The Importance of Evaluating Serum Levels of Tumor Markers M2-PK and Inhibin A in Patients Undergoing Colonoscopy

Abstract

Despite the use of colonoscopy to detect colon cancer due to its aggressiveness, high cost, and lack of patient compliance, the use of laboratory tests with high accuracy and sensitivity, such as tumor marker M2-PK and Inhibin A is recommended and can be effective for early diagnosis and screening of patients in the early stages. We studied 46 patients admitted it the gastrointestinal ward of Amir al Momenin Hospital and 45 normal (age and sex-matched) subjects as a control group (case-control and retrospective studies). Before the colonoscopy, the level of tumor marker M2-PK in the stool sample and the serum level of Inhibin A were evaluated in patients and the control group. The level of tumor marker M2-PK was significantly higher in the group with hyperplastic polyps and colon cancer (P < .001) than in the control group. At the same time, there was no significant difference in Inhibin A level (P = .054). In the hyperplastic polyps group 73% and in the colorectal cancer group 27% had a positive immunochemical fecal occult blood (IFOBT) result, significantly higher than the control group (P < .001). Evaluation of the level of tumor marker M2-PK in the stool sample in association with the three-time iFOBT test method may be suggested as a quick and noninvasive method for screening and diagnosis of polyps and early stages of colon cancer.

Introduction

Colon cancer is the third most common cancer worldwide, with 875,000 new cases each year. ¹ According to the condition of the disease and its history, early diagnosis, screening and prevention are essential and most colon cancers originate from benign polyps and turn into adenocarcinoma as the histology progresses step by step. ²

Colon cancer is the third leading cause of death in Iran, with a prevalence of eight in every 10,000 people. ³ High prevalence of the disease, it is necessary to use laboratory screening tests with high sensitivity and specificity to diagnose the condition in the early stages, and also consider the critical role of these tests in the early diagnosis of polyps before they become cancerous and the diagnosis in the early stages increases the response to treatment is related to the chances of survival and recovery of patients.^4,5

Even though sigmoidoscopy and colonoscopy are considered ideal screening methods, due to the noninvasiveness and noncompliance of the patient, as well as the limited capacity and conditions of its results, in some areas, requesting and performing noninvasive screening tests is of great interest.^6,7

One of the noninvasive laboratory tests is determining the serum level of pyruvate kinase isozyme m2 in the stool sample.^8–10 M2-PK is a protein that binds to phosphotyrosine and causes the activation of catenin's in the nucleus of cells. This isozyme is expressed in proliferating cells and is generally in tetramer form. This isozyme loses its tissue specificity in the cancer cell and is expressed as a dimer.^11–13

Also, considering the importance of tyrosine kinases in the phosphorylation of adaptor proteins of signaling pathways and tumor growth, Inhibin A is a ligand of the transforming growth factor beta family expressed in many cancers and acts as an angiogenesis regulator.^14,15

The rationale for the investigation was the evaluation of serum levels of tumor markers M2-PK and Inhibin A in patients undergoing colonoscopy and the suggestion as a quick and noninvasive paraclinical and laboratory test for screening and diagnosis of polyps in early stages of benign and malignant colorectal anomaly.

Patients and Method

forty-six patients with gastrointestinal disorders were recruited from July 2019 to March 2021 in the gastrointestinal ward of Amir al Momenin Hospital, (Table 1). The study was case-control and retrospective.

Table 1.

Main Characteristics of the Study Subjects.

Summary of study population	Patient	Control	Colon cancer patient	Hyperplastic polyps patient
Number	46	45	3 (6.5%)	43 (71.6%)
Age (mean years)	56.4 (45-66)	57.2 (44-67)
Sex	32 M; 14 F	31 M; 14 F
Total	Male 63(68.7%) female 28 (31.3%)

The sample size was calculated by comparing two means with alpha = 0.05, beta = 0.2, and effect size = 0.59 using the G*Power (v3.1,7). Informed consent was to participate and used their results and laboratory samples in this study were obtained from every patient in accordance with the Deceleration of Helsinki.

Eligibility criteria for the included patient were randomization, any intestinal signs, and symptoms of abdominal pain, occult bleeding, chronic constipation or diarrhea, and other intestinal problems its necessary to screen for malignancy, while Patients who had previously undergone colon resection surgery or chemotherapy, as well as IBD, ulcerative colitis and Crohn's patient were excluded from this study.

In this study we followed relevant equator guidelines and the reporting of this study conforms to standards for reporting diagnostic accuracy studies (STARD) guidelines. ¹⁶

Forty-five normal subjects (age- and sex-matched) as the control group were also selected for immunoassay analysis.

Stool samples were collected from all patients for testing before the bowel preparation regimen for colonoscopy. A plastic container was used to collect the stool sample and a small amount of stool was used to perform the iFOBT/M2-PK test. The stool sample was extracted from the stool sample according to the standard protocol and performed by the enzyme immunoassay method of the German company ScheBo^®, and the positive results were defined as M2-PK > 4 U/ml. Also, venous blood samples were taken from all patients, and their serum was prepared and frozen at −20°C temperature until the samples were analyzed.

Subsequently, the serum level of Inhibin A was measured using the enzyme immunoassay method of Diagnostic system laboratories, Inc., Webster, TX, USA. The test was done in one step and once, but in a complete and standard way we are sure that de-identified patients’ details and the identity of any person may not be ascertained in any way.

Statistical Analysis

Statistical analysis was done using SPSS (Statistical Package for Social Science) version 23: (Chicago, IL, USA); for windows statistical software package. For quantitative data, the mean and standard deviation were reported, and for qualitative data, the number and percentage were reported.

