Microsatellite instability screening in colorectal carcinoma: lmmunohistochemical analysis of MMR proteins in correlation with clinicopathological features and Ki 67 protein expression.

doi:10.21203/rs.3.rs-3088761/v1

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Microsatellite instability screening in colorectal carcinoma: lmmunohistochemical analysis of MMR proteins in correlation with clinicopathological features and Ki 67 protein expression.

https://doi.org/10.21203/rs.3.rs-3088761/v1

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published 25 Oct, 2023

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Background

Defects in mismatch repair (MMR) system or microsatellite instability (MSI) and detected in colorectal carcinoma (CRC); both in sporadic and more frequently in hereditary cases. Immunohistochemistry (IHC) is the most frequent method for MMR protein deficiency screening in CRCs.

Methods

In this study, we evaluated by immunohistochemical expression of MMR proteins including MSH6, MSH2, PMS2 and MLH1 in 50 resection materials with colorectal carcinoma. Their expression is correlated with clinicopathological features of patients together, with Ki-67 protein expression in attempt to screen the most significant predictor of microsatellite instability.

Results

Of the 50 cases of cancer colon, 28% were classified as MSI-H, 20% were MSI-L and 52% were MSS. The most frequent pattern in MSI-H tumors was concurrent loss of MSH-6 and PMS-2 proteins. Significant correlation between MMR proteins expression pattern with tumor size, grade, T classification and stage (0.015, 0.0515, 0.0162 and 0.0391) respectively. MSI-H tumors were located more frequently in right colon, early TNM stage, poorly differentiated and infrequent distant metastases. There was significant correlation between Ki-67 high expression and MSI status patterns suggest in their common biological aspects distinct from MSI-negative tumors.

Conclusions

Mismatch repair defective colorectal carcinoma have characteristics clinicopathological features different from MSS tumors. The role of immunohistochemistry (IHC) for MSI evaluation is the easiest and effective way for evaluation of MMR deficiency in colorectal carcinoma

Colorectal carcinoma- mismatch repair

microsatellite instability-Ki67

Colorectal carcinoma (CRC) is the third most common cancer in men, the second most frequent in women and second cause of cancer –related deaths globally [1]. Almost 60% of patients are present in developed countries [2]. The most cases of cancer colon are sporadic, while about 5%to 10% are hereditary [3]. Multiple genes are associated with hereditary cancer colon, but gene mutations associated with Lynch syndrome (LS), the most frequent cause of hereditary type, also known as colorectal cancer with inherited non-polyposis [4]. Familial adenomatous polyposis (FAP) is another form of hereditary cancer colon. Another category that show familial cancer cluster in a family with a recognized hereditary syndrome and also referred to familial colorectal cancer, however its genetic basis is still unknown [5].

Approximately, 15% of colon cancer show mismatch repair pathway (MMR) defect that leading to microsatellite instability (MSI). Pathogenesis of MSI colon cancer is caused by mismatch repair systems flaw. It’s important clinically to distinguish MSI cancer from MMS malignancies. This is due to the fact that MSI cancers have more favourable prognosis than MSS tumors and respond differently to chemotherapy. It is also desirable to recogenise Lynch syndrome patients [6].

About 15–20% of random colon cancer cases and 90% of LS patients have MSI [7]. Four genes have a part in correcting errors in mismatch repair or replication are MSH2, MSH6, PMS2 and MLH1. Defects in these genes leads to microsatellite instability (MSI), which may observed in some malignancies including colon cancers especially LS (Lynch Syndrome) [8]. MSI is seen in cancer of endometrium and stomach but it may also be seen in many other types of tumors [9]. In Lynch syndrome, the major underlying mechanism responsible for development of the tumor with MSI is a germline mutation in one allele of an MMR gene, together with the presence of somatic mutation that result in loss of function of corresponding non-mutant allele. However in sporadic cases of cancer colon, tumors occur due to aberrant methylation of promoter region of MLH1 gene that leads to transcriptional suppression and also undetectable protein production [10].

A distinct clinicopathological parameters used by pathologists including anatomical site, age, tumor infiltrating lymphocytes, degree of histological differentiation and other clinicopathological features that important for MSI prediction and had a correlation with MMR genes mutations [11].

Molecular testing using the polymerase chain reaction (PCR) could identify and assess the MSI phenotype, but several studies recognized use of immunohistochemistry for analyzing MMR mutation genes because of its simplicity, reproducibility, equally informative an availability nature [11, 12]

Ki-67 is a proliferative marker that has been widely used to evaluate proliferative activity of cancer cells and its expression in colorectal cancer varies [13].

