Expression of minichromosome maintenance proteins in the exfoliated cells supplement sputum cytology in the diagnosis of lung cancer

INTRODUCTION

Lung cancer is the leading cause of cancer-related deaths worldwide, with approximately 2.2 million new cases and 1.8 million deaths reported in 2020.^[1] It represents a group of heterogeneous entities in terms of histology and molecular profile.^[2] Primary lung cancers are classified into two main histological types: Non-small cell lung carcinoma (NSCLC) and small cell lung carcinoma (SCLC), representing 85% and 15% of all cases, respectively. NSCLC can be further divided into subtypes, including lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), and large-cell carcinoma.^[2] Low survival rates for both NSCLC and SCLC have been linked to late diagnosis and subpar response to conventional chemotherapy and radiation. This emphasizes how crucial it is to identify novel biomarkers and possible therapeutic targets to enhance the prognosis of lung cancer patients. Despite all the NSCLCs have molecularly characterized pre-invasive lesions, they are often asymptomatic and undetectable by the current noninvasive methods. Lots of researches are in progress to identify lung cancer early detection methods. Unfortunately, most of them are imaging technology-based ones and low-dose computed tomography has been identified as a sensitive technique for the early detection of stage 0 lung cancers, and this has been suggested as a screening method. Sputum is a desirable prospective source of lung cancer biomarkers due to its noninvasive nature and propensity to represent the site of injury.^[3] Various research teams have examined sputum cytology as a potential technique for early lung cancer detection.^[4] However, none of these studies were found fruitful as sputum cytology suffers a lack of sensitivity to be used for early detection. The reason for the lower sensitivity was the lack of adequate laboratory techniques to fish out the whole cell content without any morphological distortion. Furthermore, it was challenging to distinguish between reactive benign cells and malignant cells due to the morphological changes brought about by laboratory processing techniques, as well as the inherent reactive and degenerative changes caused by the exfoliated cells clogged on mucus. There were no other methods available to enhance the morphological assessment. Hence, the cytologist used to report the same as to contain atypical cells. An average of 20–30% of sputum samples are being reported to have such atypical cells without a definite diagnosis in the pathology division of our hospital.^[5] However, now, the liquid-based cytology (LBC) methods offer better technology to extract the whole cell content of sputum samples without much distortion. Furthermore, there are ancillary techniques to supplement the conventional method. We recently developed a modified sputum processing method that preserves cells for additional molecular analyses and enables the characterization of specific proteins to complement conventional morphological evaluation.^[5]

Initiating DNA replication and cell proliferation requires the presence of minichromosome maintenance proteins (MCMs), a group of proteins involved in minichromosome maintenance. They are referred to as the DNA replication-licensing factors by joining the origin recognition complex with additional licensing factors to form the pre-replicative complex, which regulates DNA replication and only permits it to take place once every cell cycle.^[6] In addition, they play a role in the cohesion, condensation, transcription, and recombination of DNA molecules and are necessary for replication elongation. A trimeric core complex made up of the MCM4, MCM6, and MCM7 subunits is coupled with a peripheral dimer of the MCM3 and MCM5 to produce a hexameric MCM complex.^[7] Throughout the cell cycle, all MCM proteins are invariably present in the nucleus. Because MCM proteins are essential for initiating DNA replication in proliferating cells but are absent in quiescent cells, they serve as reliable markers of cell proliferation.^[8] In a number of cancers, such as lung, ovarian, colon, urothelial, and oral cancers, the expression level of MCMs has been linked to important clinicopathological parameters and demonstrated considerable diagnostic and prognostic value.^[9-11] In addition, MCMs may serve as indicators for recurrent and precancerous illnesses, according to a number of lines of research, and abnormal expressions of MCMs have been linked to the onset and development of several cancers.^[9,12] Particularly in humans and other eukaryotes, MCM2 and MCM7 are engaged in a variety of biological processes.^[13] MCM2 is highly expressed during the proliferation of premalignant lung cells and is considered a potential diagnostic marker for the early detection of malignant pulmonary lesions, whereas MCM7 expression in bronchial brushings of NSCLC patients is associated with prognosis.^[14] However, the application of MCM7 protein evaluation in sputum cytology still remains unfathomed.

