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Purpose: Breast cancers are often classified on the presence/absence of hormone receptors, and growth factor oncogenes (estrogen receptor, progesterone receptor, HER2). Triple-negative breast cancers, negative for these markers, do not benefit from targeted therapy. We compared clinicopathologic parameters and immunohistochemical markers of prognostic and/or predictive significance, and outcome between African American and Caucasian triple-negative breast cancer patients., Methods: Invasive triple-negative breast cancers from African American (n=94) and Caucasian (n=68) patients were studied. Clinicopathologic features (age, tumor size, grade, lymph node status, angiolymphatic invasion, visceral metastases) and survival (overall and progression free) were compared. Marker expression (CK5, CK7, CK8, CK14, CK18, CK19, vimentin, CD44, c-Kit, epidermal growth factor receptor, p-cadherin, p53, p63, topoisomerase II, androgen receptor, Ki-67) was assessed in tissue microarrays., Results: Significant differences between African American and Caucasian women were observed for mean age and tumor size. African Americans had a trend toward greater lymph node involvement than Caucasians. The following markers were found in significantly different frequencies between the 2 groups: CK5, CK8, CK19, c-Kit, androgen receptor, and high Ki-67. African Americans show shorter overall and progression-free survival. Other clinicopathologic parameters, markers, and outcome were present at similar frequencies., Discussion: African American triple-negative breast cancers were more aggressive, occurring at a younger age, being larger, with higher proliferation, patients more frequently dying of disease, and with a trend toward positive lymph node status. Heterogeneity of marker expression suggests variation in the genetics of breast carcinomas in different races.

Prognosis of breast cancer patients has been determined traditionally by lymph node status, tumor size, and histologic grade. In recent years the Oncotype DX recurrence score (RS) assay has emerged as an expensive adjunct prognostic tool. Markers of proliferation play a large role in determination of RS, and we have shown previously that immunohistochemical expression of proliferation markers Ki-67 and phosphohistone H3 (PPH3) correlates with RS. Our current goal is comparison of the hematoxylin and eosin (H&E) mitotic score, defined by the Nottingham grading system, with anti-PPH3 mitotic figure labeling assessed by both visual and automated image analysis and correlation of mitotic score results with RS. Estrogen receptor-positive breast carcinomas from 137 patients with Oncotype DX testing were selected. A representative H&E-stained tumor section was evaluated. Mitoses were counted per 10 high-power fields and tumors graded using the Nottingham criteria by 1 pathologist in accordance with College of American Pathologists-recommended mitotic count cutoffs for a field diameter of 0.55 mm. An additional section was immunostained with PPH3 antibody. PPH3 mitotic scores were determined visually and by automated imaging system. Statistical analysis was performed using univariate tests and Spearman coefficient. There was a statistically significant positive correlation among the 3 methods of mitotic score assessment. Specifically, correlation of tumor grades obtained using visual and automated methods of assessment of mitotic activity with PPH3 stain was the strongest and most statistically significant (weighted κ value 0.84, P<0.001; Spearman coefficient 0.89, P<0.001). There was a statistically significant positive correlation between H&E mitosis score and RS (P<0.001, Spearman coefficient 0.30) and between visual PPH3 mitotic score and RS (P<0.001, Spearman coefficient 0.28). In conclusion, mitotic score by any of the 3 methods studied may be useful in assessing tumor grade, proliferation, and prognosis.