In this study, we aimed to investigate the relation between clinicopathological characteristics, Ki-67 proliferation marker and immunohistochemical expression of MMR proteins including MSH6, MSH2; PMS2; MLH1 in total of 50 resected materials with colorecatal adenocarcinoma.

Specimens and ethical approval:

In our study, 50 cases of CRC were examined. They were retrieved consecutively from archives of private laboratories. This study was approved by ethics committee of National Research Center (Approval No.16/308).

Histopathology:

The histopathological characters were reviewed. The histopathological diagnosis was verified by two pathologists The clinical and pathological information were retrospectively collected from records and are summarized.

Immunohistochemical interpretation:

Formaline-fixed, paraffine embedded tissue sections were cut to generate 4µm unstained charged slides, including center of tumor and tumor invasion front. The slides were incubated all night at 37°C for accurate adhesion of tissue on slides. Deparaffinization, rehydration and epitope retrieval step in citrate buffer was performed. The staining step and incubation time were programmed in autostainer link software. A four-antibody panel of MMR proteins, including MLH1; MSH2; MSH6; PMS2 and Ki67 were conducted. Used primary antibodies were: anti MLH-1 (DS-0301-A;Mouse Monoclonal Primary Antibody; Concentrated diluation; 1:100) (Diagnostic BioSystems; 6616 Owens Drive; Pleasanton; CA), anti MSH2 (DS-0633-B;Mouse Monoclonal Primary Antibody Concentrated dilution; 1:200) (Diagnostic BioSystems,6616 Owens Drive; Pleasanton;CA), anti MSH6 (DS-0307-B; Mouse Monoclonal Primary Antibody; Concentrated dilution; 1:100) (Diagnostic BioSystems;6616 Owens Drive; Pleasanton;CA) and anti PMS2 (DS-0161-B, Mouse Monoclonal Primary Antibody;Concentrated dilution; 1:100) (Diagnostic BioSystems;6616 Owens Drive; Pleasanton; CA), anti Ki-67 (DS-0276-B; Rabbit polyclonal Primary Antibody; Concentrated dilution; 1:100) (Diagnostic BioSystems;6616 Owens Drive;Pleasanton; CA). Tissue was then blocked with Ab for 1.5 hours at 22°C (Thermo Fischer Scientific). An HRP conjugated goat anti-rabbit antibody was used as secondary. Marker is considered positive if staining cell membrane and cytoplasm and its negative expression was considered with total complete loss of immunohistochemical stain.

According to CAP guidelines for immunohistochemical interpretation, any nuclear staining, no matter how patchy, is seen as “no loss of expression”. Only complete absence of nuclear staining was regarded as “loss of expression” provided that positive internal controls. Hence, when nuclear staining was lacking for at least one protein, carcinoma was termed MSI [14].

When revealed that, two or more markers are unstable, MSI-H is taken into account (lost expression). If only one marker is unstable, the MSI- low is taken into account (lost expression). When there is no loss of expression and no unstable markers, MSS occurs. We adhere to the MSI definition used in the Bethesda-approved Fujiyoshi et al., 2017 study. [15].

Immunohistomorphometric Quantitative Analysis of Ki67 expression:

Under the Olympus CX-41 light microscope, the immunostaining was observed and captured on camera using a DP-12 Olympus digital camera (Olympus Optical Co. Ltd, Tokyo, Japan). Pathological diagnoses had been established for all specimens. At the pathology lab, MRCE unit, National Research Centre, computerized morphometric analysis was carried out using Leica Qwin DW3000 image analysis system (LEICA Imaging Systems Ltd, Cambridge, England), that consists of a Leica DM-LB microscope with JVC color video camera attached to computer system. We selected five tumor fields with highest expression and assessed the area percentage of the positive cells at high magnification (×200). We detected the positively stained cells, which were masked automatically by a blue mask called binary image. We used the measure field software program that automatically measures the area percentage of the detected features in the binary image and then displays the results in a table form. Both processing-related artifacts and any areas of necrosis were neglected.

Immunohistochemical analysis:

The area percentage of Ki67 positively stained cells was calculated using interactive measuring software of the system as area per field in micrometre square, area percentage, and area fraction. Table with total, standard error, mean, standard deviation as well as minimum and maximum areas measured immediately flashed on the monitor as results. Proliferation index (PI) of Ki-67 was computed as follows: Ki-67 > 25% was considered to have a high PI, while Ki-67 25% was regarded to have a low PI. [16].

