Immunohistochemical Study of ALDH in Colorectal Adenocarcinoma

Conclusion and recommendations

In conclusion, high ALDH1 expression was associated with colonic tumors more than the rectal ones, lymphovascular invasion, advanced tumor staging, and lymph node metastasis in CRC. ALDH1 as a stem cell marker may play an essential role in tumor invasion and lymph node metastasis and may be a valuable marker for CRC prognosis, since being associated with poor prognostic features and shorter survival outcome. Inclusion of large number of young age in the studied cases is recommended to determine the correlation between young age and tumor’s aggressiveness. Larger number of studied cases, including pre malignant lesions, benign lesions and carcinoma insitu, is recommended to confirm the correlation between ALDH1 expression and different clinicopathological data and as a poor prognostic indicator. Further studies using different molecular methods on ALDH1, are suggested to detect the mechanisms by which ALDH1 may contribute to the aggressiveness, invasion, and progression of CRC.

Introduction

Colorectal cancer (CRC) is considered as one of the most common tumors that has a remarkable increase in frequency, and mortality, especially in developing countries⁽¹⁾. The global burden of CRC has been progressing with population, modifications in demographics, and westernization of daily lifestyle practices. In 2018, according to the World Health Organization (WHO) GLOBOCAN database, more than 18 million and 9 million new cancer cases and deaths that caused by CRC were documented, respectively⁽²⁾.

In Egypt, CRC represents 35% of total gastrointestinal system malignancies and 6.49% of all malignancies ⁽³⁾. CRC is the 7^th most common cancer in Egypt, representing 3.47% of the male tumors and 3% of the female tumors. The estimated number of colon cancer patients (excluding rectal cancer) in 2015 was slightly more than three thousand ⁽⁴⁾. The median age is 24 years in Egypt compared to 37 years in US ⁽⁵⁾.

Aldehyde dehydrogenase 1 (ALDH1) is an enzyme responsible for catalyzing the synthesis of retinoic acid and is involved in the oxidation of several exogenous and endogenous aldehydes into their carboxylic acids ⁽⁶⁾. ALDH1 is present  on chromosome 12, and it   has different isoforms, various cellular functions and tissue distributions ⁽⁷⁾. ALDH1 is detected in the cytoplasm of normal non-cancerous epithelium of the large intestine and is localized in the bottom of normal crypts where stem cells are present. High epithelial expression of ALDH1 is strongly accompanied with ulcerative pathological lesions. This finding might add an evidence that ulcerative form is correlated with unfavorable prognosis. ALDH1 stromal cells might act as local guardians or protectors of tumor cells decreasing progression of tumor as they are significantly associated with smaller size of tumors. ALDH1 plays an important role as a prognostic and therapeutic factor for CRC ^(6,8,9).

The significance of ALDH1 protein expression in CRC remains unclear, and the present study aims to evaluate the immunoexpression of ALDH1 in CRC and to study the correlation between immunohistochemical expression of ALDH1 with variable clinicopathological parameters in patients with CRC.

Material and Methods

1. Tissue specimens

The present study comprised 60 formalin-fixed and paraffin embedded tissue blocks of cases of primary colorectal adenocarcinomas which were retrieved from the archives of histo-pathological laboratories of Minia University Hospital and Minya Oncology Center (in the period between December 2018 and January 2021). The cases included, 40 cases of conventional adenocarcinomas, 12 cases of mucinous adenocarcinoma and 8 cases of signet ring cell adenocarcinoma. The grade of differentiation in the cases with CRC was low grade in 15 cases and high grade in 45 cases. The available clinicopathological data included:  patients' age, sex, tumor localization, tumor size, tumor type, tumor grade, stage, and lymph node status. Tumor type and grade were evaluated according to WHO criteria ⁽¹⁰⁾. Tumor stage was estimated by TNM staging ⁽¹¹⁾ and modified Dukes staging ⁽¹²⁾.

2. Immunohistochemistry

Primary antibody against ALDH1: Polyclonal mouse antibody (100 μl, concentrated, Lab Vision Laboratories), diluted at (1:300). A positive and negative control slide were performed with each run. Positive control was normal hepatic tissue.

3. Scoring of Immunostaining

ALDH1 was expressed in the cytoplasm. Ten high power fields were counted per section in each case and the average of counted fields was calculated. The staining frequency of ALDH1 was semi-quantitatively scored based on the percentage of positive tumor cells immunoexpression using a four-score grading system. Cases with < 5% ALDH1-positive cells were given a score of (0), those with 5-20% ALDH1-positive cells were given a score as (1), those with 20-50% positive cells were scored as (2), and those with >50% positive cells were scored as (3). For statistical analysis, cases were divided into two groups: Low expression (scores of 0 & 1) and high expression (scores of 2 & 3) ⁽¹³⁾. The median value (55%) was taken as a cut off point for ALDH1 expression, and the expression was divided into 2 categories; low expression above the median value and high expression below the median value ^(14-17). For statistical purposes, negative cases are included among low expressions and considered as one category. Interestingly, we found no differences in the results between the two systems of scoring.

Statistical analysis

         Statistical analysis was done using the Statistical Package for Social Sciences (SPSS software version 16). The Chi- square and Fisher's exact tests were used to compare categorical features.

Results

1. Clinicopathological Features

Data regarding different clinical and histopathological features for colorectal adenocarcinoma patients are summarized in Table (1).

Discussion

In the present study, 75% of CRC cases showed high cytoplasmic ALDH1 expression, while 25% showed low expression. These results were close to Yang et al. and van der Waals et al., ^(18,19).

In the current study, there was a statistically significant correlation between ALDH1 expression and tumor localization (P= 0.03). This was in concordance with a study done by Goossens-Beumer et al., who found that high ALDH1 expression was associated with colonic tumors rather than the rectal tumors⁽¹⁴⁾.

Interestingly, the present study has showed a statistically significant correlation between ALDH1 positivity and lymphovascular invasion (P=0.001). This result was similar to a study in CRC that performed by Holah et al., who detected that there was strong statistical correlation between ALDH1 expression and vascular invasion ^(13).

In the present study, we found a significant statistical correlation between ALDH1 expression and lymph node metastasis, (P=0.011) and such result was in agreement with that of other studies done by Holah et al., Said et al., who found that 42.9%, and 66% of cases with nodal metastasis showed positive ALDH1 expressing cells respectively ^(13-20).

In our study, there was a highly significant statistical correlation between ALDH1 expression, TNM (P = 0.043), and modified Dukes stages (P = 0.001). (80.6%) of cases with stage III TNM, (100%) of C1, and (84.2%) of C2 showed high expression. These results were in line with those of ⁽²¹⁾. On the contrary, a previous study performed by Wangshuo et al., indicated no significant relationship between ALDH1 expression and TNM stage. Such variation in the results may have emerged from the different populations, variable diet habits, different sample size, age range, different used antibodies, variable scoring methods, as well as the possible ALDH1 gene mutations in the studies ⁽¹⁹⁾.

Regarding TILs, we found a strong statistical correlation between the TILs and ALDH1 expression, in which marked TILs are associated with low ALDH1  expression, (P=0.001) and this agreed with ⁽²¹⁾. It was documented that a high level of TILs is an independent prognostic factors for favorable clinical outcome in breast cancer. These findings suggest that TILs are the key component for pathological complete response (pCR) and chemosensitivity⁽²²⁾.