), 59.4% (19 of 32), and 81.3% (26 of 32) of cases, respectively. In stomach adenocarcinomas, CDH17 positively stained 64.0% (112 of 175) of tissues, compared to CK20 and CDX2, where staining was observed in only 24.6% (43 of 175) and 46.9% (82 of 175), respectively. In esophageal adenocarcinomas, CDH17, CK20, and CDX2 stained 38.7% (12 of 31), 25.8% (8 of 31), and 29% (9 of 31) of specimens, respectively. Low or no expression was observed in other neoplastic tissues, except pancreatic cancers, where CDH17 displayed higher expression than CK20 and CDX2. CONCLUSIONS: -CDH17 is a specific and more sensitive marker in the gastrointestinal tract than CK20 and CDX2. CDH17 may be especially valuable when gastrointestinal tumors are suspected in cancers of unknown primary.

mASH1 is Highly Specific for Neuroendocrine Carcinomas: An Immunohistochemical Evaluation on Normal and Various Neoplastic Tissues.

CONTEXT: -High-grade neuroendocrine carcinomas and carcinoids can arise in different sites such as lung, gastrointestinal tract, prostate, and skin. Classic neuroendocrine markers such as CD56, synaptophysin, and chromogranin cannot distinguish carcinoids from high-grade neuroendocrine carcinomas. Recently, mouse monoclonal mASH1 has been shown to help discriminate carcinoids from high-grade neuroendocrine carcinomas in various neoplastic sites. To date, there have been no comprehensive immunohistochemistry studies with mASH1 on nonneuroendocrine neoplasms. OBJECTIVE: -To evaluate the specificity and sensitivity of mASH1 in various normal and neoplastic tissues, including lung cancers. DESIGN: -Formalin-fixed, paraffin-embedded tissue microarrays consisting of normal tissues and various neoplastic tissues were immunohistochemically evaluated with mASH1. RESULTS: -In normal tissues (n = 30), mASH1 (nuclear staining) was sparsely expressed in the molecular cell layer, white matter, and granular cell layer of cerebellum; C cells in thyroid; and epithelial cells in thymus. In lung cancers, mASH1 stained 1.1% (1 of 93) of adenocarcinomas, 0.9% (1 of 111) of squamous cell carcinomas, 0% (0 of 30) of large cell carcinomas, 66.7% (6 of 9) of large cell neuroendocrine carcinomas, and 82.5% (94 of 114) of small cell carcinomas. In various other neoplastic tissues (n = 1114), mASH1 was expressed in thyroid medullary carcinomas, thymic carcinomas, and brain cancers; mASH1 was also expressed in a very low percentage of breast carcinomas, ovarian cancers, and pancreatic neuroendocrine tumors. All typical carcinoids of various sites were negative (0 of 11), however, in lung atypical carcinoids, mASH1 was expressed in 42.9% (9 of 21). CONCLUSIONS: -Although not organ specific, mASH1 is highly specific for high-grade neuroendocrine carcinomas versus carcinoids and other nonneuroendocrine neoplasms.

Folate Receptor α Expression Level Correlates With Histologic Grade in Lung Adenocarcinoma.

