Colorectal Cancer (CRC) is the third most common cancer and the fourth most common cause of cancer-related death, with its incidence increasing year-by-year Michael and Smith [1].

Iraq has a low CRC incidence rate but with a steady increase overtime and a total of 7,246 cases of CRC were registered in the cancer registry for the period 2002-2011 and 706 cases in between 2012 and 2014, also the ratio varied between male to female from 1.17 :1 to 1.28 :1 and about 40%-46% of cases were diagnosed in the age group of 40-60 years,  also the adenocarcinoma constituted 84.0% of all cases and 95% were diagnosed by histology of primary site [2,3].

In addition, the degree of differentiation was moderate in 56% of cases and about 26% of cases were localized and 16% were in distant metastasis, so CRC is significant disease in Iraq, where the middle age patients presented were the highest percentage and the trend of CRC showed an accelerated increase after 2007, [2,3,4].

Hsp70 assist the proteins in their folding, stability, assembly into multi-protein complexes, protection against protein aggregation and transport across cellular membranes.as well as regulation of apoptosis and cell survival Rina et al. [6].

Potential prognostic tumor markers have been identified to improve the efficiency and survival rate; however, the prognosis of most cancers is relatively low, therefore, high/positive tissue Hsp70 expression could be related to the prognosis of colorectal carcinoma patients, furthermore, previous studies show that Hsp70 expression has biological and clinical significance Guangyu et al. [5].

Patients and Controls

The study involved (146) men and women who were clinically diagnosed with CRC by the general surgery doctor. The patient age range between 36 and 80 years. This study designs as prospective study, all samples were taken from patients in multicenter during the period from December 2021 to December 2022.  

For histopathological and immunohistochemical analysis, the tissue samples are collected from patients with CRC who undergoing surgical resection (colectomy) and colonoscopy. All data were collected throw direct interview with the patients as a questioner. 

The collected samples were kept in (10%) buffered neutral formalin for histopathological analyses, the biopsy samples were subjected to histopathological of CRC patients. The biopsy samples were collected after surgical operations were kept in (10%) buffered neutral formalin for (24) hours and transferred to (70%) ethanol then processed to embedded in paraffin blocks until the decided examination in this study, so formalin-fixed, paraffin-embedded tissue samples of (52) patients with colon or rectal cancer were chosen for histopathological analysis and staining. 

The tissue specimens of CRC were blocked into a thickness of 3 or 4 mm and fixed in 10%neutral buffered formalin for 24 hours,  then the tissues were dehydrated by series of alcohols and cleared in xylene, after that infiltration by molten paraffin at 60°C was done,  meanwhile the tissue paraffin blocks were cut into sections of 4 μm and placed on Fisher-brand positively charged slides and then placed on the racks and then dried at room temperature overnight, then The slides were stained with Mayer's hematoxylin and eosin for examination Launa [9].

Immunohistochemical (IHC) Staining Analysis

The protocol of IHC will be according to Ramos-Vara and Miller [7].

Deparaffining and rehydrating the tissue sections, that incubated with 3% H2O2. Antigen retrieval was done in sodium citrate buffer (pH 6.0) for 20 min at 97°C. Then, the slides were covered with (100 µl) of Hsp70 antibody (ready to use) at room temperature for 15 minutes. Then the slides were covered with secondary antibody, after that the slides were immunostained with 3,3′-diaminobenzidine chromogens and then counterstained with Mayer's hematoxylin. In addition, positive tissue sections were covered with Phosphate-Buffered Saline Solution (PBS) instead of the primary antibody for the negative controls.

The intensity of Hsp70 immunostaining, therefore the intensity was scored as (negative immunostaining), weak, moderate and strong immunostaining Karina et al. [11].

Demographic features

Age Distribution

The (Table 1) showed the distribution of (146) total patients in our study according to their age groups, the highest percentage recognized in age group (55-65) years old was (27.4%), while the lowest percentage was (4.7%) for the age group (35-44).

Family History

The study found that (19) cases (13.01%) had positive family history of (CRC), while (127) cases (86.98%) had negative family history of (CRC), there is a significant difference between two groups (P≤0.05), (Table 2).

Gender Distribution

The study found that incidence of CRC was more in men than in women for all stages and for all age groups, (Table 3).

Histologic Staging

Pathological Grading

 In this study grading system can be applied only to 52 cases and we find that 5 case (9.62%) were diagnosed as grade I (well differentiated), 34 cases with (65.38%) are grade II (moderately differentiated) and 13 cases with (25 %) recorded as grade III (poorly differentiated), there were a significant difference among different stages at (P<0.05), (Table 5).

