Since there are no systematic clinicopathological studies on CK7 and GATA3 negative breast tumors and limited studies characterizing the prognostic utility of GATA3 expression in breast cancer, the current study provided detailed information that is lacking in the literature.
CK7 staining is not routinely used in the primary breast cancer diagnosis except in rare cases when clinical and pathological evidence prompt the need to exclude a secondary cancer source. CK7 negative breast tumors may be diagnostically challenging in metastatic tumors and cancers of unknown origin. A comprehensive characterization of CK7 negative breast cancer may be scientifically and diagnostically useful.
In the current study, we found that as high as 13% of Grade 3 breast cancers and 30% of metaplastic cancers were CK7 negative. CK7 expression was not significantly associated with age, stage, receptor status and molecular subtype. Among the 11 histologic types included in the study, loss of CK7 expression was only seen in ductal or metaplastic tumors.
Based on Grade 3 breast cancers, survival analysis showed CK7 expression had no impact on overall outcomes (Figure 2). Given the fact that Grade 1 and 2 tumors are more likely to retain CK7 expression and associated with better prognoses, survival analysis of breast tumors of expected grade distribution (about 34% of Grade 3 instead of 55% in this series, see below) will likely show significant association of CK7 expression loss with poor patient outcome. Indeed, if Grade 1 and Grade 2 tumors were included in the current series, a significant association were seen between worse overall survival and CK7 expression loss patients (P=0.0084) (Supplemental Figure 1A). Loss of CK7 in the tumor could indicate initiation of a different differentiation pathway, presence of epithelial to mesenchymal transition, dedifferentiation, or enhanced tumor “stemness”. More studies should be carried out for further investigations.
As opposed to CK7 negative tumors, GATA3 negative breast cancer has gained more attention from both diagnostic pathologists and cancer biologists. The percentage of GATA3 negative breast cancers vary from study to study, ranging from 0 to 46% in ER positive tumors and from 17% to 97% in estrogen negative cancers . In the current study, the negative rates were 1.7% in ER positive and 48.6% in ER negative tumors. Among the published studies in which the same antibody and the same positivity cut-off were used as in this study, GATA3 negative rates ranged from 17% - 56%, consistent with our finding of 17.1%. Reported GATA3 negative rates in metaplastic cancers ranged from 44% to 82%, which was consistent with the 70% (7 out of 10) in the current study. In addition we found that none of the lobular tumors and those with mixed lobular and ductal features was GATA3 negative, which was compatible with most published studies .
GATA3 is required for ER-dependent cellular processes and GATA3 and ER participate in positive feedback loops, each stimulating the expression of the other . Forced expression of GATA3 in mouse mammary cancer model showed a protective effect with improved prognosis . Mutations of GATA3 in breast cancer are relatively common. Based on analyses performed through cBioPortal [23-24] on the largest publicly available breast cancer dataset, Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) , 11.5% (250/2173) of breast tumors harbored somatic mutations of GATA3. Out of the 250 mutations, 193 (77.2%) were truncating, 52 (20.8%) were missense and 5 (2%) were inframe mutations. GATA3 mutations were more frequently seen in mixed invasive and mucinous cancers (P = 0.0124) and more likely to be estrogen receptor positive (P < 0.0001), Her2 negative (P = 0.0002), with lower grades (P < 0.0001), and with lower pT stages (Grade 1 and 2) (P = 0.0254) (Supplemental Table 1). GATA3 mutated tumors were associated with better patient outcomes (P = 0.0018) (Supplemented Figure 2). Mutant GATA3 proteins mainly interfere with the DNA binding but the transactivation domains are largely intact . GATA3 antibody we used (clone L50-823) was raised against peptide segment between transactivation and DNA-binding domains and presumably captures both wild-type and mutant protein versions. In the current study, GATA3 was positive in half of the ER negative tumors, consisting of 28% of all Grade 3 tumors (Table 3). Further investigations should be focused on the mechanisms how GATA3 maintains its expression without activating ER.