CONTEXT: -Folate receptor α (FRA) is a glycosylphosphatidylinositol-anchored high-affinity folate receptor that localizes to the apical surface of epithelia when it is expressed in normal tissue. Unlike normal tissues, FRA may localize to the basolateral side in tumors. These features make FRA an attractive drug target, and several FRA-targeted drugs have been developed and are in phases of clinical testing. Folate receptor α protein expression shows intertumoral variability that may correlate with response to therapy and to clinicopathologic parameters. Using immunohistochemistry, a recent study of breast carcinomas found FRA protein expression was associated with triple-negative status and high histologic grade in breast cancer. Although a prior study of lung adenocarcinomas found the expression level of the gene encoding FRA, FOLR1, was significantly increased in low-histologic-grade tumors compared to high-histologic-grade tumors, the relationship between FRA protein expression and histologic grade has not been reported for lung adenocarcinomas. OBJECTIVE: -To investigate the relationship between FRA protein expression level and clinicopathologic parameters in lung adenocarcinomas, including histologic grade, by performing immunohistochemistry for FRA on a cohort of non-small cell lung carcinomas. DESIGN: -High-density tissue microarrays constructed from 188 non-small cell lung carcinomas and used in prior studies were immunostained with FRA-specific antibody clone 26B3. Folate receptor α membranous staining intensity was given a semiquantitative score from 0 to 3+ for triplicate cores of tumor and averaged for each tumor. An average semiquantitative score from 0 to 1.4 was considered low expression, and an average semiquantitative score greater than 1.4 was considered high expression. RESULTS: -The majority (60 of 78; 77%) of lung adenocarcinomas and a minority (4 of 41; 10%) of lung squamous cell carcinomas were positive for FRA. Folate receptor α expression in lung adenocarcinomas compared with squamous cell carcinomas was statistically different (P < .001, χ(2) test). In lung adenocarcinomas, FRA expression level correlated with histologic grade (P = .005, χ(2) test for trend), but no other clinicopathologic parameter. The majority (23 of 27; 85%) of grade 1 adenocarcinomas had high FRA protein expression, whereas approximately half of grade 2 (10 of 19; 53%) and grade 3 (12 of 25; 48%) adenocarcinomas had high FRA protein expression. Out of adenocarcinomas with lepidic growth pattern, 16 of 20 (80%) showed high FRA protein expression. Out of adenocarcinomas with solid growth pattern, 2 of 6 (33%) showed high FRA protein expression. In lung adenocarcinomas, FRA expression level did not correlate with thyroid transcription factor 1, napsin A, or survival. CONCLUSIONS: -Folate receptor α protein was expressed in the majority of lung adenocarcinomas and a minority of lung squamous cell carcinomas. Folate receptor α protein expression correlated with histologic grade for lung adenocarcinomas, and the greatest difference was observed between grade 1 and grade 3. Our results indicate that poorly differentiated adenocarcinomas or focuses of poor differentiation in a heterogeneous tumor may lack FRA protein expression and be more likely to be resistant to FRA-targeting drugs.

A combination of p40, GATA-3 and uroplakin II shows utility in the diagnosis and prognosis of muscle-invasive urothelial carcinoma.

Recently developed antibodies against uroplakin II and ΔNp63 (p40) show utility in diagnosing primary bladder urothelial carcinoma, although application in metastatic bladder cancer and patient outcomes has been less well defined. We evaluated these antibodies by immunostain intensity and H-score, in conjunction with GATA-3 immunoreactivity, in 89 patients with muscle-invasive urothelial carcinoma and 35 paired metastasis. The maintenance of immunoreactivity in metastatic lesions and the association to disease recurrence and survival was assessed. Bladder urothelial carcinoma showed immunoreactivity for GATA-3 in 99% (88/89), p40 in 87% (77/89) and uroplakin II in 80% (71/89) of cases, with a positive correlation between GATA-3 and uroplakin II H-score (0.44; p < 0.0001). All lesions were positive for at least one marker, reinforcing the use of these markers as a panel. In 35 patients with paired lymph node metastasis, uroplakin II and GATA-3 were retained in 90% and 75% of patients, respectively, suggesting these markers may have relatively high sensitivity in diagnosing metastatic urothelial carcinoma. High intensity p40 immunostain (3+), however, was significantly associated with reduced patient survival (p = 0.03). These results suggest that whereas GATA-3 and uroplakin II may be most useful in the diagnosis of urothelial carcinoma metastasis, p40 may be additionally suited as a prognostic marker.

A newly developed mouse monoclonal SOX10 antibody is a highly sensitive and specific marker for malignant melanoma, including spindle cell and desmoplastic melanomas.