Histopathological Observation

 Mucinous adenocarcinoma is characterized by large collections of extracellular mucin set within the lumen of intestinal glands. The glandular epithelium cells loss their polarity, with evidence of abnormal mitotic figure with increasing of lymphatic infeltration. The cells’ pleomorphic indicate the existence of different clone cells within the same tumor (Figure 1). 

The tumor glands presented either an empty lumen or a lumen that was occupied by necrotic cells of cytoplasmic fragments, nucleus residue and fibrous material, making which is called “dirty necrosis” with evidence of abnormal mitotic figure (Figure 2).

Table 1: Illustrated age group

Table 2: Distribution of patients according to family history

Table 3: Distribution of patients according to sex distribution

Table 4: Types of tumor histologic staging in (CRC) patients

Table 5:  Distribution of patients according to grades

Immunohistochmical expression of (Hsp70) in CRC Patients

Histopathological sections from (CRC) patients were stained with immunohistochmical staining to determine the staining intensity reaction of (Hsp70) to grades of (CRC) lesions, the results showed that Hsp70 expression with CRC moderately differentiated (grade I1) was 59.6% in total staining reaction, followed by the CRC poorly differentiated (grade III) 24.9%, while CRC (grade I) the expressions of (Hsp70) have been reported (7.69%) respectively (Table 6).

According to the staging of CRC, the study included 5 patients with stage I, (15) patients with stage II, 23 patients with stage III and 9 patients with stage IV.

The reaction for Hsp70 expression in (stage I) was weak (7.69%), while in (stage II) the weak reaction was (5.7 %), the moderate was (17.3%), while in the stage III the weak reaction was (3.84) and the moderate was (25%) and strong reaction was (15.3%), while in stage IV the moderate was (11.53%) and strong reaction was (5.7%), (Table 7).
 

Table 6: Reaction intensity of Hsp70 expression according to the grades of CRC adenocarcinoma in IHC assay

P<0.05 (High significance level)

Table 7: Reaction intensity of Hsp70 expression according to the stages of CRC adenocarcinoma in IHC assay

P < 0.05 (High significance level)

The statistical analysis showed a high significance level with Probability value <0.05 for both Hsp70expression according to different stages and grades.

According to (IHC) examination (7.7%) from the total examined samples were diagnosed as negative for (Hsp70) when sections did not show any sign of staining at all (Figure 3).

Sections from high grades of CRC related to patients shown crowding cells, enlarged nuclei with hyperchromatic and loss of polarity, the neoplasia may appear as moderate and high expression (grade 2, grade 3), the cases that diagnosed as high grade showed strong positive intensity and the (Hsp70) expression was increased with increasing grades and stages of CRC (Figures 4-6).

In this study the cases with high grade of (CRC) showed moderate reaction intensity, while in low grade of (CRC) cases the reaction intensity was weak, moderate or negative. This comes in consistent with a study carried out by Guangyu et al. [5], that observed a severity of lesion correlated with the reaction intensity, also the study confirmed that the reaction intensity of Hsp70 was usefull compared with other parameters and it’s considered as an indicator for the expression of CRC also, Hsp70 overexpression can predict poor survival in colorectal cancer patients.

Our data showed a high level of (Hsp70) with signification difference in all (CRC) patients compared with its concentration in healthy people,  this may be to the role of (Hsp70) with (CRC) and the cancerous cells that produce it in addition t, this results in agreement with other studies by Guangyu et al. [5] which showed that the patients with CRC had showed high level of Hsp70 and more less than in healthy group.

Also, our study agreed with other study made by Steele et al. [8] who observed an association of levels Hsp70 in CRC patients, the high Hsp70 expression in most patients without chemotherapy due to the production of cancer cells and cells surrounding the tumor tissue.

Hence, inhibition of Hsp70 may be considered as a striking chemotherapeutic avenue [10] (Mohammed et al., 2020). The expression of Hsp70 became positive in all adenocarcinoma cases and it could be used to distinguish between benign tumor and the malignant tumor, therefore our study is disagreement with study done by (Gu et al.,2004), which indicate that Hsp70 expression is suppressed under induced oxidative stress conditions in malignant tissues of patients with CRC.

After analyzing 52 CRC samples, Hsp70 was found to be significantly associated with CRC independently of the tumor stage.

The biomarker is aberrantly overexpressed in CRC and high expression of Hsp70 biomarker induced by associated with high grades of CRC, in contrast the low expression level associated with low grade of CRC.

Conflict of Interest

All authors declare that there is no conflict of interest.

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