To summarize our results, we have shown that BLCs and other UTNC of the breast frequently demonstrate a distinctive immunophenotype characterized by loss of expression of the Rb protein and diffuse immunoreactivity for the p16 protein (Rb −/p16+). This immunophenotype is similar to that of the HPV-related SCCs of several specific sites (oropharynx, vulva, and penis), which BLC resemble morphologically. In latter sites, the diffuse immunoreactivity for p16 is thought to reflect the consequences of HPV E7 protein inactivation of Rb. In BLC and UTNC, Rb expression is frequently absent, but we show herein that HPV is not detectable by in situ hybridization. We demonstrate functional consequences of inactivation of the Rb protein in BLC, in that loss of this mediator of G1 cell cycle arrest is associated with the higher proliferation rates seen in BLC and UTNC than high-grade HER-2-positive IDCs. Moreover, Rb−/p16+ BLC have a higher proliferation rate than those BLC lacking this immunophenotype. This difference was magnified when we considered those Rb −/ p16+ BLC with aberrant p53 overexpression, a useful though clearly imperfect surrogate for p53 mutation in these cancers. The latter cancers mimic even more closely HPV-related SCCs at the molecular level, as HPV E6 and E7 protein inactivate p53 and Rb, respectively. Finally, we demonstrate that the Rb−/p16+ phenotype often occurs at the stage of DCIS.
Our data are concordant with most of the prior literature regarding expression of p16, Rb, and p53 in breast carcinomas. Aberrant p53 overexpression and p53
mutations have been correlated with high-grade IDCs (including BLC), so our finding of frequent aberrant p53 overexpression in BLC, UTNC, and HER-2 IDC is not unexpected.8,25,39
Most (but not all) prior studies have shown that loss of Rb protein or overexpression of p16 protein is associated with breast carcinomas having poor prognostic factors.1,9,12,13,20,21,29,30 Importantly, virtually all of these studies occurred before the entity of BLC was established by gene expression profiling in 2001
. Since the recognition of the entity of BLC, the p16 and Rb status of BLC has not been systematically assessed. One study34
indicated that BLC associated with BRCA1
gene inactivation express lower
levels of p16 than carcinomas associated with inactivation of BRCA2
, which were more frequently ER positive. In addition, a review of BLC and BRCA1
-associated tumors also states that BLC are characterized by lower
levels of p16 than the typical breast carcinoma.43
In contrast, Gauthier et al,17
in the setting of a study of DCIS, examined their published GEP data and noticed that BLC show high levels of p16 transcripts and low levels of Rb transcripts. Analyzing a subset of primary tumors representing each of the 5 molecular subtypes of breast cancer, they showed that p16 IHC labeling correlated well with mRNA levels in BLC, though they did not statistically compare their p16 results in BLC with those of the other different molecular subtypes of breast cancer. They hypothesized that “loss of functional p16/Rb signaling may play a defining role in the biology of this tumor subtype.” Our results correlate perfectly with their hypothesis.
We suspect that the Rb−/p16+ phenotype is biologically distinctive, as supported by several observations. First, we have shown that this phenotype is almost always restricted to TNC (BLC and UTNC). Second, we demonstrated that this phenotype correlates with higher proliferation rates among BLC. Such correlation makes biologic sense, in that inactivation of Rb results in inactivation of the G1
-S cell cycle checkpoint, and promotes unblocked entry into the cell cycle. Indeed, another type of carcinoma in which Rb is consistently inactivated, small cell neuroendocrine carcinoma, is characterized by strikingly high Ki-67 indices compared with other carcinomas.3
It is now generally recognized that BLC defined by either gene expression or IHC are heterogeneous, and one challenge is to identify a more homogenous subset to which targeted therapy can be applied.5,22,35,36,42,44
We suspect that the Rb−/p16+ immunophenotype may define a more homogenous subgroup of BLC. Some cases, which otherwise considered UTNC but which share this phenotype may also belong to this group. Although our Ki-67 proliferation index results support this assertion, correlation with other clinical parameters, such as response to chemotherapy and outcome, and association with BRCA1
gene inactivation, would be required to substantiate this possibility.
