['This study was aimed at understanding the clinicopathological significance of cystatin M loss, and investigating possible factors responsible for cystatin M loss in breast cancer.', 'The expression of estrogen receptor (ER), progesterone receptor (PR), HER2, HER4, and cystatin M was retrospectively analyzed using immunohistochemistry in 117 patients with ductal carcinoma in situ (DCIS) and in 175 patients with invasive breast cancer (IBC). The methylation status of CST6 gene encoding cystatin M was evaluated using methylation-specific polymerase chain reaction (PCR) in formalin-fixed paraffin-embedded tissues from 292 participants and using pyrosequencing in fresh-frozen tumor and matched normal tissues from 51 IBC patients.', 'Cystatin M loss was found in 9 (8%) of 117 patients with DCIS and in 99 (57%) of 175 with invasive breast cancer (IBC) (P < 0.0001). Cystatin M loss was found in 58 (57%) of 101 HER2-negative IBCs and in 41 (55%) of 74 HER2-positive IBCs, and this difference was not statistically significant (P = 0.97). However, cystatin M loss was significantly associated with the loss of ER (P = 0.01), PR (P = 0.002), and HER4 (P = 0.003) in IBCs. Cystatin M loss occurred in 34 (76%) of the 45 HER4-negative IBCs and in 65 (50%) of the 130 HER4-positive IBCs. Multivariate analysis showed that cystatin M loss occurred at a 3.57 times (95% CI = 1.28 to 9.98; P = 0.01) higher prevalence in the triple-negative IBCs of ER, PR, and HER4 than in other subtypes, after adjusting for age. The quantity of CST6 methylation was associated with ER loss (P = 0.0002) in IBCs but not with the loss of PR (P = 0.64) or HER4 (P = 0.87).'] Is cystatin M loss associated with the losses of estrogen receptor , progesterone receptor , and HER4 in invasive breast cancer?