Statistical Methods:

Using the Windows version of SPSS 20.0, statistical analysis was carried out (New York, IBM, USA). In order to examine association between protein expression and clinicopathological indices, Fisher's exactor Pearson Chi-square tests were utilized. Using these tests perform a univariate study comparing lymph node metastasis, distant metastasis, and clinicopathological indices was carried out. Expression of these factors was correlated, and Spearman's correlation analysis was used to assess this. Sex, age, differentiation, location, depth of invasion, distant metastasis, lymph node metastasis and expression of Ki67 were included as covariates. For analysis, beta coefficients and 95% confidence intervals (CI) were utilized. P value was regarded as statistically significant, when its value is ≤ 0.05

Clinicopathological Features of CRC:

In this study, 50 colorectal carcinoma patients were examined. Of all patients, 30 (60%) were female. Age range was 20-71 years, These age groups consisted < 50 years 36% of patients and ≥ 50 years were 32 (64%). Based on pathological report of surgical specimens, 36 cases with tumor size larger than 5 cm. Regarding site of tumor involvement in pathology report: patients categorized in: right colon, left colon and rectum. The most common site of involvement was left colon with 40%of reported cases. Thirty patients were classified as grade II and twenty patients as grade III. Approximately 84% of the tumors tested were stage II and III counting 18 and 24 respectively. Table 1

Table (1): Clinicopathological features

No. of patients

(50)

Clinicopathological features

20 (40%)

30 (60%)

Gender

Male

Female

18 (36%)

32 (64%)

Age (yrs)

<50

≥50

14 (28%)

36 (72%)

Tumor size

< 5

≥ 5

16 (32%)

20 (40%)

14 (28%)

Tumor site

Right

Left

Rectum

30 (60%)

20 (40%)

Tumor grade

III

4(8%)

8 (16%)

24 (48%)

14 (28%)

T-classification

22 (44%)

18 (36%)

10 (20%)

N-classification

44 (88%)

6 (12%)

M-classification

4 (8%)

18 (36%)

24 (48%)

4(8%)

Stage

III

6(12%)

16 (32%)

24 (48%)

4 (8%)

Duke´s classification

A (6)

B (16)

C (24)

D (4)

Correlation of Clinicopathological features with MSI status patterns:

Of the 50 cases, 14 (28%,) were categorised as MSI-H (Figure 1), 10 (20%) were MSI-L (Figure 2), and 26 (52%) were MSS (Figure 3), by MSI analysis. The correlations of clinicopathological parameters of CRC patients with MSI status patterns were illustrated in (Table2) Colorectal carcinomas with MSI exhibited different clinicopathological characteristic than those with MSS. The differences were statistically significant especially for tumor size, grade, T classification and stage as demonstrated in (Table 2). When compared to MSS, MSI-H were more frequently seen in right colon, poorly differentiated, at relatively early TNM stage as well as infrequent distant metastasis. On the other hand the clinicopathological features of MSS tumors had no significant differences in comparison to MSI-H or MSI-L in gender, tumor site, age, lymph nodes metastasis nor Duke`s classification.

Table (2): Correlation of Clinicopathological features of colorectal carcinoma patients with MSI status patterns:

P-value

MSS

(26)

MSI-L

(10)

MSI-H

(14 )

Clinicopathological features (No. of cases)

0.789 (NS)

Gender

Male (20)

Female (30)

0.208 (NS)

Age (yrs)

<50 (18)

≥50 (32)

0.015 (S)

Tumor size

< 5 (14)

≥ 5 (36)

0.186 (NS)

Tumor site

Right (16)

Left (20)

Rectum (14)

0.0515 (S)

Tumor grade

II (30)

III (20)

0.0162 (S)

T-classification

T1 (4)

T2 (8)

T3 (24)

T4 (14)

0.295 (NS)

N-classification

N0 (22)

N1 (18)

N2 (10)

0.777 (NS)

M-classification M0 (44)

M1 (6)

0.0391 (S)

Stage

I (4)

II (18)

III (24)

IV (4)

0.365 (NS)

Duke´s classification

A (6)

B (16)

C (24)

D (4)

Correlation between lack of MMR protein expression and MSI status patterns:

