The results of the present study demonstrate that HSPA1A is elevated and the p62 level is decreased in PBMCs from women with breast cancer as compared to their levels in PBMCs from women with benign breast lesions. We also found that the p62 level was decreased in PBMCs from patients with invasive carcinoma as compared to those with in situ carcinoma. This paralleled our observation that p62 was lower in PBMCs from breast cancer patients with a positive sentinel lymph node biopsy as compared to those with a negative sentinel lymph node biopsy.
The influence of HSPA1A levels on autophagy has been investigated previously. Doklandy et al.  in an in vitro study clearly demonstrated that the extent of HSPA1A expression was inversely related to the level of autophagy in tumor cell lines. The mechanism involved activation of Akt kinase and the mammalian target of rapamycin (mTOR), a kinase that blocks autophagy induction. The authors proposed that an inverse correlation might exist between HSPA1A and autophagy in tumor cells. Additionally, Kanninen et al.  in an in vitro study provided data suggesting that the intracellular HSPA1A concentration in PBMCs from pregnant women and autophagy induction were inversely proportional. Other studies in yeast cells demonstrated that the chaperone activity of HSPA1A might be the main mechanism to prevent accumulation of misfolded proteins in the endoplasmic reticulum and, thus, limit autophagy [32, 33].
The mechanism(s) responsible for decreased p62 levels and elevated HSPA1A production in PBMCs in women with a malignant mammary tumor remains to be determined. It is likely that tumor-related antigens as well as intracellular macromolecules released from lysed cells appear in the circulation following malignant transformation of mammary cells. Their presence would signal the need for immune system stimulation and an elevated level of autophagy and HSPA1A activation in PBMCs. A probable subsequent increased elevation of these and likely other compounds in the systemic circulation as tumorigenesis progresses is consistent with our observation of decreased p62 and increased HSPA1A in PBMCs as breast cancer became more invasive. Therefore, based on findings from our study, we hypothesize that elevated intracellular levels of HSPA1A and decreased p62 levels in PBMCs could be markers of breast cancer and for advanced disease.
A strength of our study is its unique focus on PBMCs rather than on tumor cells, and specifically on alterations in intracellular levels of compounds associated with elevated immune activation. However, the findings must be interpreted in the context of several limitations. First, the sample size of the study population, especially the group with benign breast lesions, was very small and, therefore, subject to selection bias. The majority of women with benign lesions do not typically undergo surgery which limited our ability to recruit more subjects in this category. In addition, our study was not designed to detect differences in HSPA1A and p62 levels based on histology and stage of breast cancer. Therefore, our study was under- powered to evaluate these differences and the findings must be regarded as preliminary and hypothesis-generating. Further studies are warranted to compare the preoperative and postoperative levels of these markers, since HSPA1A and p62 levels may also fluctuate according to physical conditions and stresses other than breast malignancy. Lastly, utilization of p62 as the sole measure of autophagy is not definitive and optimally requires independent confirmation by measurement of other autophagy-associated biomarkers [31, 34].