CONTEXT: Recent immunohistochemical studies have demonstrated Sry-related HMG-Box gene 10 (SOX10) expression in malignant melanomas, malignant peripheral nerve sheath tumors, a subset of breast carcinomas, and gliomas. SOX10 has shown important clinical utility in its ability to detect desmoplastic and spindle cell melanomas. To date, most publications have employed a research use-only goat polyclonal SOX10 antibody for immunohistochemical staining. OBJECTIVE: To describe the development of a new mouse monoclonal SOX10 antibody (BC34) and evaluate its immunohistochemical staining profile in a wide range of normal and neoplastic tissues, with an emphasis on melanoma. DESIGN: SOX10 antibody was optimized for staining using a polymer detection system and visualization with diaminobenzidine. RESULTS: In normal tissues, SOX10 was expressed in skin melanocytes and eccrine cells, breast myoepithelial and lobular epithelial cells, salivary gland myoepithelial cells, peripheral nerve Schwann cells, and central nervous system glial cells. SOX10 was expressed in 238 of 257 melanomas (92.6%), including 50 of 51 of both spindle cell and desmoplastic melanomas (98%). SOX10 was expressed in 100% of nevi (20 of 20) and schwannomas (28 of 28). In other neoplasms, SOX10 was expressed in 18 of 109 invasive ductal breast carcinomas (16.5%). All other carcinomas were negative for SOX10. SOX10 was identified in 25 of 52 central nervous system neoplasms, primarily in astrocytomas (22 of 41; 53.7%), and in 4 of 99 various sarcomas examined (4.0%). CONCLUSIONS: The newly developed mouse monoclonal SOX10 antibody BC34 is highly sensitive and specific for malignant melanoma, including desmoplastic and spindle cell variants, and appears highly suitable for clinical use.

Uroplakin II (UPII), GATA3, and p40 are Highly Sensitive Markers for the Differential Diagnosis of Invasive Urothelial Carcinoma.

Distinguishing between invasive urothelial carcinoma from other genitourinary lesions such as prostatic and renal carcinomas can be difficult, and may require highly sensitive immunohistochemical markers. GATA-binding protein 3 (GATA3) has been reported in a high percentage of urothelial and breast carcinomas. Mouse monoclonal uroplakin II (UPII) and p40 antibodies have recently been developed and demonstrated high specificity in urothelial carcinoma. This study evaluated the immunohistochemical staining sensitivities of UPII, GATA3, p40, and p63 in the detection of invasive urothelial carcinoma. UPII, GATA3, and p40 were further tested for specificity in lung, breast, colon, kidney, and prostate cancers. In all invasive urothelial carcinoma cases, UPII, GATA3, p40, and p63 exhibited sensitivities of 77.7%, 83.5%, 85.4%, and 80.6%, respectively. The combination of UPII, GATA3, and p40 antibodies stained 94.2% (97/103) of all invasive urothelial carcinoma cases, including 92.2% (71/77) of grade 2-3 urothelial carcinomas. In addition, GATA3 and UPII showed negative staining in lung squamous cell carcinomas and p40 showed negative staining in breast infiltrating ductal carcinomas. The combination of UPII, GATA3, and p40 showed negative staining in lung adenocarcinoma, colon adenocarcinoma, and renal carcinomas. In conclusion, UPII, GATA3, and p40, when used in combination, are highly sensitive in the differential diagnosis of invasive urothelial carcinoma.

Use of p63/P504S monoclonal antibody cocktail in immunohistochemical staining of prostate tissue.

