Relationship among mismatch repair deficiency, CDX2 loss, p53 and E-cadherin in colon carcinoma and suitability of using a double panel of mismatch repair proteins by immunohistochemistry

Introduction

Colon carcinomas (CCs) are commonly observed worldwide, and represent the most common cancer of the gastrointestinal system. Environmental and genetic factors play important roles in their etiology [1, 2]. Genetic susceptibility in CCs has been observed ranging from well-defined hereditary syndromes such as familial adenomatosis polyposis to syndromes in which heredity cannot be demonstrated [1, 2]. Understanding the molecular mechanisms in colon tumorigenesis has led to the development of new treatment strategies and new molecular tests. In molecular classification, various factors have been described, such as chromosomal instability, point mutations, and microsatellite instability (MSI) [3, 4, 5, 6]. In recent years, some promising biomarkers have been found in molecular-level studies of CCs, and they have been used in clinical studies [7]. A defect in at least one of the set of DNA mismatch repair (MMR) genes (MLH1, MSH2, MSH6, and PMS2) that abrogate protein function is described as MMR deficiency. MSI testing with polymerase chain reaction and/or MMR protein immunohistochemistry are used to identify MMR deficiency in tumor tissue sections in which expression of MMR genes is lost while their expression in healthy adjacent tissue sections is intact [8].
In some centers, a double-antibody panel (MLH1 + MSH2) is used for detecting MMR deficiency in CCs because of its sensitivity [8, 9, 10]. However, a four-antibody panel (MLH1, MSH2, MSH6, PMS2) has attracted increased interest in many centers for mismatch repair protein deficiency in both germline and sporadic CCs [8, 9, 10]. Although the diagnostic importance of MMR protein deficiency is known, lately there has not been clear use of single, double, or a greater number of markers in immunohistochemical (IHC) studies. In addition, there is an ongoing debate on the relationships between pathological risk factors and deficiencies in these proteins.
CDX2 is a nuclear homeobox transcription factor that is related to the CDX homeobox gene [11, 12]. In the last decade, CDX2 has been considered as a specific and sensitive biomarker in IHC studies, especially in colorectal adenocarcinomas [12, 13]. It was shown that p53 is an independent prognostic indicator of presence of metastasis and the later stages of CCs [14]. In addition, several studies have shown that inactivation of the P53 gene has a key role in the development of colorectal cancer [7, 15, 16]. It was reported that reduction in the expression of the E-cadherin molecule, one of the biomarkers used in our study, leads to the invasion of the cancer due to a decrease in intercellular adhesion in CCs and an increase in metastatic capacity [17, 18]. However, the relationships of CDX2, p53, and E-cadherin with each other, with MMR deficiency and with classical pathological risk factors in CCs are still not clear.
Pathological analysis provides histological and molecular information in terms of appropriate treatment and prognosis. The aim of this study is to investigate the relationship among MMR proteins, CDX2, p53, E-cadherin and pathological risk factors in CCs using IHC staining. Moreover, we aimed to determine the practical and clinical usability of studying MMR proteins, regardless of sporadic or familial origins, in a single or double panel instead of a quadruple panel with IHC staining.

Material and methods

Patient data

One hundred and eleven patients who had been diagnosed with colon adenocarcinoma in the Erzincan University School of Medicine and Ds¸kap Yldrm Beyazt Training and Research Hospital between 2008 and 2014 were included in our study. Rectal cancers were excluded. Ethical consent was obtained from the local ethics commission for both groups. Because the nature of the study is retrospective, the status of cases – whether sporadic or inherited – was not known. The patients were reevaluated in terms of the parameters of histological differentiation (well/moderate/poor), invasion depth (submucosa, muscularis propria, and serosa), lymphatic vessel invasion (positive/negative), lymph node metastasis (positive/negative), perineural invasion (positive/negative), and vascular invasion (positive/negative). Slides of these cases obtained from the archive were reevaluated under light microscopy (Olympus BX53, Tokyo, Japan). We tried to select a paraffin-embedded tissue block for each case containing tumoral and preferably adjacent normal mucosa.

Immunohistochemistry

Four-micron sections were taken for positive charged slides by selecting appropriate formalin-fixed, paraffin-embedded tissue sections. Then, MLH1, MSH2, MSH6, PMS2, CDX-2, E-cadherin and p53 biomarkers (Table I) were studied using a fully automated IHC staining device (Leica Bond-Max, Melbourne, Australia).

Microscopic evaluation

When evaluating immunostaining, nuclear staining of tumor cells was taken into account for MMR proteins and CDX2. For these markers, nuclear staining was assessed as absent: 0 and present: 1+ (without evaluation of the percentage of staining). MMR deficiency was interpreted as a lack of at least one of the four proteins. In addition, tumor cells that showed no staining for nuclear staining for p53 and membranous staining for E-cadherin were considered 0, those that indicated less than 5% staining were considered as 1+, those that showed 5–50% staining were considered as 2+, and those that indicated more than 50% staining were considered as 3+. For all the biomarkers, staining of normal colonic glands or inflammatory cells was used as a positive control. Negative controls were tested with omission of the primary biomarkers.