We determined the proportion of tumors lacking a specific mismatch repair protein for each category of microsatellite instability (MSI-H, MSI-L, or MSS) (MSH6, MSH2, MLH1 and PMS2) (Table 3). Among MSI-H tumors (Figure 1); 8 (16%) exhibited loss of MSH2, 10 (20%) exhibited loss of MSH6, 2 (4%) exhibited loss of MLH1 and 12 (24%) exhibited loss of PMS2. Among MSI-L tumors 4 (8%) exhibited loss of MSH2 (Figure 2); and 3 (6%) exhibited loss of PMS2, without any loss of MSH6 or MLH1. Of total 12 tumors that showed loss of MSH2 expression, six were negative for PMS2 expression and four cases were negative for MSH6. Eight cases lacked expression of both MSH6 and PMS2. None of examined 50 tumors for MLH1 showed loss of its expression except two cases.

Table (3): Correlation between MMR protein lack expression and MSI status patterns:

MSI status patterns	MMR protein expression				P-Value
MSI status patterns	Loss of MSH2 No.(%)	Loss of MSH6 No.(%)	Loss of MLH1 No.(%)	Loss of PMS2 No.(%)	P-Value
MSH-H	8 (16%)	10 (20%)	2 (4%)	12 (24%)	0.0719 (S)
MSH-L	4 (8%)	0	0	3 (6%)	1 (NS)
MSS	0	0	0	0	0
					0.6039(NS)

Correlation between ki67 expression and MSI status patterns:

According to the appropriate cut-off values of Ki67 indicated above, all included patients were divided into two groups: Low expression and high expression. Quantitiave assessment of Ki67 expression area is measured by image analysis system dividing them into low expressed group <25% and high group ≥ 25% (Figure4). Twenty eight cases (56%) have low expression of Ki67 ranged by 0.193-10.478 and 22 (44%) of cases have high expression with range of 25.351-78.504.

Among the Low expression of Ki67 tumors, 8 cases were MSI-H with mean area percentage of expression 4.291±0.59, 10 cases were MSI-L with mean area percentage of expression 2.887±0.531, 10 cases were MSS with mean area percentage of expression 3.711±0.79. On the other hand the high expression of Ki67 tumors were assessed as 6 cases of MSI-H with mean area percentage of expression 32.218±1.235 and 16 cases were MSS with mean area percentage of expression 44.545±2.273. There was negative correlation between Ki67 low expression and all of MSI status patterns. The high expression of Ki 67 showed a positive correlation with MSI status patterns(Table 4).

Table (4): Correlation between ki67 expression and MSI status patterns:

Ki67 Area % Expression	MSI status			P-Value
Ki67 Area % Expression	MSH-H	MSH-L	MSS	P-Value
Low	28(56%)			0.867 (NS)
No.	8	10	10
Range	2.471-7.634	0.193-5.907	1.443-10.478
(Mean ±SE)	4.291±0.59	2.887±0.531	3.711±0.79
High	22(44%)			0.055 (S)
No.	6	0	16
Range	25.351-32.218	0	26.084-78.504
(Mean ±SE)	32.218±1.235	0	44.545±2.273

Colorectal carcinoma is considered the second most common cause of cancer –related deaths worldwide [17]. Colon carcinoma caused by defects in DNA mismatch repair system commonly show microsatellite instability, which is observed in both sporadic and more frequently inherited cases of colorectal carcinoma [3]. PCR amplification of microsatellite genes is gold standard approach for screening the microsatellite status, however it is expensive, technically tricky and not predictable in routine pathology laboratories [11]. Immunohistochemistry IHC was validated as another method for mutation screening being sensitive, available (lost expression) inexpensive [18]. Our study aimed to assess CRC cases, IHC expression of MMR proteins in correlation with patients clinicopathological features and also correlation between them and Ki67 expression.

In the present study most patients (64%) were 50 or over 50 years with no noticeable sex dominance for CRCs. Mean age of incidence in the literature is 62; and it is rare to be screened under 40 years unless there are predisposing factors [19]. Our results complied with studies of Karahan et.al., [20] which showed that mean age of CRC patients was 66 years and only 4.8% of cases were under 40 however with male predominance for CRCs.

Many studies reported that rectosigmoid and proximal colon are common locations for CRCs [21], however other studies showed that the tumor was mainly localized in sigmoid colon with no statistical significance [20]. In our study, the commonest site of involvement was left colon with no statistical meaning, this could be as a result of few cases.

In 15–17% of patients with colorectal carcinoma, microsatellite instability pathway is responsible for the pathogenesis [3, 8]. In both hereditary and sporadic cancer; molecular phenotype of colorectal carcinoma is tightly correlated with its biological behavior, clinicopathological features, prognosis and even patient response to treatment. both [21].