The current work sought to characterize malignant cells in sputum using MCM2 and MCM7. Through the identification of these proteins, the research aimed to improve traditional sputum cytology. Furthermore, the expression of these proteins was investigated in relation to various clinicopathological characteristics.

MATERIAL AND METHODS

RESULTS

Intensity of MCM expression in different tumor types

The expressions of MCM2 and MCM7 were confined exclusively to the nuclei, with no background staining observed. A positive MCM2 marker was detected in 62.9% of the subjects (61 out of 97), while overexpression of MCM7 was observed in 72.2% of the subjects (70 out of 97). The intensity of expression varied among different tumor types. Notably, LUAD samples exhibited the highest expression of MCM7, with a mean H-score of 184.3 [Table 2 and Supplementary Figure 1].

Table 2: Mean H score/SD of markers for different lung lesions in sputum samples.

Diagnosis	Number of specimens	MCM 2 mean H score (SD)	MCM 7 mean H score (SD)
LUAD	19	123.9 (70.3)	184.3 (66.3)
LUSC	23	93.1 (53.5)	155.2 (68.9)
NSCLC	10	77 (37.6)	113 (39.2)
Atypical cells	24	22.5 (10.7)	53.5 (30.9)
NMC	21	0 (0)	0 (0)

MCM2: Minichromosome maintenance protein 2, MCM7: Minichromosome maintenance protein 7, LUAD: Lung adenocarcinoma, LUSC: Lung squamous cell carcinoma, NSCLC: Non-small cell lung carcinoma, NMC: Negative for malignant cells, SD: Standard deviation, H Score: Histology score

SUPPLEMENTARY FIGURE

Download

The patterns of MCM2 and MCM7 expression showed varying degrees of intensity, including mild, moderate, and dense expressions in tumor cell nuclei. With a few exceptions, where weak localized expression was detected in a minimal set of cells, neither marker was expressed in samples that were negative for malignancy [Figure 1]. Hematoxylin and eosin (H&E) staining demonstrated the cellular morphology of LUAD, LUSC, NSCLC, atypical cells, and non-malignant cells ([Figure 1a, d, g, j, m], respectively). Moderate nuclear expression of MCM2 [Figure 1b] and dense nuclear expression of MCM7 [Figure 1c] were observed in adenocarcinoma cells. In squamous cell carcinoma cells, moderate nuclear expression of MCM2 and MCM7 was noted [Figure 1e and f]. Mild nuclear expression of both markers was seen in NSCLC cells [Figure 1h and i], while atypical cells exhibited weak or mild nuclear expression of MCM2 and MCM7 [Figure 1k and l], though this was limited to a small number of cells. In contrast, non-malignant samples were negative for both MCM2 and MCM7 [Figure 1n and o].

Close

Figure 1:

(a-o) Cellblock sections of sputum samples of lung cancer patients showing MCM2 and MCM7 expressions. (a, d, g, j, m) H&E, magnification 20x of sputum cell blocks in LUAD, LUSC, NSCLC, Atypical and NMC subtypes. (b) Moderate nuclear expression of MCM2 in Adenocarcinoma cells. (c) Dense nuclear expression of MCM7 in Adenocarcinoma cells. (e and f) Moderate nuclear expression of MCM2 and MCM7 in squamous cell carcinoma cells. (h and i) Mild nuclear expression of MCM2 and MCM7 in NSCLC cells. (k and l) Weak/Mild nuclear expression of MCM2 and MCM7 in atypical cells. (n and o) MCM2 and MCM7 negative in non-malignant samples (Scale bar- 100 µm). The arrow points the malignant cells in the cellblock. (H&E: Hematoxylin and eosin, MCM2: Minichromosome maintenance protein 2, MCM7: Minichromosome maintenance protein 7, LUAD: Lung adenocarcinoma, LUSC: Lung squamous cell carcinoma, NSCLC: Non-small cell lung carcinoma, NMC: Negative for malignant cells).