Statistical analysis

Categorical variables were compared using the chi-square test. The degree of agreement of MMR deficiency with MLH1, MSH2, MSH6, PMS2, PMS2 and/or MLH1, PMS2 and/or MSH2, and PMS2 and/or MSH6, P53 and CDX2 tests was evaluated with the Cohen kappa () test. A p-value < 0.05 was considered as statistically significant. Analysis was conducted using R 3.1.0 (www.r-project.org).

Results

In the 111 patients with colon adenocarcinoma, the average age was 64.5 and 30.6% were female. There was lymph node metastasis in 62 (55.9%) cases, lymphatic vessel invasion in 64 (57.7%) cases, perineural invasion in 49 (44.1%) cases and vascular invasion in 41 (40.5%) cases. There was invasion limited to the submucosa in 6 (5.4%) cases, muscularis propria invasion in 27 (24.3%) cases, and serosa invasion in 80 (70.3%) cases.
In IHC staining of all colon adenocarcinomas, there was MLH1 loss in 13 (11.7%) patients, MSH2 loss in 9 (8.1%), MSH6 loss in 10 (9%), and PMS2 loss in 28 (25.2%) (Fig. 1–4). There were only seven cases with a concordant nonstaining pattern with four MMR proteins. Isolated MSH6 loss was seen in only one case, while isolated PMS2 loss was seen in 12 cases. There was no isolated MLH1 or MSH2 loss. The degree of agreement between MMR protein deficiency and the loss of MMR proteins (alone and paired) from strong to poor was as follows: PMS2–MSH6, PMS2–MSH2, PMS2, PMS2–MLHI, MLH1, MSH6, and MSH2 (Table II).
With CDX2 there was no staining in 11 (9.9%) cases (Fig. 5). Staining with p53 was not detected in 19 (17.1%) cases, while 18 cases (16.2%) had 1+ staining, 40 cases (36.0%) had 2+ staining, and 34 cases (30.6%) had 3+ staining (Fig. 6). Staining with E-cadherin was detected in all carcinomas, with 29 cases (26.1%) having 1+ staining, 26 cases (23.4%) having 2+ staining, and 56 cases (50.5%) having 3+ staining (Fig. 7). Tumor histological differentiation was found to be statistically related to perineural invasion, vascular invasion, p53, CDX2, and E-cadherin (p < 0.05) (Table III). In addition, there was a statistically significant relationship between the MMR proteins, such as only MLH1 loss, only PMS2 loss, loss of PMS2 paired with other MMR proteins, and MMR deficiency presence with histological differentiation (p < 0.05). However, there was no significant relationship among sex, lymph node metastasis, lymphatic vessel invasion, tumor depth, only MSH2 loss, or only MSH6 loss with histological differentiation (p > 0.05).
Twenty-eight patients with PMS2 loss within the 29 patients with MMR deficiency covered all 11 patients (100%) with CDX2 deficiency while covering only 5 of 19 cases (26.3%) with P53 loss. Unfortunately, because of staining of all cases this association has not been evaluated with E-cadherin.
On the other hand, there were statistically significant relationships and strong cohesion among MMR status, only MLH1 loss, only MSH2 loss, only MSH6 loss, only PMS2 loss, PMS2 and/or MSH6 loss, CDX2 loss, and E-cadherin expression when each of these was compared individually to the others (p < 0.05). There was no significant relationship between these biomarkers and sex, lymph node metastasis, lymphatic vessel invasion, perineural invasion, vascular invasion, or p53 expression (p > 0.05).