Diagnosis of prostate needle biopsies can be challenging, particularly when the atypical areas of interest are very small. The utility of immunostains for high-molecular-weight cytokeratin (34betaE12) to highlight prostatic basal cells in these cases is well established, and recent reports also document the utility of immunostains for p63 (a marker that stains the nuclei of prostate basal cells) for this purpose. Several investigators have demonstrated that immunostaining for P504S, a cytoplasmic protein that is overexpressed in a high percentage of prostate cancers and in many cases of high-grade prostatic intraepithelial neoplasia (PIN), can also be of use in the diagnosis of prostate biopsies. Because of the cytoplasmic localization of P504S and nuclear localization of p63, the authors hypothesized that a cocktail of these two antibodies might allow simultaneous demonstration of P504S and p63 using a single immunostain. In this report the authors describe the successful use of a cocktail of p63/P504S for immunohistochemical staining of prostate tissue. Two different staining approaches were investigated, with essentially identical results. This cocktail localizes P504S in the cytoplasm of prostate carcinoma cells and high-grade PIN and demonstrates the nuclei of prostatic basal cells, providing information on both the status of P504S and the presence or absence of basal cells with a single immunostain. This cocktail can be of great utility in the examination of diagnostically challenging prostate specimens.

An immunohistochemical analysis of a newly developed, mouse monoclonal p40 (BC28) antibody in lung, bladder, skin, breast, prostate, and head and neck cancers.

CONTEXT: Immunohistochemistry is important to the pathologist for accurate diagnosis of lung cancer. In recent studies, a rabbit polyclonal p40 (RPp40) antibody demonstrated equivalent staining versus anti-p63 in lung squamous cell carcinoma, and superior specificity because it stains a lesser percentage of lung adenocarcinoma. OBJECTIVES: To develop an anti-p40 mouse monoclonal antibody (MMp40) for immunohistochemistry, and to evaluate its sensitivity and specificity in normal and neoplastic tissues, with emphasis on lung cancer. DESIGN: The MMp40 (BC28) antibody was developed and tested for specificity and sensitivity on normal (n = 34) and neoplastic tissues (n = 493). Staining of MMp40, p63, and RPp40 were directly compared in lung cancers, and MMp40 was evaluated in breast, bladder, skin, prostate, and head and neck cancers. Benign prostate glands and prostatic intraepithelial neoplasia were also tested in a direct comparison of MMp40 versus p63. RESULTS: The MMp40 provided equivalent staining versus RPp40 and p63 in lung squamous cell carcinoma, but stained a lesser percentage of lung adenocarcinoma than p63 did. The MMp40 staining was observed in a greater percentage of urothelial carcinomas, squamous and basal cell skin cancers, and head and neck cancers of squamous cell origin. No breast-infiltrating ductal carcinomas or prostatic adenocarcinomas were stained. The MMp40 expression in basal cells of prostate glands and prostatic intraepithelial neoplasia were almost identical to those of p63. CONCLUSION: The MMp40 (BC28) monoclonal antibody is a high-quality screening antibody for determining squamous cell carcinoma in lung cancers, skin cancers of squamous or basal cell origin, squamous cell head and neck cancers, and urothelial carcinomas.

A new rabbit monoclonal E-cadherin antibody [EP700Y] shows higher sensitivity than mouse monoclonal E-cadherin [HECD-1] antibody in breast ductal carcinomas and does not stain breast lobular carcinomas.

Immunohistochemical studies have shown E-cadherin to be expressed in breast carcinomas showing a ductal histology, with a corresponding loss of expression in tumors with a lobular histology. As a result, mouse monoclonal anti-E-cadherin [HECD-1] has been used by pathologists to differentiate between ductal and lobular carcinomas, with currently published sensitivity and specificity rates of approximately 90%. Rabbit monoclonal antibodies may combine the best properties of both mouse monoclonal antibodies and rabbit antisera. Therefore, this study compares the staining sensitivity and specificity of a new rabbit monoclonal E-cadherin and the standard mouse monoclonal E-cadherin [HECD-1] in breast ductal carcinomas, and evaluates a cocktail of rabbit monoclonal E-cadherin and p120 catenin in the discrimination of ductal from lobular carcinomas. The rabbit E-cadherin showed sharper staining and increased sensitivity (80/81, 99%) than the mouse E-cadherin (75/81, 93%). The rabbit E-cadherin achieved a score of 3+ in 85.2% (69/81) of cases as compared with a 3+ in only 21.0% (17/81) of cases stained with mouse E-cadherin. All lobular carcinomas (n=37) were confirmed by the absence of E-cadherin and the diffuse cytoplasmic expression of p120 catenin. Although both the single mouse E-cadherin and dual stain can differentiate ductal from lobular lesions, the dual stain is helpful in challenging cases because of its bright pink p120 catenin and dark brown rabbit E-cadherin staining. The highly sensitive rabbit E-cadherin antibody is the preferred antibody for evaluating ductal carcinomas and for distinguishing ductal versus lobular lesions, and the dual stain was superior to the single E-cadherin stain.

