Neoadjuvant chemotherapy is now considered as one of the standard options in the treatment of locally advanced and also for primary operable breast cancers [5–8]. Morphological classification systems have been established to assess the pathological response and prognosis after systemic primary therapy. These consider pathological features such as the percentage of residual tumour cells, the presence or absence of in situ component with or without considering nodal status [3, 5–8]. Reliable biomarkers to assess response to primary systemic therapy are not yet available. Connexins may function as gap junctions or hemichannels in the cell membranes or through intra-cytoplasmic protein interactions and play fundamental roles in cell homeostasis including cell cycle control [11, 16]. In this study we analysed the expression of four connexin isotypes Cx26, Cx32, Cx43 and Cx46, and Ki67 for cell proliferation prior to and after neoadjuvant chemotherapy in breast cancer in correlation with clinicopathological parameters, overall survival and pathological response based on current classification systems.
Our main results suggest that increased Cx46 positivity either pre- or post-chemotherapy and reduced Cx26 expression post-chemotherapy may separate prognostically more favourable subgroups within the intermediate categories of the classifications including Miller-Payne G2-3, Sataloff TB, EWGBSP TR2b and CPS-EG 4 (Figure 5B). In addition, pre-chemotherapy only Cx46 expression appeared to correlate with overall survival. Others have also observed that biomarkers such as PR, HER2, grade, or tumour size did not necessarily correlate with prognosis either before or after chemotherapy . In agreement with those who showed elevated Cx26 expression to contribute to carcinogenesis in pancreatic and prostate carcinoma [32, 33] we also found a significantly reduced Cx26 expression after chemotherapy in association with better overall survival in breast cancer (p=0.011). Moreover, decreased Cx26 expression after chemotherapy could be more promising for prognosis than classical features such as HER2 expression, grade, Ki67 index according to our results. Interestingly, loss of Cx26 expression has also been implicated in reduced survival in primary gastric and colorectal carcinomas, suggesting an organ specific biological significance of Cx26 [34, 35].
The role of connexins in breast cancer development, progression and metastatic growth has been the subject of only few previous studies all focusing on primary breast cancers [21–23, 36]. The transitional loss of Cx43 has been reported where re-expression of Cx43 might sensitize breast cancer cell lines for chemotherapeutic agents . Primary breast cancers exhibiting elevated expression of Cx26, Cx32 and Cx43 have been found more often in node positive than node-negative breast cancers [21, 22]. Although, Cx46 has been implicated in protecting MCF-7 breast cancer cells from hypoxia, apart from our study, there has been no data revealed on the prognostic role of Cx46 in breast cancer . Increased Cx26 and Cx43 levels have also been associated with breast cancer metastasis . However, the prognostic and predictive role of connexins in relation to neoadjuvant chemotherapy has not been investigated before.
Gap junctions are thought to inhibit cell cycle though most findings relate to Cx43 . Forced Cx43 expression can block G1/S phase transition or delay G2/M transition through increasing p21waf1 and reducing Cdk2 levels . Also, Cx43 expression and gap junction coupling can be reduced through phosphorylation by Cdk1/cyclinB complex at G2/M transition . Therefore, Cx expression may dynamically change during cell cycle. In breast cancer cell lines low levels of Cx26 at G1/S phase, increasing levels during late S/G2 phase transition and down-regulation in M phase was observed .
The correlations we have revealed between Cx expression and hormone receptor, HER2 or Ki67 levels suggest that connexin isotypes are differentially involved in the regulation of breast cancer cell functions. In concordance with published data we found a positive correlation between Cx43 expression and hormone receptor levels both before and after treatment in primary breast cancer . However, either Cx43 or Cx26 protein can be elevated again in the lymph node metastasis of invasive breast cancer . We also found a positive correlation between Cx32 and HER2 expression and between Cx32 and Ki67 expression; and a negative correlation between Cx43 and HER2 levels and between Cx46 and Ki67 expression. As expected, Ki67 expression showed a negative correlation with hormone receptor expression and a positive correlation with HER2 levels. These results may imply potential tumour suppressive functions for Cx43 and Cx46 in primary breast cancer as opposed to Cx26 or Cx32 expression which may be tumour protective as well. The positive correlation between Cx46 levels and nodal status may be explained by the lack of correlation between nodal status and overall survival in our cohort and that the majority of our cases were ER positive.
In this study, reduced Cx26 and Cx32 levels and elevated Cx46 expression upon chemotherapy may also reflect the efficiency of chemotherapy. The exact role of connexins in carcinogenesis and metastasis formation is controversial or more probably context dependent.
Several previous studies addressed the changes of biomarker expression after primary systemic therapy in breast cancer [40–43]. Discordance in hormone receptor status has been reported to vary from 2.5% to ~50% [41–43]. The same has been found for HER2 status with altered immunophenotype found in up to 43% of cases especially when immunohistochemistry was the choice of assay [42, 43]. Although not analysed in detail here, we found discordant ER/PR/HER2 status in only <10% of our studied patients after neoadjuvant chemotherapy.
The predictive role of hormone receptors and HER2 status in view of partial and complete pathological response in breast cancer has also been extensively investigated [44–49]. HER2 positive, ER negative breast cancers with poor histological differentiation without nodal involvement have achieved better complete pathological response compared to those with lymph node metastasis [44, 46–50]. In our cohort, ER positive HER2 negative tumours dominated representing prognostically intermediate categories. Therefore, we did not correlate hormone and HER2 expression with partial and complete pathological response to neoadjuvant therapy.
We have classified clinicopathological parameters in breast cancers according to five current systems and could establish a significantly improved overall survival in cases scoring 1–2 against those scoring 3–5 only when using the CPS-EG classification. The lack of correlation with overall survival in the rest of classifications (NSABP, Miller-Payne, Sataloff and EWGBSP) is probably due to the fact that the majority of our cases fell into the prognostically intermediate categories. As we are aware, comprehensive assessment of therapy response using the major classifications as we tested here has not been done so far by others in breast cancer treated with neoadjuvant therapy.