Laryngeal cancer is most frequently seen between the 5. and 7. decades of life in the world [1]. Laryngeal cancers include 1.1% of all malignancies worldwide and this rate is 1.9% in males, and 0.3% in females [4]. In our study similar to literature, the mean age of the patients was 56 ± 8.1 and male/female ratio was 8.
Heat shock proteins are chaperon proteins that have a protective function for the cell by normally creating an adaptation mechanism and increasing against environmental and pathophysiological stressors, and they make up an important step in the defense system. HSP70t and HSP70-2 (HSPA-2), unlike other HSP70 chaperon proteins, are expressed in the testicular tissues and are not seen in other tissues or are rarely seen in them [19]. In a study conducted on rats, Zhu et. al. stated that when they caused HSPA-2 defects, the primer spermatocytes got stuck at meiosis I, and other HSPA proteins cannot compensate for it. According to these results, they have argued that HSPA-2 is a molecular chaperone for CDC2/cyclin B1 complex formation, and that their interaction is necessary to ensure the G2/M-phase transition during mitosis and meiotic cell cycles [20]. In a different study, it has been noted that the G1/S phase arrest occurred, and malign cell proliferation decreased after the HSPA-2 gene was suppressed in the lung adenocarcinomas. It was shown that after the HSPA-2 decreased; the ERK1/2 cascade having functions such as cell survival rate, proliferation stimulation, cell differentiation, cell aging and apoptosis was affected [21].
It has been reported that HSPA-2 is effective in many malignancy carcinogenesis. While it is not expressed in normal tissues, it is excessively expressed in malign cells [14]. The excessive expression of HSPA-2 has been shown in malignancies in the pancreas [11] , cervix [12], bladder [13], lungs [14], esophagus [15], nasopharynx [16] and liver [17]. All of these studies show that HSPA-2 is effective in the formation and development of tumors. In our study we have confirmed that HSPA-2 is expressed in LSCC. In all of the cases, HSPA-2 positivity was determined in tumoral areas (%100). The positive expression rate of HSPA-2 in the adjacent non-cancerous tissues was 48/114 (%42). There was significant correlation between adjacent non-cancerous tissues and tumoral areas. HSPA-2 expression was only observed in the basal layer of adjacent squamous epithelium not containing dysplasia in all of the cases. In well-differentiated tumors expression was observed predominantly in the periphery of tumor solid islands, and a more intense expression was observed in a more diffused way in areas where differentiation loss was distinct in tumors cells. A diffused and strong expression being observed in moderately and poorly differentiated tumors while weak staining seen in the adjacent epithelial basal and the periphery of well-differentiated tumor islands have suggested that HSPA-2 might have a role in tumor cell migration, invasion and carcinogenesis.
 Zhang et. al. reported that while they did not observe staining with HSPA-2 in the normal esophagus epithelial, they observed poor staining in areas adjacent the tumor and squamous epithelium basal layer, and stronger staining in infiltrative areas [15]. In their study, Scıeglnska et. al. reported observing positive HSPA-2 in keratinocytes in the adjacent epidermis basal layer and in the infiltrative areas of all the skin tumors. On the other hand, they reported that they observed positivity with HSPA-2 in infiltrative areas in half of their cases, while no positivity was observed in the normal epithelium in the areas adjacent to the tumor in SCC in the head and neck region [14].
A different point that in our study we have observed important differences with staining rate and intensity in the low-grade epithelial dysplasia and high-grade epithelial dysplasia areas of the adjacent epithelium.  In all of our cases in low-grade epithelial dysplasia areas, positivity was found in only 1/3 of the basal part of the epithelium, while a full coat strong staining was seen in high-grade epithelial dysplasia areas. With this different staining pattern, we think that the HSPA-2 expression pattern can be extremely helpful in differential diagnosis when distinguishing epithelial dysplasia in laryngeal biopsies. No study has been conducted in the literature on this subject, and we think that new studies should be conducted with a larger number of cases.