A newly developed uroplakin II antibody with increased sensitivity in urothelial carcinoma of the bladder.

CONTEXT: Uroplakin II is a 15-kDa protein component of the urothelial plaques that enhance the permeability barrier and strength of the urothelium. Studies have shown uroplakin II messenger RNA to be expressed in bladder cancer tissues and peripheral blood of patients with urothelial carcinoma. Little is known about the protein expression of uroplakin II in urothelial carcinoma, possibly because of the absence of a commercially available uroplakin II antibody. Pathologists have used the uroplakin III antibody (AU1) to identify tumors of urothelial origin; however, the use of AU1 is limited because of its poor sensitivity. OBJECTIVES: To evaluate a newly developed mouse monoclonal uroplakin II antibody (BC21) in urothelial carcinoma and to compare it with previously developed mouse monoclonal uroplakin III (BC17 and AU1). DESIGN: Uroplakin II and III antibodies were optimized for staining using a horseradish peroxidase-polymer detection system and were visualized with 3,3'-diaminobenzidine. RESULTS: BC21, BC17, and AU1 demonstrated sensitivities in urothelial carcinoma of the bladder of 79% (44 of 56), 55% (31 of 56) (P = .002), and 34% (19 of 56) (P < .001), respectively. Subsequently, the increased staining sensitivity and intensity of BC21, compared with BC17, was validated in a larger study (134 of 174; 77% and 94 of 174; 54%, respectively) (P < .001). BC21 was found to be highly specific when evaluated in various normal and neoplastic tissues, including prostatic and renal carcinomas. CONCLUSIONS: The mouse monoclonal uroplakin II antibody (BC21) demonstrated superior sensitivity and specificity in urothelial carcinoma, compared with uroplakin III (BC17 and AU1), suggesting its advantages in the differential diagnosis of urothelial carcinoma and in the detection of tumors of unknown origin.

Folate receptor α associated with triple-negative breast cancer and poor prognosis.

CONTEXT: Folate receptor α (FRA) has been shown to be selectively expressed in several types of human cancer, including breast cancer. Currently, several FRA target therapies are under intensive study. OBJECTIVE: To investigate the expression pattern of FRA in a large cohort of patients with breast cancer and analyze its relationship with different clinicopathologic features, with expression of several key biomarkers, and with clinical outcome. DESIGN: Four hundred forty-seven cases of infiltrating ductal carcinoma diagnosed between 1997 and 2008 at the University of Rochester Medical Center were identified and reviewed, and 25 blocks of tissue microassays were constructed. The association between expression of FRA and clinicopathologic features; expression of estrogen receptor (ER), progesterone receptor (PR), HER2/neu, and Ki-67; and clinical outcome of these tumors were evaluated. RESULTS: The expression of FRA was significantly associated with tumors with high histologic grade, higher nodal stages, ER/PR negativity, and high proliferative activity (Ki-67 ≥ 15%), and was independent of HER2/neu overexpression. In all, 74% of ER/PR-negative and 80% of triple-negative breast cancers expressed FRA. The expression of FRA was significantly associated with a worse disease-free survival. CONCLUSIONS: Our data demonstrate that a significant subgroup of ER/PR-negative and triple-negative breast cancers express FRA, and its expression is associated with worse clinical outcome.