International guidelines recommended screening of MMR deficiency for all cases of colorectal carcinoma, without respect to age at diagnosis [22]. Accurate patient identification with Lynch Syndrome (LH) is extremely important as it can increase survival and enhance quality of live [23, 24].

In our study, 28% of cases were categorized as MSI-H, 20% were MSI-L and 52% were MSS by MSI analysis. Clinicopathological features of MSI tumors were different from MSS ones, especially for tumor size, grade, T-classification, stage and differences were statistically significant. Compared with MSS tumors, MSI-H cancers were more frequently located in right colon, poorly differentiated tumors, early TNM stage as well as infrequent distant metastases. On the other hand the clinicopathologic characteristics of MSS tumors had no significant differences compared either to MSI-H or MSI-L CRC in gender, age, site of the tumor, lymph nodes metastases or Duke`s classification. In study of Karahan et.al., [20] and in respect to our study, cases of CRCs that were localized in right colon, did not express at least one MMR marker, and had poorly differentiating carcinoma morphology. On the other hand, Soliman et. al study ,[25], showed significant correlation between MMR expression and lymphovascular emboli, tumor grade, N stage, T stage, tumor infiltrating lymphocytes, signet ring component and peritumoral lesion. They also showed, that right sided location, lower grade, higher nodal stage and marked infiltrating lymphocytes were chosen as MSH-H colorectal carcinoma predictors.

Study of Garcia et. al., [26], showed significant association of MMR protein expression with right sided colon location, poor differentiation and mucinous histology but not with gender, age, stage, lymphocytic infiltrate or lymphovascular invasion.

Our Study in accordance with Karahan et al., [20] none of MMR markers used had statistical significant relationship with lymph node metastases. Also in another study of Ramezani et al.,[3] which demonstrated that MSH6 had insignificant correlation with lymph node metastases but with better prognosis.

In our study, Patients with MSI-H and MSI-L colon carcinoma have relatively less advanced stage compared to those with MSS tumors. This is similar to previous researches of Malesci et al., Ogino et al., and Yuan et al., [27, 28, 21], These results explain relative better prognosis of MSI- correlated carcinoma compare with MSS ones.

In our current study we investigate expression of the four MMR proteins using IHC, including MSH-6, MSH-2,MLH-1,PMS-2. Of 50 tumors with abnormal MMR protein expression, 14 were classified as MSI-H, 10 were MSI-L and 26 were MSS by MSI analysis. Among 14 MSI-H tumors, abnormal MRR protein expression is present, with the most frequent expression pattern was concurrent loss of MSH-6 and PMS-2 proteins. Among MSI-L tumors 8% exhibited loss of MSH-2 and 6% exhibited loss of PMS-2 without any MSH-6 or MLH-1 loss. Of total 12 tumors that showed loss of MSH-2 expression, six were PMS-2 negative and four cases were negative for MSH-6. Eight cases lacked expression of both MSH-6 and PMS-2 while only 2 cases of 50 tumors examined showed loss of MLH-1 expression. In Yuan et. al., study [21], concurrent loss of MLH-1 and PMS-2 is the most frequent. This is followed by concurrent loss of expression of MSH-2 and MSH-6. This study detected also that isolated MSH-6 loss was present in 8.3%, followed by isolated negative expression of PMS-2 (1.4%).

The results from another study demonstrated that loss of MLH-1 and PMS-2 expression are more common than those of MSH-2 and MSH-6 in colorectal carcinoma [29]. Garcia et. al., [26] also showed loss of MMR proteins expression in 8.7% of patients of caner mostly MLH-1 and PMS-2. These findings are consistent with the molecular properties of MMR proteins, as researches in vivo and vitro proved that MMR gene products in the cells are always present as heterodimers complex. Also, MSH-2 and MLH-1 are obligatory patterns, combined with their secondary patterns MSH-6 and PMS-2 respectively. So, if respective MMR gene is mutated and causes degeneration of the earlier patterns, the latter patterns will no longer exist [26].