Kolmogorov–Smirnov and Shapiro–Wilks tests were used to check the assumption of normality. Between-group tests were evaluated for quantitative variables using the Kruskal–Wallis test, and association for qualitative variables was assessed using Fisher's exact test at a significance level of 5%.

Results

Table 2 shows a summary of results for patient and control groups.

Table 2.

Median ± IQR Values and Fisher's Exact Test of m2-PK, Inhibin A, Hemoglobin and iFOBT in Control and Patients Group With Colon Cancer and Hyperplastic Polyps.

	Colon cancer patient	Hyperplastic polyps patient	Control	P -value
M2-PK IU/mL	68 ± 3.53	5.3 ± 4.15	2.2 ± 1.95	.0001
Inhibin A pg/mL	4.1 ± 1.42	6.1 ± 3.48	5.1 ± 4.25	.054
Hb g/dL	10.1 ± 1.50	15.2 ± 2.65	15.4 ± 2.65	.010
iFOBT Positive (%)	3 (27)	8 (73)	0 (0)	.0001

Values are expressed as median ± IQR.

Abbreviations: g/dL, grams per decilitre; Hb, hemoglobin; iFOBT, immunochemical fecal occult blood; IQR, interquartile range; IU/mL, International units per milliliter; M2-PK, M2-pyruvatkinase; pg/mL, picograms per milliliter.

The median levels of stool m2-PK were higher in the patient group (colon cancer patient and group of hyperplastic polyps patient) compared with the control group (P < .001).

However, the median serum levels of Inhibin A no significant difference in the patient group (colon cancer patient and group of hyperplastic polyps’ patient) compared with the control group (P < .054).

According to the description of the kit used and the manufacturer level of M2-PK less than or equal 4 U/ml is negative and the level of M2-PK greater than 4 U/ml is positive.

According to the description of the kit used and the manufacturer A reference concentration of 4 U/ml corresponds to a sensitivity of 80% for diagnosing colorectal cancer with stool sample.

Discussion

The increase in tumor marker M2-PK level is considered an indicator of the high level of anaerobic glycolysis during the first stages of polypogenesis and tumorigenesis. The dimer isoform of M2-PK is the predominant form in the early stages of tumorigenesis, and it is metabolically inactive and has a lower affinity to phosphoenol pyruvate.^17–19 Therefore, it accumulates in cells and is related to increased cell proliferation.

The observed high stool levels of M2-PK concentration indicate the presence of a benign or malignant generation process. The occurrence of an increase in M2-PK level status is frequently observed in patients with cancer (both solid and hematological malignancies) which could be resulted from the effect of the tumor cell activity on the glycolytic and metabolic system.^20–22 The findings of the high level of M2-PK state in this study agree with previous reports on colorectal cancer patients.^23–26

An increased amount of inflammatory cytokines, released in response to the tumor and changing the metabolic process of transformed cells in the early stages of polypogenesis and tumorigenesis play an important role in the metabolic system activation in the early stage of colorectal cancer patients.^27,28

In the studies conducted, M2-PK serum level is high in the bladder, pancreas and kidney tumors, and its status in serum, urine, and stool samples is associated with high sensitivity.^29–32 Also, the combined use of examining m2-PK levels with the other tumor markers such as carcinoembryonic antigen, CA19-9, lactate dehydrogenase, and CA72-4 can be used to differentiate the presence of any tumoral or hyperproliferative lesion in the gastrointestinal system.^33,34

Despite the positive role of Inhibin A serum level in examining pregnancy status and diagnosing chromosomal disorders such as Down's syndrome and gynecological and adrenal disorders, in our study, no significant correlation was observed between its serum levels in the patient group and the control group. Since colonoscopy is currently used as a method with high cost and time consuing to evaluate the initial classification of polyps and tumoral lesions in the intestine. according to the change in the metabolic process of transformed cells in the early stages of tumorigenesis, the presence of M2-PK in stool warrants further evaluation for diagnosis and those with the positive result of iFOBT and M2-PK test should undergo complete diagnostic evaluation.

The features of the fecal M2-PK test is highly sensitive, fast, and easy to use in clinical and paraclinical settings and is deemed suitable as a primary screening tool and a complimentary test for polypoid lesion and colorectal cancer.‏

Selection bias is a particular problem inherent in case-control studies, where it gives rise to noncomparability between cases and controls. In case-control studies, controls should be drawn from the same population as the cases, so they are representative of the population which produced the cases. In this study, we tried to minimize the information and confirmation bias by carefully examining the patients and the results obtained from the paraclinical. And by strictly observing the include and exclude criteria of the study, we have minimized the bias of selecting patients and the control group.

Limitations of the Study

The limitation of this study is the smaller sample size, patients were recruited from a single center and high cost of laboratory kits and equipment and the late provision of research funding. Thereby, further study with patient recruits from multiple centers and with more sample size is needed.

Conclusion

Our data propose evaluating M2-PK isoenzyme levels may be as a suitable tumor marker in both stool and serum samples as a noninvasive, rapid, and low-cost paraclinical method for the initial screening of polypoid lesions and colorectal cancer.

Abbreviations

IFOBT

immunochemical fecal occult blood

M2-PK

M2-pyruvatkinase

IU/mL

international units per milliliter

pg/mL

picograms per milliliter

g/dL

grams per decilitre

IQR

interquartile range

STARD

standards for reporting diagnostic accuracy studies

U/mL

units per millilitre

CA

cancer antigen

CEA

carcinoembryonic antigen

LDH

lactate dehydrogenase

Hb

hemoglobin

TGF-β

transforming growth factor beta

SPSS

statistics is software for advanced statistical analysis