Use of immunohistochemical versus microsatellite analyses as markers for colorectal cancer

Introduction

Colorectal cancer (CRC) is one of the most frequent causes of cancer-related mortality [1] and is the third most common malignancy in Turkey [2]. Hereditary non-polyposis colorectal cancer or Lynch syndrome (LS), the most common hereditary form of CRC, is an autosomal-dominant syndrome predisposing the development of 3%–4% of all CRCs [3].

Patients with LS are prone to malignancies due to germ line heterozygous mutations in one or more of the mismatch repair (MMR) genes [4]. Following the loss of the other allele in somatic cells, the defective MMR gene loses its function and the neoplastic process starts. At least four MMR genes are found to be mutated in LS: mut L homolog 1 (MLH1), mut S homolog 2 and 6 (MSH2 and MSH6) and post meiotic segregation increased 2 (PMS2) [5].

Frequently, DNA replication errors causing microsatellite instability (MSI) occur in LS patients’ tumor cells. Microsatellites are short (1–6 bases) and repeating DNA sequences that are distributed throughout the human genome. During DNA replication in a LS tumor cell, the defective MMR protein results in altered allele lengths of microsatellite regions due to deletions or insertions, which lead to MSI [3]. Thus MSI is a surrogate marker for MMR defects. MSI-polymerase chain reaction (PCR) analysis and MMR protein detection by immunochemistry (IHC) are the proposed screening methods for MMR defects and LS [6].

The diagnosis of LS depends on family history, pathologic findings, IHC, MSI analysis and detection of germline MMR gene mutations. Family history is still an important parameter for the diagnosis, and the Amsterdam I, Amsterdam II and the revised Bethesda Criteria rely heavily on family history to identify individuals at high risk for LS [7]. Moreover, early detection of LS leads to 65%–70% decrease in morbidity and mortality. Additionally, various prophylactic surgeries can be recommended in the case of mutation carriers [8]. However, MSI and MMR gene mutation analyses are costly and time-consuming, making these tests a financial burden for developing countries. Therefore, there is a need for a cheap, quick, and reliable screening algorithm for LS.

MSI is present in approximately 10%–13% of sporadic CRCs [8] and similarly MMR genes are involved in the 10%–15% of the sporadic CRCs [9].

In the present study, we performed a four antibody-panel IHC in parallel with MSI-PCR analysis. Our hypothesis was IHC can identify Lynch syndrome suspected patients’ MSI status well enough for using as a first line screening method for Lynch syndrome especially in developing countries. Our aim was to determine how well IHC analysis identified colon cancer patients with microsatellite instability (MSI) and thus suspected LS in a population of Turkish patients.

Materials and methods

This study has been conducted according to the principles established in Helsinki. This study is approved by Ethics Committee of Ankara University (approval no: 29-603).

Results

Immunohistochemistry

Pathologists had consensus in all samples. As shown in Table 3, two patients (14.3%) in the sporadic CRC group, five patients (35.7%) in the familial group and 10 patients (71.4%) in the suspected LS group had at least one MMR protein deficiency. A statistically significant difference was detected between the three groups (p=0.008). In paired comparisons, a statistically significant difference was detected between the suspected LS group and the sporadic CRC group (p=0.002). Representative sections of MMR staining loss are shown in Figure 1. Of the 10 patients with MMR loss in the suspected LS group, six patients showed loss of expression for both MLH1 and PMS2, two patients showed loss of expression for both MSH2 and MSH6, one patient lacked only MSH6, and one patient lacked only PMS2. Of the five patients with MMR loss in the familial CRC group, three patients showed loss of staining for MLH1 and PMS2 and two showed only loss of staining for MSH6. Of the two patients with MMR loss in the sporadic CRC group, one patient showed loss of expression for MSH2 and MLH1 and another showed loss of expression only for MSH6.

Figure 1:

Representative sections of mismatch repair (MMR) staining loss.

(A) Normal staining of a MMR protein in tumor cell nuclei. (Familial colorectal cancer group case – MSH6). (B) Loss of less than 75% of nuclear staining for the MMR proteins immunohistochemistry (IHC) evaluation. (Sporadic colorectal cancer group case – MSH6). (C) Loss of more than 75% nuclear staining for any MMR protein was categorized as “MMR protein loss” in the IHC evaluation. (Familial colorectal cancer group case – MSH6). (D) Complete loss of a MMR protein in tumor cells. (Suspected Lynch Syndrome group case – MLH1).

Table 3:

Mismatch repair protein loss as determined by immunohistochemistry and microsatellite instability as determined by polymerase chain reaction in patient groups.

Groups	MMR loss ratio (%)	MSI ratio (%)	High MSI n	Low MSI n	MSS n
Sporadic CRC	2/14 (14.3%)	1/14 (7.1%)	1	0	13
Familial CRC	5/14 (35.7%)	2/14 (14.3%)	2	2	10
Suspected LS	10/14 (71.4%)	9/14 (64.3%)	9	0	5

1. MMR, Mismatch repair; MSI, microsatellite instability; MSS, microsatellite stabile; CRC, colorectal cancer; LS, Lynch syndrome; n, number.

Discussion

Contemporary molecular medicine enables the optimization of treatment strategies according to the molecular profiles of CRC subtypes. However, identification of CRC subtypes at the molecular level requires advanced infrastructure, which is not possible to implement in every healthcare unit. The most accurate diagnosis of LS is via MMR gene mutation analysis, however, MSI is often used as a surrogate marker for MMR gene mutation and LS. However, both of these methods are expensive and may not be affordable in many healthcare settings. In this study, our aim was to determine how well immunohistochemical analysis, which is a relatively simple and inexpensive testing method, identified CRC patients with MSI and thus suspected LS in Turkish patients. We found that the four-panel IHC identified 35/42 (83.3%) patients correctly using MSI-PCR as our comparator. Of the seven patients with IHC results discordant with the MSI-PCR results, six were false positives and one was a false negative.