Export to PPT

DISCUSSION

Replicative immortality and sustained proliferative signaling are the well-established hallmarks of cancer.^[15,16] DNA replication is a key factor in cell proliferation, which occurs more frequently in malignant cells compared to healthy ones. Cancer cells have aberrant expression of several proteins involved in DNA replication. One example is the replication factor MCM7, which forms the pre-replication complex during the progression of the cell cycle and attaches to the double-stranded DNA at the replication origins in the late G1 phase.^[17] It is highly expressed in various malignancies and may serve as a potential marker for cell proliferation.^[18,19] Further, MCM7 has been demonstrated as a proliferation marker in several cancers, including lymphoma, ovarian cancer, prostate cancer, lung cancer, etc., equivalent to the established proliferation markers like Kiel-67 (Ki-67) and proliferating cell nuclear antigen.^[10]

MCM proteins are more sensitive markers of proliferation than Ki-67 because MCM proteins can identify a broader range of proliferative cells, including those that may not be detected by Ki-67, especially in the early G1 phase.^[20] MCM proteins have been studied in various types of lung cancers, including NSCLC and SCLC. Their expression is often higher in more aggressive forms. Ki-67 is also used across different lung cancer types, but its expression can differ significantly among diverse tumor types and even within distinct areas of the same tumor.^[21] A meta-analysis by Martin et al. examined the prognostic value of Ki-67 expression in lung cancer, providing a comprehensive overview of its role in predicting patient outcomes.^[22] Another study by Stoeber et al. investigated the diagnostic potential of MCM proteins in cancer, suggesting broader applications beyond lung cancer.^[23] The use of MCM proteins in clinical practice is still evolving, but they offer potential as diagnostic and prognostic markers, possibly complementing or even surpassing Ki-67 in certain cases.

MCM7, unlike Ki-67, is expressed throughout the entire cell cycle (except G0), making it a more sensitive marker for detecting cells that are actively proliferating. This is particularly important in lung cancer, where the early detection of proliferative cells can significantly influence prognosis and treatment strategies. Williams G. H. and Stoeber K. emphasized the broader sensitivity of MCM proteins, including MCM7, in identifying proliferative cells across different phases of the cell cycle, which could improve the accuracy of diagnosis of lung cancer. In addition to its diagnostic and prognostic value, MCM7 may also serve as a therapeutic target. Since MCM7 plays a crucial role in DNA replication, targeting it could inhibit cancer cell proliferation, providing a novel approach to lung cancer treatment.^[24] Samad et al. discussed the potential of targeting MCM7 in lung cancer therapy, noting that its inhibition could disrupt tumor growth by interfering with DNA replication processes.^[25]

Cytological analysis of sputum increases the lung cancer detection rate, and hence, it is considered a poor man’s bronchoscopy.^[26] Veena et al., in 2019, suggested an advanced technique for sputum processing to address the shortcomings of sputum cytology and the uncertainty of atypical samples.^[27] Recently, there has been a lot of focus on finding certain molecular markers to add to the traditional morphological study of sputum to increase the technique’s sensitivity and specificity. LBC of sputum samples provide monolayered smear without much mucus and other debris in the smear background, which reduces the likelihood of diagnostic errors. Furthermore, the cells clogged in mucus can be obtained in the smear without much reactive changes. Computerized screening of sputum cytology slides may be possible with LBC. Cell blocks prepared in cell buttons processed in the LBC method provided clear sections without much background materials of mucus and inflammatory cells. In the current study, we have noticed a higher expression of MCM7 protein in all the lung cancer sputum samples analyzed, which was followed by MCM2 expression which supports the previous reports. MCM2 was found to be an independent predictor of survival in patients with NSCLC and a prospective diagnostic marker for premalignant lung lesions.^[28,29] According to Toyokawa et al., both the messenger ribonucleic acid (mRNA) and protein levels of MCM7 expression were significantly greater in various cancer tissues compared to normal tissues.^[10] However, we could not find MCM7 expression in benign respiratory epithelium in any of the sputum samples. The observation of higher MCM7 H score values being positively linked to tumor type in lung sputum samples is in consistence with other earlier reports in lung tissue samples from Liu et al.^[30]

Our investigation also demonstrated a noteworthy correlation of MCM2 with tumor cytology and MCM 7 with tumor stage and cytological type. The prognosis can be predicted using this observation. These results corroborate earlier research that revealed MCM7 markers could be utilized to forecast tumor growth and patients’ prognosis with NSCLC.^[10] A substantial association between MCM2 expression and a poor prognosis in NSCLC patients was also discovered by Yang et al., indicating that tumor proliferation may be a crucial factor in determining the prognosis of NSCLC.^[31] The poor survival rate for NSCLC patients is associated with the late-stage presentation of patients with unresectable tumors because there is no validated screening method for early diagnosis, making small biopsies/cytology samples more essential.^[32]

