Micronucleus and Its Significance in Effusion Fluids.

BACKGROUND: Micronucleus (MN) is an extranuclear body within the cell formed due to failure of incorporation of whole chromosomes or their fragments during cell division. MN scoring can be done to identify malignant effusions. AIMS: This study aimed to score micronuclei to distinguish malignant effusion from benign effusions and to correlate MN score with type of malignant effusion. METHODS AND MATERIALS: A retrospective study was conducted on 30 malignant and 30 benign effusions. The number of micronucleated cells per 1,000 cells was counted in effusion smears stained with Papanicolaou stain under oil immersion (1,000×).
RESULTS: The mean MN score in malignant effusions was 3.77 with standard deviation (SD) of 2.13. The mean MN score in benign effusions was 0.50 with SD of 0.57. The difference in MN score between malignant and benign effusions is statistically significant (P < 0.001). A cut-off MN score of 6.5 was seen to distinguish malignant and benign effusions with 100% specificity and 100% sensitivity in this study.
CONCLUSIONS: MN score is higher in malignant effusions when compared with benign effusions. This can be used to differentiate malignant effusions from benign effusions in low resource setting. Copyright:

INTRODUCTION

An effusion is an excessive accumulation of lymphatic fluid in the body cavities, which can be exudates or transudates.[1] Effusion fluids are obtained by simple punctuation of serous cavities and aspiration. The cytological analysis is an inexpensive method which can be utilized for assessing the cause of effusions.[2] The diagnostic yield of effusion fluid cytology depends on histological type of malignancy, transportation time, experience of cytopathologist, use of fixative solutions, and cell blocks in addition to smear.[34]

A malignant pleural effusion is most commonly associated with adenocarcinoma of the lung and malignant ascitic effusion is associated commonly with colorectal or ovarian carcinoma. The diagnostic yield of pleural fluid cytology ranges from 40% to 87% and that of ascitic fluid cytology ranges from 56.7% to 60%.[5678] The parameters looked for in an effusion fluid sample are type of cells present, any abnormal increase in number of cells present and their arrangement, cell sizes, and its variability including cytoplasmic features – mucin or keratin formation; nuclear features – hyperchromasia, nuclear cytoplasmic ratio, mitoses, mesothelial cells.[9]

Distinguishing reactive from malignant cells is of great importance for management of cancer and better prognosis. The cell block technique involves fixation of fluid sample followed by centrifugation. The sections are prepared from the cell pellet obtained and are stained accordingly.[10] The use of immunocytochemical panels with monoclonal antibodies is done to identify and to type malignant cells but is expensive.[11]

Micronucleus (MN) is an extranuclear body present within the cell formed due to failure of incorporation of whole chromosomes or their fragments into the main nucleus during anaphase stage of cell division as they lack centromere. It is round to oval in shape with a mean diameter less than one-third of primary nucleus.[12] A close relationship was found between number of micronuclei and presence of chromosomal abnormalities, mutagen activity.[13] Cytological analysis of presence of MN within cells marks genomic instability, aneuploidy and has been associated with cancers, e.g., by studying cervical smears, exfoliated buccal cells, peripheral lymphocytes to detect chromosomal damage.[121415] Limited studies have been done on effusion fluids to distinguish malignant effusions from benign ones.[16171819] MN can also be identified alternatively by DNA cytometry to objectively assess genomic instability.[20] Fluorescence in situ hybridization (FISH) can also be used to detect micronuclei containing whole chromosome, with centromeric DNA pulses or antikinetichore-antibody.[21]

Identification of malignant cells by costly, invasive and immunocytochemistry panels can be avoided by scoring MN in effusions for malignancy.

MATERIAL AND METHODS

A retrospective study was conducted on the effusion fluids received in the department of pathology for detecting malignant cells in 2017–2018. The effusions were processed in the laboratory, which involves centrifugation to obtain supernatant and the cell pellet. The cell pellet was used for preparing smears, which were stained with conventional Papanicolaou stain. A total of 60 cases, sent for fluid analysis were considered for the study, 30 malignant and 30 benign cases each. All pleural and ascitic effusion fluid smears with good cellularity, received for detection of malignant cells, were reviewed. Smears with necrotic cells, degenerated cells, and overlapping cells were exempted from counting and scoring.