Lindor et al. [18] used IHC for MLH1 and MSH2 in 1144 colorectal cancer patients and reported that the predictive value of normal IHC for MSS or low MSI phenotype was 96.7%, whereas the predictive value of abnormal IHC for high MSI was 100%. When mixed groups of CRC patients were considered, IHC (for MLH1 and MSH2) had 77%–100% sensitivity for the detection of high MSI and 99% specificity [15]. Generally, both low MSI and MSS are considered as a single subset, but this issue has been recently debated [19]. We found low MSI in two patients with loss of at least one of the MMR proteins as detected by IHC, suggesting that further evaluation might be needed for patients with low MSI.

Cawkwell et al. [20] showed that the expression of MLH1 or MSH2 was completely absent in the high MSI phenotype tumors in their study, and they suggested that investigation of the minor MMR enzymes (PMS1, PMS2, hMSH6/GTBP, hMSH3) would not be required. However in our study, two patients with high MSI showed defects in either MSH6 or PMS2 and one patient with low MSI showed a defect in only MSH6. Our results are in agreement with the detailed study by Lindor et al., which concluded that if IHC is performed only for MLH1 and MSH2, then some cases with MSI can be missed. In addition, Shia reported that using a four MMR antibody-panel (MLH1, MSH2, PMS2, MLH6) for IHC, instead of a two antibody-panel (MLH1 and MSH2), significantly increases the sensitivity of IHC in predicting germline mutation to a level which is virtually equivalent to MSI testing [6].

In a study by South et al., all cases of CRC were prospectively stained for these 4 MMR proteins and 57 out of 270 (21.1%) patients lacked expression in one or more of these proteins. South et al. [5] suggested that routine evaluation of CRC cases for MMR proteins is feasible regardless of age at onset or family history. In a study conducted by Karahan et al. [21], loss of expression of one or more of these four MMR proteins was found in 24 out of 186 (12.9%) CRC cases. In our study, this ratio was 40.5% (17/42 patients) when all of the patients were considered. In a study by Erdamar et al. [9], routine examination of sporadic CRC patients detected loss of MLH1 or MSH2 protein expression in 32 out of 74 patients (43.2%). In our study, we found a defect in MLH1 or MSH2 protein expression in one out of 14 sporadic CRC patients (7.1%). The difference in our ratios from literature can be explained by the patient selection criteria used in these studies as well as some technical aspects of the tissue fixation and processing.

In patients who had a pre-diagnosis of LS using the Amsterdam II criteria, MSI was detected by PCR in 10 out of 28 (35.7%) patients, and MLH1 or MSH2 protein expression defects were detected in all 10 patients [22]. In our study, we showed MSI in nine out of 14 (64.3%) patients with suspected LS, and 7/9 (77.7%) of these patients had MLH1 or MSH2 expression defects. One patient with high MSI had an expression defect only for PMS2. These differences in results can be explained by insufficient number of patients and recording errors in pedigrees in our country.

Several factors may account for the discordance found between the results of the IHC panel and the MSI-PCR analysis in our study. In general, the success of the four-panel IHC as a diagnostic tool may vary due to the fixation and processing procedures of the tissues [18]. The discordance in cases with MSS, but some apparent loss of MMR protein expression, might be explained by variations in staining quality and challenges of the IHC procedures [23] or difficulties in detecting unusually low rate of MSI due to technical problems [24]. Another cause of discordance may be whether or not the patient received neoadjuvant therapy, which can induce loss of MSH6 expression [25]. This may have been the case for patient number 39 in our suspected LS group, who received preoperative radiation and showed MSS but with loss of MSH6 expression. Patient number 40 in the suspected LS group showed high MSI, but no loss of MMR protein expression was initially seen by IHC. However, when the slides for this patient were reassessed, there was 50%–75% loss in MLH1 staining in the tumor tissue. This emphasizes the need for good standardization of the IHC evaluation criteria for MMR loss. Finally, MSI evaluation is used as a surrogate marker for more expensive MMR gene sequencing and mutation analysis. Discrepancies between MSI and MMR gene mutation could explain some of the discrepancies between MSI and MMR protein expression. If discordant test results in IHC and MSI testing occurs, patients should undergo further evaluation [26].

Our study had some shortcomings. First, this study was retrospective in nature and paraffin-embedded tissue samples were used for laboratory tests, reflecting real-life clinical practice. Second, we were not able to make a definitive diagnosis by germline MMR gene mutation analyses. Despite this, our study is one of the rare studies on this subject that has been conducted in our country.

In conclusion, it is widely accepted that all CRC patients should be tested for MSI/MMR gene defects using IHC, PCR or both at the same time regardless of any parameter [27]. Recently, Kidambi et al. [28] conducted a study addressing this issue, and they found similar outcomes between universal and selective screening for LS. In our opinion, routine expensive MSI-PCR analysis can be unfeasible for many health care units in our country or in developing countries. We suggest that if LS is suspected, then patients should be screened by a four antibody-panel IHC, and loss of nuclear staining in more than 75% of neoplastic cells can be accepted as a suggestive for LS. Molecular tests should be performed only if necessary, for example in cases where adjuvant therapy may influence the results.