In addition to HSPA-2 being excessively expressed in many types of cancer, there are many studies showing that it has an effect on tumor progression. It is indicated that tumor cells gain resistance against apoptosis related to hypoxia with an increase of HSPA-2, and that is how they stay alive [22]. In our study, a significant relationship was observed between the excessive expression of HSPA-2 and important prognostic parameters such as lymphovascular invasion, the primary tumor (pT), lymph nodule metastasis (pN), metastasis (pM), recurrence, the TNM stage and survival rate. Lymphatic metastasis is one of the most important reasons for poor prognosis and therefore low survival rate in carcinomas. The strong relationship between lymphovascular invasion and HSPA-2 expression in our study emphasized the prognostic importance of HSPA-2. Also lymph node metastasis (pN), primary tumor (pT), metastasis (pM) are the most important determinants in terms of staging and prognosis. In our study, a significant relationship was observed between all three and HSPA-2 expression. One of the most important points in our study is the link between the survival of patients and HSPA-2 expression. With the Kaplan Meier analysis of overall survival method, we found that patients with increased HSPA-2 expression at all stages had shorter survival regardless of other prognostic parameters (Table 3).  It is known that HSPA-2 migrates to the nucleus and nucleoli under stress conditions where it is localized in the cytoplasm under normal conditions. This is critical for cell migration, tumor formation and metastasis, as HSPA2 moves continuously between the cytoplasm and the nucleus in a heat shock state [23]. In our study, staining in cytoplasm and nucleus with HSPA-2 was observed in the infiltrative areas. We also observed stronger nuclear staining in patients with metastases compared to those not seen.
Fu et. al. reported that histological grade, tumor radius, clinical stage in hepatocellular carcinomas were directly proportionate to HSPA-2, thus HSPA-2 can have a role in carcinogenesis, tumor development, progression and metastasis. They indicated that they found a significant negative correlation especially between survival rate and HSPA-2 expression [17]. In their study emphasizing on esophagus SCC cases, Zhang et. al. stated that the excessive expression of HSPA-2 was associated with the primary tumor, clinical stage, lymph nodule metastasis and recurrence [15]. Garg et. al. have argued that HSPA-2 expression is related to cellular motility and growth, so HSPA-2 has a role in cancer spread during the early stages. In their study on bladder cancer, they stated that they observed a significant relationship between the clinical stage, histological grade and HSPA-2 expression [13]. In a different study conducted by Garg et. al. on cervix cancer, they obtained similar results and indicated that HSPA-2 had an effect on the progression of cervix cancer [12]. In their study, Zhai et. al. have noted that HSPA-2 is related to a poor prognosis in tumor angiogenesis and pancreatic adenocarcinoma, and has an important place in its progression [11]. Similar to our study, various studies have shown that in cases where excessive expression of HSPA-2 is seen, there are higher lymph nodule metastasis and therefore a worse prognosis [13, 24]. In their study, Scıeglınska et. al. found a significant relationship between HSPA-2 expression and the TNM stage, histological grade and prognosis in Non-Small Cell Lung Carcinomas. They have highlighted that especially in stage I and II cases, high HSPA-2 expression is related to lower survival rates [14]. In their in vivo study using a mouse model, Garg et. al. considered the matter from a different point of view and stated that they decreased tumor growth rates by 75% with HSPA-2 inhibition in rats [12]. When the results of our study and the available data in the literature were evaluated, finding a significant relationship between important prognostic parameters such as the primary tumor, lymphovascular invasion, lymph nodule metastasis, the TNM stage, metastasis, recurrence, survival rate suggested that this marker can be used in prognosis in LSCC by immunohistochemically. Especially in our study, it has been shown for the first time that HSPA-2 can be an important prognostic indicator in LSCC.
Conclusion
Our study supports that there is an association between high HSPA-2 expression levels and poor prognostic parameters. According to our results, we believe that HSPA-2 can be used as a prognostic marker in laryngeal cancer. There are different studies on this in the literature. However, there is a limited number of studies conducted with immunohistochemical method. In this respect, our study shows that HSPA-2 gives significant results with immunohistochemical methods as well.  Also, we think that HSPA-2 is important in early diagnosis due to its increased expression even in LSCC precursor conditions.