Published data regarding GATA3 as a prognostic marker are conflicting. Loss of GATA3 expression has been associated with unfavorable clinical outcome and worse survival [15, 26-27]. However, no association with outcome has been observed in other studies . In one study, GATA3 expression was found to be associated with favorable outcome in all the breast tumors in the study, while the association was lost when only ER positive cancers were analyzed . GATA3 expression is closely associated with ER and PR expression and loss of GATA3 expression is postulated to be similar to loss of ER expression prognostically. However, our study did not see similar findings in 196 Grade 3 breast cancers. Multiple factors could contribute. First, the case number was not large enough to bear sufficient statistical power. Second, ER and GATA3 are closely related in the hormonal pathway, but they could still have distinct functions as to tumor progression. Third, in the current practice, vast majority of ER positive tumors have been treated hormonally. The treatment may impact the prognosis. Four, our survival study only included Grade 3 tumors and the adding more Grade 1 and Grade 2 tumors is likely to include more GATA3 positive tumors with better prognosis and thus with a significant prognostic difference. Indeed, repeating analysis by adding Grade 1 and Grade 2 tumors in the current series showed significant worse survival of GATA3 negative cancer patients (P=0.0063) (Supplemental Figure 1B)
Our study is limited by a high proportion of Grade 3 breast cancer (55%) while it was estimated that only a third of breast cancer are Grade 3 . A significant amount of additional work would be involved if all the concurrent Grade 1 and Grade 2 tumors were included in the study. Results that would be affected by disproportional Grade 1 and Grade 2 cases include survival analyses and association studies when markers were not evenly distributed among grades, such as ER, PR, Her2 and pTNM status. Therefore, these analyses were performed only in Grade 3 tumors except association studies that directly involved the grade.
Another possible limitation is about the proportions of each special type cancer in this study. Our study included 10 metaplastic (0.28% of all tumors), 6 cribriform (1.6%), 4 micropapillary (1.1%), 5 mucinous (1.4%), 3 tubular (0.8%) and 1 apocrine cancer (0.3%). The cribriform and tubular tumors were slightly overrepresented and mucinous and apocrine tumors were slightly underrepresented, comparing to known percentages for each special type: 0.2% to 5% for metaplastic, 0.3-0.8% for cribriform, 0.9-2% for micropapillary, 2% for mucinous, 2% for tubular and 4% for apocrine cancers . However, the differences do not significantly affect the main conclusions in this study.
The mainly purpose of the study is to understand the pathobiology of the CK7 negative and GATA3 negative breast cancer. If we want to apply the findings in this study to facilitate the diagnosis of metastatic breast cancer, we need to know the CK7 and GATA3 expression in matched pairs of primary and metastasis. It certainly requires a separate study. A small portion of CK7 and/or GATA3 negative cases in our series later developed tumor metastasis; however, CK7 and GATA3 immunostains were not used to form the diagnoses of these metastases. Some of the metastases occurred before GATA3 immunostain became available. However, clinical information, microscopic morphology and other mammary gland markers were often enough to establish the diagnoses. In a pilot study, we collected 12 pairs of primary tumor and liver metastasis and 12 pairs of primary tumor and bone metastasis. There was no GATA3 expression change in primaries and liver metastases: all 10 GATA3 positive primaries retained GATA3 expression in their paired liver metastases and GATA3 expression in 2 negative primaries remained negative in the paired liver metastases. Some of the GATA3 expression was lost in bone metastasis: GATA3 expression in 8 positive primaries was only retained in 4 paired bone metastases and GATA3 expression in 4 negative primaries remained negative in the paired bone metastases. The exact reason why GATA3 expression was lost in some bone metastases is unclear. It could be true loss of expression (such as post-chemotherapy) but it is more likely to be related with tissue processing such as decalcification or sampling error due to the small size of bone biopsy. The same type of sampling error could happen to core biopsy or fine needle aspiration (FNA). Further studies are needed to explore GATA3 and CK7’s role in diagnosing metastases of special cancer types, such as metaplastic, mucinous, and apocrine tumors.
In the current series, we identified 8 cases of CK7 and GATA3 double negative breast cancer. All eight cases have been considered as primary breast cancer and pathological diagnoses were made without CK7 and/or GATA3 stains. It could have been challenging if CK7 and GATA3 immunostains were performed at the time of diagnosis. During the current study, we had retrospective chart review for these cases including any follow-up history and no non-breast primary tumor was identified. In our practice, GATA3 immunostain is routinely performed on any triple-negative cancers that do not have an in situ component. Ultimately, only history and follow-up can definitively confirm the breast origin in some cases.
In summary, this is the first study that comprehensively characterized clinicopathological and prognostic features of breast cancers missing either of two canonical markers, CK7 and GATA3. There was 13% CK7 negative and 28% GATA3 negative tumors in Grade 3 breast cancer. CK7 negative tumors showed no association with most clinicopathological features and loss of CK7 expression was likely associated with worse outcome in patients with Grade 3 tumors. Loss of GATA3 expression was associated with negative ER status and GATA3 negative tumors were enriched in the basal-like molecular subgroup. About half of the ER negative breast cancers retained GATA3 expression and no overall survival was seen associated with GATA3 negative Grade 3 tumors.