Introduction
Triple-negative breast cancer (TNBC), which accounts for about 15% of breast cancer cases, is defined as estrogen receptor (ER)-negative, progesterone receptor (PgR)-negative, and human epidermal receptor 2 (HER2)-negative disease. TNBC generally has a high-grade and aggressive phenotype with early distant metastasis after primary treatment, and the prognosis of patients with TNBC is often poor. Because of the lack of effective therapeutic targets, conventional chemotherapy is still considered the standard treatment for TNBC [
1].
Neoadjuvant chemotherapy (NAC) is a standard therapeutic strategy for TNBC. Despite their overall poor survival, a subset of TNBC patients responds well to the standard chemotherapy. The prognosis of the patients in whom pathological complete response (pCR) is achieved is quite favorable, similar to that of other breast cancer subtypes. However, TNBC patients with residual disease after NAC have significantly worse survival than those who achieve pCR [
2,
3]. Therefore, it is meaningful to investigate the factors influencing the efficacy of NAC and the prognosis after NAC, especially for patients who fail to achieve pCR.
Recent studies have shown that TNBC is a highly heterogeneous disease and is classified into several subgroups on the basis of different approaches, classical pathology, mRNA expression profile, and DNA sequencing, including analyses of copy number variations and structural rearrangements and other molecular methods [
4‐
7]. Phenotypes related to the epithelial–mesenchymal transition (EMT) and cancer stemness are also characteristic of TNBC [
8]. There are several predictive factors that influence the sensitivity to NAC in TNBC, including high nuclear grade, high proliferation rate, high immune cell infiltration, alteration of DNA repair-related genes, EMT-related gene expression, and cancer stemness [
9‐
12]. Among the molecular subtypes of TNBC, the basal-like 1(BL1) subtype is associated with higher pCR rate than others [
5,
6,
13].
Among DNA repair-related genes, low expression of BRCA1 or “BRCAness” features is correlated with taxane resistance [
14‐
16] and a poor prognosis, specifically TNBC [
17,
18]. Regarding EMT, we previously reported that vimentin, the major EMT-related factors, is poor prognostic factors for TNBC [
19,
20]. However, the relationship between the expression of vimentin and the pathological response to NAC is not clear.
The adhesion molecule CD44 is expressed in cancer stem-like cells (CSCs). CD44 variant 9 (CD44v9), a splicing variant of CD44, has emerged as a novel marker of cancer stemness in a variety of solid tumors [
21‐
23]. CSCs with high expression of CD44v have an enhanced capacity for GSH synthesis and defense against reactive oxygen species, resulting in resistance to various therapeutic stresses [
23]. CD44v9 has been studied in various malignant tumors, including gastric cancers and head and neck squamous cell carcinomas (HNSCC) and urothelial cancer [
21,
22,
24]. The high expression of CD44v9 has been shown to be associated with resistance to pre-surgical treatment in HNSCC [
21]. However, there have been no reports regarding the clinical significance and the association with chemosensitivity of CD44v9 in breast cancer.
In the present study, we first would like to investigate the relationships between the expression of CD44v9 in pre-NAC tumor samples and the pathological response to NAC in TNBC. In addition, we evaluated the expression of BRCA1, vimentin, and basal phenotype, which are considered to be related to the chemosensitivity or prognosis of TNBC. The factors to be related to the prognosis of the patients, who fail to achieve pCR, were also examined.
Discussion
In this study, we investigated the clinical significance of the expressions of CD44v9, a protein related to the cancer stemness in TNBC patients who received NAC. We showed that high CD44v9 expression was significantly correlated with poor prognosis, especially in patients who failed to achieve a pCR. The NG of the residual tumor cells was significantly associated with shorter DMFS, and high CD44v9 expression prior to NAC was significantly associated with the high NG in the residual tumors. To our knowledge, this is the first report of the relationship between the CD44v9 expression and the pathological response to NAC and the prognosis in TNBC.