Ki-67 protein expression is associated with proliferation in tumor cells, and can be used as a marker for tumor aggression. Every cell cycle's active phase (G1, S, G2, and M) contained Ki-67, whereas resting phase lacked it (G0) [30]. Correlation between Ki-67 and prognosis of patients with cancer colon was still contradictory in various studies [31]. However, other reports showed that high expression of Ki-67 was associated with poor prognosis of colon cancer patients [32]. In our study, we evaluated relationship between microsatellite status and Ki-67 expression in colorectal carcinoma in order to clarify biological profile of MSI-positive tumors. Our results revealed that among low expression of Ki-67 tumors (˂ 25%), eight cases were MSI-H, 10 cases were MSI-L and 10 cases were MSS, and there was negative correlation between Ki-67 low expression and all MSI status patterns.

On the other hand, the high expression of Ki-67 (≥ 25%) were assessed in 6 cases of MSI-H and 16 cases of MSS, and the correlation is significant between Ki-67 high expression and MSI status patterns. Previous study of Takagi et. al., [33] shown that colorectal carcinoma with MSI exhibit elevated Ki-67 expression regardless of hereditary or non-familial cancer types, pointing to their shared molecular features that set them apart from MSI-negative tumors [33]. However, other study showed that MSI is not correlated with Ki-67 expression and could not improve diagnostic accuracy of colorectal carcinoma, but Ki-67 expression alone is significantly high in poorly differentiated colorectal carcinoma and correlate with presence of metastases [29, 30].

In conclusion, mismatch repair defective colorectal carcinoma has characteristics clinicopathological features different from MSS tumors. The most frequent pattern in MSI-H tumors was loss expression of MSH-6 and PMS-2 protein. This study high light the role of immunohistochemistry for screening MMR protein status in patients with cancer colon. However, results of our study must be supported with studies conducted in large series for detecting MMR deficiency in CRC. Accurate identification of MMR protein status is extremely important for colon cancer patients being increase survival and improve quality of life.

MMR

Mismatch repair

MSI

Microsatellite instability

CRC

Colorectal carcinoma

MSH6

MutS homolog 6

MSH2

MutS homolog 2

PMS2

Postmeiotic segregation increased 2

MLH1

MutL protein homolog 1

MSI-L

Low Microsatellite instability

MSI-H

High Microsatellite instability

IHC

Immunohistochemistry

Lynch syndrome

PCR

polymerase chain reaction

Proliferation index

Confidence intervals

All authors have contributed to and approved the final manuscript

Ethics approval and consent to participate:

This study was approved by the ethics committee of National Research Center (Approval No.16/308).

Consent for publication: Not applicable.

Availability of data and materials:

The datasets used and/or analyzed in the current study are available from the corresponding author on reasonable request.

Competing interests:

All authors declare that they have no competing interests.

Funding: Not Applicable

Authors` contributions:

NNY and MES designed the study. NNY, DMA and ASAS Collection of specimen blocks, Collection of data, examination of slides. NNY, MES, and DMA analyzed the data. NFA and NNY wrote the manuscript. All authors read and approved the final manuscript.

Acknowledgements: Not Applicable

Author information:

Authors and Affiliations: Pathology Department, Medical Research and clinical studies institute, National Research Centre, Cairo, Egypt. 33 El Buhouth St, Ad Doqi, Dokki, Cairo Governorate 12622

Noha N. Yassen *, Dalia M. Abouelfadl, Naglaa F. Abbas, Ahmed S.A Soliman , Marwa E. Shabana

Corresponding author:

Correspondence to Noha N. Yassen

Manuscript should be published in Bulletin of the National Research Centre Journal as it goes with aim and scope of the journal, it deals with more than one of the topics of journal interest; Anatomic Pathology also Molecular, cellular and cancer biology.

it is an original and unpublished work has not been published previously in print or electronic format as well as it is not under consideration by another publication or electronic medium.

There is no declaration of any potential competing interests.

All authors have approved the manuscript for submission.

The content of the manuscript has not been published, or submitted for publication elsewhere

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published 25 Oct, 2023

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Editorial decision: Major revision
25 Sep, 2023
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21 Aug, 2023
Reviewers invited by journal
09 Aug, 2023
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29 Jun, 2023
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Microsatellite instability screening in colorectal carcinoma: lmmunohistochemical analysis of MMR proteins in correlation with clinicopathological features and Ki 67 protein expression.

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Abstract

Background

Methods

Results

Conclusions

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Background

Material and methods

Specimens and ethical approval:

Histopathology:

Immunohistochemical interpretation:

Immunohistomorphometric Quantitative Analysis of Ki67 expression:

Immunohistochemical analysis:

Statistical Methods:

Results

Discussion

Conclusions

Abbreviations

Declarations

References

Status:

Journal Publication

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