PAX8 mouse monoclonal antibody [BC12] recognizes a restricted epitope and is highly sensitive in renal cell and ovarian cancers but does not cross-react with b cells and tumors of pancreatic origin.

PAX8 is expressed in a high percentage of renal cell and ovarian cancers; however, the current existing anti-PAX8 rabbit polyclonal (P) antibodies also recognize B cells, pancreatic cancers, carcinoids, and some soft tissue tumors. Cross-reactivity with B cells can be especially troublesome in lymph nodes when identifying tumors of unknown origin. A new mouse monoclonal (M) anti-PAX8 antibody (Clone BC12) has been developed that recognizes PAX8 expression in a high percentage of renal cell and ovarian carcinomas, whereas exhibiting no staining of B cells. PAX8 (M) was tested for specificity and sensitivity in over 1300 cases of both normal and neoplastic tissues. PAX8 (M) demonstrated superior staining sensitivity in clear cell and papillary renal cell carcinomas (88.8% vs. 84.4%) and in serous and endometrioid ovarian carcinomas (87% vs. 83%), when compared with PAX8 (P). PAX8 (M) also stained a high percentage of endometrial and thyroid cancers, 67.5% and 60.7%, respectively. PAX8 (M) demonstrated low sensitivity in cervical and bladder cancers, 2.5% and 1.4%, respectively. All other cancers including lung, breast, prostate, stomach, liver, soft tissue, pancreas, testis, brain, colon, melanoma, lymphoma, adrenal, pituitary, and rectal were negative. In normal tissue, PAX8 (P) stained lymph nodes, pancreas, and neuroendocrine cells of stomach and colon. In contrast, PAX8 (M) was negative in each of these tissues. These results demonstrate that mouse monoclonal PAX8 [BC12] stains nuclei exclusively and performs well in formalin-fixed paraffin-embedded tissues. PAX8 (M) is a highly sensitive marker for thyroid, renal, and ovarian cancers. Importantly, PAX8 (M) does not stain B cells and does not seem to recognize epitopes of pancreatic origin and neuroendocrine cells in stomach and colon; thus, providing superior specificity and making PAX8 [BC12] an excellent marker for confirming primary tumor site and for differential diagnosis.

Tissue-preserving antibody cocktails to differentiate primary squamous cell carcinoma, adenocarcinoma, and small cell carcinoma of lung.

CONTEXT: With the availability of cell type-specific therapies, differentiating primary lung squamous cell carcinomas (SCCs) and adenocarcinomas (ACAs) has become important. The limitations of small sample size and the need to conserve tissue for additional molecular studies necessitate the use of sensitive and specific marker panels on a single slide. OBJECTIVE: To distinguish SCC from ACA and small cell carcinoma (SmCC) of lung using 2 novel tissue-conserving cocktails. DESIGN: We compared two antibody cocktails, desmoglein 3 + cytokeratin 5/napsin A and p40/thyroid transcription factor 1 (Biocare Medical, Concord, California) in diagnosing SCC and ACA of the lung on tissue microarray, cytology, and surgical specimens. Both lung and nonlung tissue were evaluated on an 1150-core tissue microarray that contained 200 lung cancers. A microarray of 35 SmCCs and 5 small cell SCCs was also evaluated. RESULTS: A cocktail of desmoglein 3 + cytokeratin 5/napsin A provided diagnostic accuracy in lung cancers with a sensitivity and specificity of 100% in SCCs and a sensitivity of 86% and a specificity of 100% in ACAs. A p40/thyroid transcription factor 1 cocktail showed p40 to have a specificity of 92% and a sensitivity of 93% in SCCs, whereas thyroid transcription factor 1 had a specificity of 100% and a sensitivity of 77% in ACAs. Cell blocks of fine-needle aspiration cytology compared with corresponding surgical (n = 20) specimens displayed similar findings. The p40 was useful in differentiating bladder from prostate carcinoma with 88% sensitivity. Isolated carcinomas from nonlung tissues were desmoglein 3 + cytokeratin 5 positive. Napsin A was positive in 22% of renal tumors as previously observed. Both cocktails were excellent in differentiating SmCCs and small cell SCCs because none of the SmCCs stained with p40. CONCLUSIONS: Both antibody cocktails are excellent in differentiating primary lung ACA from SCC, as well as excluding SmCC and ACAs from all other sites on small specimens. A cocktail of desmoglein 3 + cytokeratin 5/napsin A is slightly superior compared with p40/thyroid transcription factor 1 cocktail.