The major prognostic factors after NAC are pathological response (pCR or non-pCR), the residual cancer burden (RCB), and tumor proliferation, such as mitotic counts and the Ki67 index, in the residual tumor cells [
25,
27‐
29]. The significantly poor prognosis of the patients in whom pCR was not achieved by NAC is an urgent issue to be overcome, especially for TNBC. One possible strategy for resolving this issue is the addition of further treatment after NAC and surgery. The recent Create-X study revealed the effectiveness of the adjuvant capecitabine for non-pCR patients. The disease free and overall survival were significantly longer in the capecitabine group than in the control group, especially in TNBC patients [
30].
Not only the efficacy of NAC but also the prognosis after NAC is extremely different among the TNBC subtypes [
5,
6,
31]. The basal-like 1 (BL1) subtype has been reported to be highly sensitive to NAC in TNBC. In the present study, the basal phenotype determined by IHC was significantly associated with high pCR rate, although it is difficult to distinguish between BL1 and BL2 by IHC alone.
Based on the findings of previous studies regarding CD44v9, we expected the pre-NAC expression of CD44v9 to be related to the subsequent efficacy of NAC [
21‐
23]. However, the CD44v9 expression in pre-NAC tumors was not related to the pathological response on the whole. Nevertheless, high CD44v9 expression was significantly correlated with poor prognosis, especially in the patients in whom pCR had not been achieved. Multivariate analysis could not reveal that high CD44v9 in pre-NAC samples was independent prognostic factor in the non-pCR patients. Intriguingly, high CD44v9 expression in pre-NAC tumors was significantly correlated with high NG in the residual tumor cells after NAC. This seems to have contributed to the poor prognosis.
In contrast, low CD44v9 expression before NAC was correlated with better prognosis, even in the non-pCR cases. As mentioned above, the Create-X study showed that the addition of capecitabine after surgery improved the prognosis of non-pCR HER2-negative breast cancer after NAC. Thus, high CD44v9 expression may be useful marker suggesting the need for additional treatment for patients with non-pCR tumors after NAC. Sulfasalazine (SSZ), which is used for rheumatoid arthritis (RA), inhibits glutamate–cysteine transport and has been reported to suppress the CD44v-dependent tumor growth and increase the sensitivity to cytotoxic drugs in urogenital cancer [
32]. A clinical study to confirm the utility of SSZ has already been started for several malignancies, including gastric cancer [
33]. Reducing the CD44v9 expression or CD44v-dependent tumor growth is expected to improve the sensitivity to chemotherapy and the prognosis. In this study, there was no difference in DMFS and the NG of the residual tumor cells among the patients with low and high CD44v9 expression in the residual tumor cells. The reason of this phenomenon is not clear. The mechanism of the regulation of the expression of CD44v9 is not fully elucidated. The manipulation by the chemotherapy might modulate the expression of CD44v9 irrespective of the sensitivity to NAC.
The loss of BRCA1 expression was observed in about 20% of patients and was correlated with a poor prognosis in this study. These findings are consistent with those of previous reports [
17,
18], although no association was noted between the resistance to taxanes and the loss of BRCA1 expression in this study. The EMT phenotype is considered to be related to chemoresistance. However, in the present study, there is no statistical difference in the sensitivity to NAC between vimentin-negative or positive tumors. This is probably because EMT is not defined by the expression of vimentin alone and there are many factors that determine the chemosensitivity other than EMT phenotype. Thus, in the present study, it was difficult to show the relationship between EMT evaluated based on the expression of vimentin and chemoresistance.
Several limitations associated with the present study warrant mention. The number of patients included in this study was very small, and the analysis was retrospective. To confirm the findings obtained in this study, analyses in a larger cohort are necessary.
In conclusion, the CD44v9 expression in pre-NAC tumors predicts poor prognosis after NAC in TNBC. The combined treatment of NAC and therapy targeting CD44v9 may be useful in future therapeutic regimens.