The smears were analyzed by light microscopy under oil immersion (1,000×) separately for micronuclei [Figure 1a, 1b] and score was calculated. The number of micronucleated cells per 1,000 cells was counted.

Figure 1

Micronucleus (indicated by arrow) present inside cell viewed under oil immersion (1,000×) with Papanicolaou stain in (a) ascitic effusion smear and (b) pleural effusion smear

MN can be detected by the following criteria:[12]

Diameter of MN around 1/3 to 1/16 of diameter of the primary nucleus.

Shape, color, and texture of MN similar to those of nucleus.

Should be in the same plane of focus as main nucleus.

Round to oval shaped with no actual contact with nucleus.

Intensity of staining similar to that of nucleus.

Cells lying singly were preferred.

Demographic details such as age, sex, clinical history, and other laboratory investigations details were collected from the case files. The smears of confirmed malignant and benign cases were reviewed.

The Institutional Ethics Committee issued certificate of approval for conducting the research.

Statistical analysis of data: All the quantitative variables, such as age of patient and number of micronucleated cells presented, are expressed as mean and standard deviation (SD). Differences in mean value between the groups were tested for statistical significance by student t-test. Associations of various factors were tested for statistical significance by Mann–Whitney test. Receiver operating characteristic (ROC) curve analysis was done to determine the cut-off point between the two groups.

RESULTS

The present study was done on a total of 60 effusions which included 30 malignant and 30 benign effusions, which included both pleural and ascitic effusions. Out of 60 effusions, we had 25 pleural effusions and 35 ascitic effusions. Ten of the pleural effusions were malignant and 15 were benign. From the total ascitic effusions, 20 were malignant ones and 15 were benign. The malignant effusions were associated with 7 cases of lung adenocarcinoma, 7 cases of colorectal carcinoma, 6 cases of ovarian carcinoma, 4 cases of metastatic breast cancer, 1 case of squamous cell lung carcinoma; and 5 cases with malignancy of unknown origin. There were no cases of mesotheliomas, leukemias, lymphomas, or sarcomas seen. The benign effusions were due to reactive inflammatory process. The age range was from a minimum of 20 years to a maximum of 88 years with a mean age of 54.65 years. Females had preponderance over males as 32 (53.3%) effusions were from females and 28 (46.7%) effusions were from males.

The mean MN score in malignant effusions was 3.77 with SD of 2.128 ranging from a maximum of 9 and minimum of 0. The mean MN score in benign effusions was 0.50 with SD of 0.572 ranging from a maximum of 2 and minimum of 0 [Table 1]. Student t-test scoring revealed P value <0.001 indicating that the difference in MN score between malignant and benign effusions is statistically significant. The mean MN score in malignant ascitic effusions was higher with a value of 4.85 than that of malignant pleural effusions which was 3.3. Mann–Whitney test scoring on MN score of malignant pleural and ascitic effusions revealed that the difference between them was not statistically significant. Using the ROC curve analysis, we found out a cut off of 6.5 for MN score giving a sensitivity of 100% and a specificity of 100% for these data collected.

Table 1

Result of statistical analysis between malignant and benign effusions

	Number of effusions (n)	Mean MN score	Standard deviation (SD)
Benign effusions	30	0.50	0.572
Malignant effusions	30	3.77	2.128

DISCUSSION

MN scoring has been done in various types of cancers on exfoliated buccal, cervical samples, and lymphocytes.[121415] The value of mean MN score has been high in cases of malignancy than in benign cases in all these studies conducted.