MCM protein sensitivity in sputum samples has also been examined as a potential predictor of malignant lesions. Cytology samples are more sensitive than histology samples because MCM-positive cells typically arise at the surface of the aberrant epithelia.^[33] For this reason, MCMs are extremely promising indicators for the early identification of cancer and precancerous conditions in cytology samples. In our evaluation of sputum samples from patients with various subtypes of lung cancer, we found that the MCM7 protein demonstrated a sensitivity of 92% and a specificity of 100% for diagnosing malignancy. Another study showed that cell division cycle 6 protein demonstrated the highest sensitivity of 87.7% in sputum samples for characterization of malignant cells followed by MCM5 and MCM2 with 66.67% and 58.89%, respectively.^[34]

The current study could recognize some of the samples of sputum reported with an indiscriminate diagnosis of atypical cells to a definite diagnosis of malignancy, suggesting the significance of MCM7 in the differential diagnosis of malignancy in sputum samples. This information can be used to define some alternative techniques for the detection of malignant cells in sputum, particularly for people with clinical complaints and abnormal X-ray results in low-resource environments. Incorporation of MCM7 immunocytochemistry in routine clinical practice will certainly enhance the diagnostic efficacy of sputum cytology, which is comparatively easier, cost-effective, and highly economical compared to bronchoscopy and biopsy for the diagnosis of lung cancer, and it can be repeated easily if required without any hardship to the patient. While this research offers insightful information about using MCM2 and MCM7 as biomarkers to enhance traditional sputum cytology, there are some limitations which include the relatively small sample size in each type of tumors, lack of early-stage tumors and precancerous conditions, and lack of follow-up. A multicentric study in a large cohort from a broader population with follow-up of patients is warranted to generalize the significance of MCM proteins in lung cancer diagnosis.

SUMMARY

To our knowledge, this is the first study to report MCM7 expression in sputum cells, highlighting its potential to significantly increase the sensitivity of sputum cytology. The incorporation of MCM7 protein analysis into conventional sputum cytology could greatly improve the detection of malignant cells, thus supplementing morphological evaluation methods. This is particularly valuable for samples with atypical cells, where traditional cytology alone might not provide definitive results. By increasing the sensitivity of sputum cytology, MCM7 can help ensure that more cases of lung cancer are accurately identified at an earlier stage. Furthermore, our analysis suggests that examining MCM7 expression in sputum samples from patients with biopsy-proven premalignant lung conditions could establish this protein as a useful marker for primary screening of lung cancer. This application has the potential to facilitate earlier detection and intervention, thereby improving patient outcomes. In addition to its diagnostic applications, the potential of MCM7 in forecasting the clinical behavior of lung lesions, as well as its prognostic value in terms of survival, warrants further investigation. Exploring these aspects could pave the way to the development of more precise and personalized treatment approaches for lung cancer patients, ultimately enhancing prognosis and survival rates.

AVAILABILITY OF DATA AND MATERIALS

The data analyzed in this study can be accessed through the corresponding author upon reasonable request.

ABBREVIATIONS

AUC: Area under the curve

CDC6: Cell division cycle 6

CT: Computed tomography

DPX: Dibutylphthalate polystyrene xylene

Ki-67: Kiel-67

LBC: Liquid-based cytology

LUAD: Lung adenocarcinoma

LUSC: Lung squamous cell carcinoma

MCM2: Minichromosome maintenance protein 2

MCM7: Minichromosome maintenance protein 7

MCMs: Minichromosome maintenance proteins

NMC: Negative for malignant cells

NPV: Negative predictive value

NSCLC: Non small cell lung carcinoma

PCNA: Proliferating Cell Nuclear Antigen

PPV: Positive predictive value

ROC: Receiver operating characteristic

SCLC: Small cell lung carcinoma

AUTHOR CONTRIBUTIONS

NP: Wet lab works, data collection and analysis, interpretation of results, original manuscript preparation, review and editing; AL: Supervision of wet lab works, discussion drafting; JKKM: Statistical data analysis; VVS: Immunocytochemical data analysis; KS: Conceptualization, supervision, resources, review, and editing. All authors read and approved the final manuscript.