The Oncotype DX Recurrence Score (RS) is often used in lymph node-negative, estrogen receptor-positive breast cancer to refine prognosis and direct therapy. Its utility is limited by its cost, proprietary nature, and turnaround time. Markers of proliferation factor heavily into determination of RS. Our aim is to correlate expression of proliferation markers Ki-67 and phosphohistone H3 (PPH3) with RS and other prognostic indicators. Estrogen receptor-positive invasive breast carcinomas from 133 patients with Oncotype DX testing were selected. Representative tumor sections were stained with MIB1, a monoclonal antibody that reacts against Ki-67, and antibody to PPH3. Nuclear staining was quantitated through an automated imaging system. The percentage of positive cells was scored as low (<10%), intermediate (10% to 20%), or high (>20%) for Ki-67, and low (<2%), intermediate (2% to 5%), or high (>5%) for PPH3. Expression of both markers was compared with RS and clinicopathologic parameters including grade, tumor size, lymph node metastasis, and angiolymphatic invasion. Ki-67 and PPH3 expression were both significantly associated with RS (P=0.02 and P=0.027, respectively) and grade (P<0.001 and P=0.002, respectively). Ki-67 expression correlated with angiolymphatic invasion (P=0.01) but not with tumor size or lymph node metastasis; PPH3 expression showed no association with any of these 3 parameters. Expression of proliferation markers Ki-67 and PPH3 by immunohistochemistry is significantly correlated with RS and tumor grade. This observation suggests that immunohistochemical assessment of markers of proliferation may provide useful prognostic information, at lower cost than RS testing.

Although podoplanin expression in breast myoepithelial (ME) cells has been reported to be readily distinguishable from that of lymphatic endothelium (LE), we recently encountered a case in which attenuated expression led to an incorrect interpretation of lymphatic invasion. This prompted us to further analyze the pattern and intensity of podoplanin expression in LE and in ME cells surrounding both non-neoplastic and neoplastic breast epithelium in 42 cases of breast carcinoma. In addition, cases with presumed lymphatic invasion (tumor cells within podoplanin-positive lymphatic-like structures) were further characterized on the basis of histologic review and results of additional endothelial (CD31 and/or CD34) and ME (p63+/-calponin) immunostains. Normal LE always displayed a strong (3+) linear podoplanin expression pattern, whereas ME cells surrounding non-neoplastic breast epithelium had a granular, branching membranous staining pattern that was either moderate (2+) or strong (3+) in 13 (34%) and 25 (66%) foci, respectively. ME cells surrounding ductal carcinoma in situ (DCIS) displayed weaker (1+ to 2+) podoplanin expression with 26 (72%) foci showing only a residual thin/discontinuous pattern of expression, whereas the other 10 (28%) foci showed a pattern similar to that around non-neoplastic epithelium. Further evaluation of 10 foci of presumed lymphatic invasion confirmed the presence of lymphatic invasion in 6 (60%) cases, whereas 4 displayed surrounding ME cells and were thus interpreted as DCIS. One case, in which both DCIS and lymphatic invasion were unequivocally present, displayed a few nests of tumor cells surrounded by both podoplanin-positive and p63-positive cells in different areas of the periphery and could not be readily classified. These findings represent an important caveat in the diagnosis of lymphatic invasion and, accordingly, one should always interpret results of podoplanin staining in the context of the histologic appearance and/or consider evaluation of additional endothelial or ME immunostains, especially when the characteristically strong linear pattern of expression of LE is not evident.

One of the problems with immunohistochemistry is that storage of unstained slides can have a detectable negative effect on the intensity of staining for various antigens. This has not been well studied for the p63 antigen. A time-course study was performed to evaluate the effect of storage on the intensity of p63 immunohistochemical staining. Slides from various breast lesions/foci were stained with a p63 antibody on day 0 and after storage at room temperature for 2, 4, and 8 weeks. A p63 intensity score, ranging from 0 to 300, was calculated using the intensity (0, 1, 2, or 3+) and percentage of peripheral myoepithelial cell staining (0% to 100%). There was a progressive decline in the p63 intensity score with time with the differences between day 0 and 2 weeks and 2 and 4 weeks being statistically significant (P=0.037 and 0.001, respectively), whereas that between 4 and 8 weeks was not (P=0.075). These findings confirm a time-dependent decline in p63 immunostaining intensity evident as early as with 2 weeks of storage and still present after 8 weeks. This should be considered in the evaluation of p63 immunostains performed on stored unstained sections, both in diagnostic surgical pathology and in translational research studies.