We note that the correlation of the Rb −/p16+ and p53 overexpression immunophenotypes with higher Ki-67 indices was present and significant only within the BLC group, and not within the UTNC group. Several nonmutually exclusive explanations are apparent. First, the BLC group (21 cases) in this study was larger than the UTNC group (12 cases), making statistical comparisons more robust in the BLC group. Second, and perhaps more importantly, the BLC group is likely more homogeneous than the UTNC group. UTNC lacks a specific defining positive IHC marker (such as CK 5/6 or EGFR expression in BLC) and therefore is a diagnosis of exclusion. The UTNC category therefore likely is a heterogeneous “mixed-bag” of cases, which includes carcinomas related to and unrelated to BLC. Third, cell type may influence the effects of the Rb−/p16+/p53 overexpression phenotype. The high molecular weight CK 5/6 immunoreactivity seen in BLC may reflect early stages of squamous differentiation, making these cancers immunophenotypically closer to HPV-related squamous carcinomas (also CK 5/6 positive) that the BLC resemble. One can postulate that the effects of Rb and p53 inactivation are most distinctive and robust in cells showing squamous differentiation, resulting in higher Ki-67 indices.
Our observation that the Rb−/p16+ immunophenotype occurs at the DCIS stage of BLC is also concordant with the literature. Indeed, DCIS with a basal-like immunophenotype (ER −, PR −, HER-2 −, CK 5/6+, or EGFR+) has recently been described.7,10,28
It is known that BLC frequently have only a minimal DCIS component, and that pure DCIS with a basal immunophenotype (10% of DCIS) is less common than basal-like invasive carcinoma (20% of IDC). Therefore, it has been postulated that DCIS with the basal-like immunophenotype rapidly evolves toward IDC. Perhaps the frequent (4/6 cases, 67%) inactivation of Rb protein within these DCIS lesions, by promoting unchecked proliferation and further accumulation of genetic alterations, contributes to this rapid evolution to IDC. The data of Gauthier et al17
support this hypothesis. These authors found that DCIS demonstrating a high p16/high Ki-67 phenotype, which reflects an abrogated response to cellular stress (and likely reflects Rb inactivation), recur more frequently than DCIS demonstrating a high p16/ low Ki-67 index profile, which reflects a physiologic response to stress.
Finally, our study also highlights the often underappreciated power of morphology in elucidating the molecular pathogenesis of cancers. We chose to examine the Rb/p16 pathway and p53 in BLC of the breast because of their distinct morphologic similarities to HPV-related poorly differentiated SCCs of the oropharynx, penis, and vulva. On the basis of similar morphology, we hypothesized that similar genes may be inactivated in these neoplasms, albeit by different mechanisms (genomic or epigenetic alterations in breast cancer, HPV in SCCs). As surgical pathologists, it is gratifying that our study, triggered by analysis of morphology (“traditional pathology”), has recently17
been supported by analysis of GEP data (“molecular pathology”). Our findings also suggest that one may define a family of BLCs of certain anatomic sites in which distinctive basal-like morphology correlates with inactivation of Rb protein and diffuse p16 expression. Poorly differentiated SCCs of the oropharynx, penis, and vulva are established members of this family, and are linked by the presence of high-risk HPV, which inactivates Rb protein. Parenthetically, this correlation of morphology with HPV status does not hold in the cervix or anus, where virtually all SCCs are HPV positive and diffusely express p16 protein. Our study suggests that BLC of the breast is an additional member of this family, representing a non–HPV-related carcinoma in which basal-like morphology predicts inactivation of Rb protein and diffuse p16 expression. We note with interest that basaloid lung carcinomas have recently been demonstrated to more frequently harbor inactivation of Rb protein than other non-small cell carcinomas of the lung, suggesting that this carcinoma may represent another member of this family.6
We also note that aberrant diffuse p16 and p53 protein expression by IHC now has shown to be characteristic of 2 carcinomas, BLC of the breast (this study) and high-grade serous carcinoma of the ovary,33
which both are associated with germline BRCA1
gene mutations. This observation suggests that inactivation of BRCA1
may mediate carcinogenesis in concert with alterations in the Rb/p16 and p53 pathways.