Limited studies have been done on MN scoring of effusion fluids. They include studies conducted by Kaur et al., Tyagi et al., Ganesan et al., and Kokenek et al.[16171819] Kaur et al. studied ascitic effusions with metastatic adenocarcinoma stained with MGG stain that included 20 malignant cases and 15 benign cases. They observed a mean MN score of 21 in malignant effusions and 2.93 in benign effusions.[16] The study conducted by Tyagi et al. on ascitic effusion fluids with use of MGG stain including 60 benign and 40 malignant fluids showed mean MN score in malignant group to be 13.2 and 0.57 in benign group.[17] Ganesan et al. studied 30 benign and 30 malignant effusions with use of Leishman staining for effusion smears. The mean MN score in malignant cases was 15.7 and 1.87 in benign reactive effusions.[18] The study conducted by Kokenek et al. with MGG stain was on 78 effusions with 48 ones with malignant outcome and 30 effusions with benign outcome. The mean MN score was 24.8 and 6.7 in effusions with malignant and benign outcome, respectively.[19]

Our results show increased MN score in malignant effusions, as also observed in other effusion studies, but not as high as seen in them [Table 2]. The cut-off MN score that can be used as a diagnostic tool with 100% sensitivity and 100% specificity as proposed by Ganesan et al. was 5.5 and that by Kokenek et al. was 11.[1819] We got a cut-off MN score of 6.5 for the same, whereas a score of 5.5 for 100% specificity and 76.7% sensitivity. These variations in mean MN score and cut-off value could be due to following factors. First, each type of malignancy is associated with different amounts of chromosomal aberrations. The histogenetic types of primary site of malignancies in our study were mainly adenocarcinoma and squamous cell carcinoma of the lung, colorectal adenocarcinoma, adenocarcinomas of the ovary, and ductal carcinoma of breast. The studies by Tyagi et al., and Kaur and Dey were only on ascitic effusions with adenocarcinoma, while study by Kokenek et al. was only on pleural effusions.[161719] The difference in the type of malignancy associated with the effusions can account for the difference in the cut-off values observed. Second, difference in fluid processing and staining – MGG stain and Leishman stain were used in other studies, while we have used Papanicolaou stain for staining effusion smears. Although MN count was higher with the use of Papanicolaou stain as shown in study conducted by Arul et al. on exfoliated buccal cells we got a lower score compared with MGG stained smears in other effusion studies.[14] A study conducted by Metgud et al. found that the mean micronuclei score in exfoliated buccal mucosal cells with DNA-nonspecific stain such as Giemsa stain was significantly higher than with DNA-specific stains like Feulgen stain, acridine orange.[22] Further studies using specific stains is recommended. Third, difference in fluid cellular composition due to variable mixing and overlapping of cells could have increased MN score in other studies.

Table 2

Comparison of MN score with previous effusion studies

Study	Sample size	Mean MN score	Range (min-max)	Cut-off value obtained by data
Kaur et al.	M-20	21	9-34
	B-15	2.93	0-10
Tyagi et al.	M-40	13.2	1-58
	B-60	0.57	0-5
Ganesan et al.	M-30	15.7	6-50	5.5
	B-30	1.87	0-5
Kokenek et al.	M-48	24.8	11-60	11
	B-30	6.4	1-10
Present study	M-30	3.77	0-9	6.5
	B-30	0.50	0-2

MN=Micronucleus; M=Malignant effusions; B=benign effusions

In our study, we have not included other markers of chromosomal instability, such as nuclear buds and nucleoplasmic bridges as done in study conducted by Tyagi et al.[17] Further studies need to be done to establish the most predictive marker of chromosomal instability in malignant effusions including DNA-cytometry.

We did not find any significant difference in MN score of malignant pleural and ascitic effusions neither with respect to age or gender.

The difficulties we came across while identifying MN were mostly due to various mimicking structures. These include apoptotic bodies [Figure 2], stain deposits, nuclear debris, overlapping cells, and other cells such as platelets and lymphocytes. It is important to differentiate these structures from MN for getting correct MN score. Overlapping cells are excluded from counting as they may give a false impression of MN. Platelets and lymphocytes can be identified by cell size and difference in cytoplasmic staining. Apoptotic bodies can be identified by absence of main nucleus of cell and stain deposits will not have any chromatin inside them. These clues help us to avoid false recognition of MN.

Figure 2

Apoptotic bodies – mimicker of MN (indicated by arrow)

We have not considered physical appearance of fluid, biochemical parameters of effusion fluid in relation to MN score, or malignancy in our study. Further studies are recommended on combining MN score with these parameters of effusions to increase overall diagnostic yield of effusion fluid analysis in cases of malignancy.

Our study shows that MN score can be used in diagnosis of malignant effusions, both pleural and ascitic, in a low resource setting.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.