Interobserver agreement and assay reproducibility of folate receptor α expression in lung adenocarcinoma: a prognostic marker and potential therapeutic target.

CONTEXT: Lung cancer is the leading cause of cancer deaths in the United States and globally. Folate-targeted drugs are among the promising new targeted therapies for lung cancer, provided predictive biomarkers can be identified for optimal patient selection. OBJECTIVE: To evaluate the interobserver agreement and reproducibility of an immunohistochemistry assay for folate receptor α as a potential predictive marker for folate-targeted therapies. DESIGN: Immunohistochemistry using anti-folate receptor α antibody 26B3 was performed on formalin-fixed, paraffin-embedded tissues. The M-score, a semiquantitative measure of staining intensity and proportion of tumor cells staining, was determined for each specimen. Interobserver agreement was assessed using lung adenocarcinoma specimens stained at a single site and evaluated by 3 independent pathologists. Interinstrument reproducibility assessed 20 specimens stained by 3 different automated stainers. Interlaboratory agreement was determined on 5 specimens, repeatedly stained on each of 5 days, at 3 different study sites. RESULTS: Folate receptor α expression was identified in 39 of 54 cases of lung adenocarcinoma (72%) and 4 of 37 cases of lung squamous cell carcinoma (11%). Agreement among 3 pathologists was found in 24 of 26 cases (92%). Interinstrument reproducibility was observed in 19 of 20 cases (95%). Agreement among 3 laboratories was found for 49 of 50 specimens (98%). CONCLUSIONS: Immunostaining of folate receptor α in lung adenocarcinomas is reproducible across staining platforms and among laboratories. Agreement among pathologists is achieved using a semiquantitative scoring method. An accurate and convenient method for determining folate receptor α expression offers a potentially invaluable tool for selecting patients for folate-targeted therapies.

A 6-antibody panel for the classification of lung adenocarcinoma versus squamous cell carcinoma.

Non-small cell lung cancer can be classified into several histologic subtypes, most commonly lung adenocarcinoma (LADC) or squamous cell carcinoma (SqCC). With the introduction of targeted therapies that can result in dramatically different outcomes based on subtype, the importance of accurate classification has been amplified. Six antibodies (Napsin A, Desmoglein 3, TTF-1, CK5, p63, and tripartite motif-containing 29) were selected for evaluation on cases of LADC of lung SqCC. Guided by the sensitivities and specificities determined for individual antibodies, a protocol was developed using a sequential series of 2-antibody cocktails that resulted in the classification of 93% of cases with 100% specificity. Importantly, the initial step in this method, a napsin A+Desmoglein 3 antibody cocktail classified >85% of cases, resulting in <15% of cases requiring further evaluation beyond a single test. Two new antibodies specifically developed and optimized for the diagnosis of LADC and lung SqCC, a rabbit polyclonal Napsin A and a mouse monoclonal Desmoglein 3 [BC11], were the key elements of the antibody panel. Most importantly, the described protocol uses routine interpretation methods and an uncomplicated algorithm for classification. Given the increased difficulty of diagnosing poorly differentiated tumors, the ability of this 6-antibody panel to classify 96% and 87% of moderately and poorly differentiated cases, respectively, is of particular value, especially when limited tissue for molecular testing is an issue.