Discussion

There have been few studies specifying the use of dual protein panels in the literature, although the quadruple panel has been used to identify the lack of MMR proteins as one of the influential factors in colon carcinoma tumorigenesis [8, 9, 10]. However, studies on the correlation and coherence of MMR proteins with each other as a product of different perspectives via IHC staining are very limited. For instance, Shia et al. claimed that the dual panel (PMS2 and/or MSH6) can be used instead of the quadruple panel [8]. Moreover, Hall et al. found that the dual panel is 100% sensitive and specific when compared with the quadruple panel [9]. In the present study, we found that MMR deficiency had a moderately statistically significant relationship with only MSH2 loss, only MSH6 loss, and only MLH1 loss, while it had a high statistically significant relationship with only PMS2 loss and PMS2 and/or MSH6 loss. The rates of PMS2 and/or MSH6 loss were compared with MMR deficiency (100%), and the results were compatible with the findings of Hall et al. [9]. In addition, it was remarkable that the cases with PMS2 loss comprised 98% of the cases with MMR deficiency. On the other hand, when MMR profiles were compared with each other, the results were statistically significant. According to these data, it has been concluded that single or double panels do not affect diagnosis, treatment or prognosis.
Hall et al. suggested that using a double panel instead of a quadruple one is more beneficial in terms of time and economy [9]. Similar to the mentioned study, our findings support studying a single (PMS2) or double panel (PMS2 and MSH6) with automated immunohistochemistry instead of a quadruple panel. On the other hand, they reported MMR deficiency of 30.2% and an isolated PMS2 loss rate of about 1% in CCs. In the present study, MMR deficiency was found to be 26.1% regardless of sporadic or familial origins. In addition, isolated PMS2 deficiency was detected in 41.4% of all MMR deficiency cases. The high isolated PMS2 deficiency found in our study compared to the study by Hall et al. may reveal the sensitivity and importance of this protein.
While CDX2 expression was found in 98–100% of CCs in some studies, it was observed in 63–86% in others [13, 19, 20, 21]. In a similarly high rate to their data, in our study, while CDX2 expression in CCs was 90.1%, which is comparable with the literature, the staining rate in normal colon mucosa was 99%. On the other hand, it is quite interesting that the group of patients with PMS2 loss covered all patients with CDX2 deficiency. According to our extensive literature research, no study has detected this correlation before.
In the literature, some studies have been conducted using pairwise comparison of CDX2, MMR proteins, E-cadherin, and p53 biomarkers using IHC staining in CC patients [19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33]. According to our literature search, there have been no studies evaluating all of these markers together via IHC staining. Loss of CDX2 has been associated with high-grade tumors in colon tumors and advanced tumor stage [13, 22, 23, 24, 25, 26]. It has also been noted that there is a relationship between poor differentiation and loss of E-cadherin expression [27, 28, 29]. Moreover, there is a reported relationship between poorly differentiated tumors and MMR protein loss [30, 31]. According to our data, MMR protein deficiency, loss of CDX2, and loss of E-cadherin were associated with decreased tumor histological differentiation, and it is considered that these biomarkers may be associated with differentiation. However, there was no statistical relationship between MMR, CDX2, or E-cadherin and the prognostic risk factors other than differentiation. Although the relationship between CDX2 loss and MMR protein deficiency has been reported in other studies, in accordance with our research [11, 13, 22, 23, 32], there have been other studies reporting no relationship between the two [25, 33, 34, 35]. In addition, Funakoshi et al. claimed in a study investigating the relationship between CDX2 and E-cadherin that the former plays a regulatory role in the expression of the latter [36]. In another study of colorectal cancer with MMR proficiency and MLH1 loss, increased expression of nuclear beta-catenin and membranous E-cadherin loss were reported as independent negative pathological risk factors [37]. In our study, there was also a relationship between E-cadherin expression and CDX2 and MMR protein loss using IHC staining.
In some research, p53 protein expression was reported to be associated with poor prognosis in CCs [7, 15, 16]. While a relationship of p53 expression with increased stage and reduced histological differentiation of tumor and increased depth of tumor invasion has been shown in some studies [38, 39], no relationship has been found in other studies [40, 41]. In our research, p53 expression increased with decreased tumor differentiation and increased tumor invasion depth. Except for these parameters, there was no significant relationship of p53 with other prognostic pathological factors. It has been stated that mutation of p53 as a tumor suppressor gene is effective in the later stages of tumor development [14]. It has been also reported that CDX2 suppresses many genes that are effective in processes such as tumorigenesis, cell proliferation, and cell migration [42]. In addition, it has been proposed that CDX2 is a tumor suppressor molecule in the mouse model [42]. Baba et al. found a correlation between p53 and CDX2 loss [25]. Moreover, Valentini et al. found a relationship between p53 hyperexpression and MMR protein deficiency [43]. However, in our study, there was no relationship between p53 expression and MMR proteins, E-cadherin, or CDX2 expression.
The results of the present study highlight the association and correlation of MMR deficiency with CDX2 loss and E-cadherin expression in patients with CC. Additionally, it is interesting that the patients with PMS2 loss covered all patients with CDX2 loss. These findings confirm that it is necessary to conduct further genetic and pathological studies focusing on these two markers together. On the other hand, we presume it is important evidence that only PMS2 or the combination of PMS2 with MSH6 may be sufficient in the detection of MMR protein deficiency with IHC staining. Studying the remaining MMR proteins may be more suitable in terms of time (average process time: 4 hours), cost, and labor when deficiency is not detected with PMS2 or especially in combination with MSH6.

The authors declare no conflict of interest.

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Address for correspondence

Ilyas Sayar
Departmant of Pathology
Faculty of Medicine
Erzincan University
Erzincan Turkey
e-mail: drilyassayar@hotmail.com