Mammaglobin vs GCDFP-15: An Immunohistologic Validation Survey for Sensitivity and Specificity

Rohit Bhargava, MD1; Sushil Beriwal, MD2; David J. Dabbs, MD1


Am J Clin Pathol. 2007;127(1):103-113. 

In This Article

Materials and Methods

We stained 29 matched primary and metastatic (within lymph node) invasive breast carcinomas (ductal, 14; lobular, 14; mixed, 1) with a mammaglobin antibody cocktail and GCDFP-15.

The tissue microarray (TMA) was constructed from 64 randomly selected, well-characterized, in-house breast carcinomas. Of these 64 cases, 52 were ductal, 9 were lobular, 1 was metaplastic, and 2 were mixed ductal and lobular carcinoma. Three 0.6-mm tissue cores were obtained from 1 to 2 tissue blocks on each case. Four-micrometer TMA sections were stained with the mammaglobin antibody cocktail and GCDFP-15.

TMAs (US Biomax, Rockville, MD) for 12 organ systems were examined. These included 12 TMA slides prepared from formalin-fixed, paraffin-embedded normal and tumor tissues without using tape-transfer technology. Each core's diameter measured 2 mm. The slides were stained with the mammaglobin antibody cocktail.

Slide details are as follows: (1) melanoma array with 80 primary and metastatic melanomas from different sites; (2) ovarian array (80 cores): papillary serous carcinomas, 38; mucinous and clear cell carcinomas, 1 each; and benign ovary samples, 40; (3) endometrial array (63 cores): endometrioid adenocarcinoma, 59; and invasive mole, proliferative endometrium, secretory endometrium, and benign smooth muscle, 1 each; (4) uterine cervical array (80 cores): squamous cell carcinoma, 41; and benign cervical tissue samples, 39; (5) lung array (100 cores): squamous cell carcinoma, 27; adenocarcinoma, 18; and normal lung tissue samples, 55; (6) stomach array (80 cores): adenocarcinoma, 27; undifferentiated carcinoma and signet-ring cell carcinoma, 4 each; mucinous carcinoma and carcinoid tumor, 2 each; and benign stomach tissue samples, 41; (7) colon array (80 cores): adenocarcinoma, 38; signet-ring cell carcinoma, 1; and benign colonic tissue samples, 41; (8) kidney array (80 cores): clear cell carcinoma, 37; undifferentiated carcinoma, 2; B-cell lymphoma, 1; and benign kidney cortex samples, 40; (9) bladder array (80 cores): urothelial carcinoma, 37; adenocarcinoma, 2; squamous cell carcinoma, 1; and benign bladder tissue samples, 40; (10) salivary gland tumor array: adenocarcinoma not otherwise specified and mucoepidermoid carcinoma, 3 each; adenoid cystic carcinoma, basal cell adenocarcinoma, epithelial-myoepithelial carcinoma, pleomorphic adenoma, and undifferentiated carcinoma, 2 each; and acinic cell carcinoma, adenosquamous carcinoma, malignant fibrous histiocytoma, malignant myoepithelioma, mucinous adenocarcinoma, mucus adenocarcinoma, myoepithelioma, sarcomatoid carcinoma, and Warthin tumor, 1 each; (11) skin tumor array: basal cell carcinomas, 13; dermatofibrosarcoma protuberans, 9; malignant schwannoma, 3; squamous cell carcinoma, 14; fibrosarcoma, leiomyosarcoma, and sebaceous adenocarcinoma, 1 each; and sweat gland carcinoma, 10; and (12) pancreas tissue array: pancreatic adenocarcinoma, 67.

Owing to the absence of endocervical adenocarcinomas in the tissue arrays, 20 cases of invasive and adenocarcinoma in situ (AIS) were also included in the study.

Four-micrometer-thick formalin-fixed, paraffin-embedded sections were immunostained on the Benchmark XT automated stainer (Ventana Medical Systems, Tucson, AZ). The protocol consisted of a pretreatment with CC1, pH 8.0 (Ventana), followed by incubation with mammaglobin mouse (clone 304-1A5) and rabbit (clone 31A5) monoclonal cocktail (Zeta, Sierra Madre, CA) at a 1:25 dilution. In our initial validation, the mammaglobin antibody cocktail demonstrated "crisper" staining without increasing the background noise and, therefore, was preferred over a single antibody for this study. A similar pretreatment protocol was followed for the GCDFP-15 antibody (clone 23A3, Cell Marque, Hot Springs, AR). Antigen-antibody complexes were detected with an IVIEW-DAB (diaminobenzidine) detection system (Ventana). Owing to the presence of melanin in the melanoma array slide, detection was performed via Ventana's "Enhanced V-Red Detection," which is an alkaline phosphatase that uses naphthol and fast red chromogen.

Immunohistochemical analysis for estrogen receptor was performed using the 6F11 antibody and IVIEW detection on the Benchmark XT (Ventana). Immunohistochemical analysis for the progesterone receptor was performed using the 1A6 antibody (Ventana) and IVIEW detection on the Benchmark XT. Any staining was considered as positive staining for estrogen and progesterone receptors. HER-2/neu protein was analyzed and scored using the CB11 antibody (Ventana) and basic DAB detection on the Benchmark XT. Scoring was performed similar to that in the DAKO HercepTest (DAKO, Carpinteria, CA) guidelines. FISH for the HER-2/neu gene was performed in 2+ cases. HER-2 positivity was defined as 3+ overexpression or amplification by FISH.

The randomly selected breast carcinomas represented on TMA were graded according to the Nottingham grading criteria. Because there was an equal mix of ductal and lobular carcinomas in the whole tissue section group, only nuclear grading was performed. Nuclear grading of tumor nuclei was performed based on nuclear pleomorphism and graded as nuclear grade 1, 2, or 3.

Statistical analysis was performed using the Arcus Quickstat software program (Longman Software Publishing, Cambridge, England). The breast whole tissue section group was analyzed separately from the breast TMA group for reasons detailed in the results section. Differences in percentages were analyzed by using the χ2 test. Differences in means between different groups were analyzed by using the paired t test.


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