Molecular testing on serous effusion: An update.

Cytological examination of the effusion fluid provides valuable information regarding the presence of malignancy. At times, it is challenging to diagnose malignant cells in serous effusion. The various ancillary techniques are available to solve the problem including immunocytochemistry, DNA ploidy, and multicolored flow cytometry. At present, the molecular tests on the effusion sample are of growing interest. The effusion sample is rich in cells and cell-free fluid that contains free DNA, cytokines, and extracellular vesicles. Molecular tests in effusion sample not only provide a diagnosis of malignancy but can also give valuable information that may be essential for the individualized therapy, management, and prognostic assessment. In this paper, we reviewed the application of the different molecular tests in the effusion sample.

INTRODUCTION

There are three major sources of serous effusions in the body: Pleural, peritoneal, and pericardial. Both neoplastic and non-neoplastic lesions may be responsible for the effusion. Out of the neoplastic causes, metastatic carcinomas are the far more frequent than mesothelioma. Cytological examination of the effusion fluid provides valuable information regarding the presence of malignancy. However, the sensitivity and specificity of cytological examination to detect malignancy are widely variable from 67% to 90% and 90 to 100%, respectively.[ It is mainly due to the overlapping cytological features of reactive mesothelial cells and malignant cells.[ The various ancillary techniques are available to solve the problem including immunocytochemistry,[ DNA ploidy, and multicolored flow cytometry. At present, the molecular tests on the effusion sample are of growing interest. The effusion sample is rich in cells and cell-free fluid that contains free DNA, cytokines, and extracellular vesicles. Molecular tests in effusion sample not only provide a diagnosis of malignancy but can also give valuable information that may be essential for the individualized therapy, management, and prognostic assessment. In this paper, we reviewed the different molecular tests that are done in the effusion sample.

CYTOLOGY SAMPLE FROM THE EFFUSION FLUID

Several different types of cytology sample preparation are available for the molecular tests from the effusion fluid that includes (1) cell block, (2) direct smear, (3) cytospin preparation, (4) cell pellet, (5) liquid-based cytology (LBC), and (6) liquid biopsy.

Cell block

It is the most widely used preparation for the molecular tests. The material for the cell block should be fixed in 10% neutral buffered formalin, and any fixative containing heavy metal should be avoided.[ The cellularity in the cell block preparation should always be assessed on the hematoxylin and eosin-stained smear. The cell block material can be used for future tests, and various immunocytochemistry can be done on cell block sections. The significant disadvantages of cellblock are as follows: (1) Immediate assessment of the cellularity is difficult as the cell block preparation takes at least 1 day time, (2) the absence of the whole nuclei and fragmentation of DNA, and (3) the formation of cross-linking of between nucleic acid and proteins.

Direct smear

In the absence of cell block preparation, the Diff-Quik stained slide or Papanicolaou’s stained slide can be used for the molecular tests. The Diff-Quik stained smears provide better quality DNA compared to Papanicolaou’s stained smear.[

The cells from the slide are collected by the scraping by scalpel or by lifting the cells with the help of Pinpoint solution.[ The direct smear has several advantages such as (1) quick assessment of cellularity, (2) less formalin interference, and (3) the dispersed tumor cells are easy to locate. However, direct smear needs the sacrifice of the slide and may have medicolegal consequence.

LBC (supernatant of effusion fluid)

Slides prepared from LBC platform provide a good source of DNA. The cells can be scrapped from the LBC slide for molecular tests or DNA can be extracted from the LBC solution.[ The CytoLyt fixative solution used in ThinPrep LBC platform (Hologic Inc., Marlborough, USA) usually provides several times the higher yield of DNA than the CytoRich fixative solution used in SurePath LBC platform (Becton, Dickinson and Company, NJ, USA).[

Hence, it is necessary to choose the proper fixative in LBC platform. The main advantages of LBC are as follows: (1) Rapid preparation of the slide, (2) direct visualization of the cells, and (3) intact cell nuclei.

Cytospin

The cytospin preparation provides multiple slides in a small predetermined area. The cells are concentrated and provide higher DNA yield.[

Cell pellet

It is the preliminary preparation for the cell block or smear preparation.

The cell pellet in the centrifuged specimen of effusion sample also contains an optimum amount of DNA material for the molecular tests.[

Liquid biopsy (supernatant of effusion fluid)

The cf-DNA may be present in the effusion sample. The DNA is derived from both the normal cells and also from the circulating cell-free tumor DNA (ct-DNA). Therefore after centrifugation, the supernatant fluid of the effusion can be used as a liquid biopsy specimen and various molecular tests such as sensitive polymerase chain reaction (PCR) and next-generation sequencing (NGS).[

Table 1 shows the relative advantages and disadvantages of the different preparation of effusion sample for the molecular tests.

Table 1:

Comparison of various types of preparation of effusion fluid for molecular tests.

Types of preparation	Advantages	Disadvantages	Comments
Cell block	(1) The cellularity and cell morphology can be visualized from the hematoxylin and eosin stained section of the cell block,(2) the nature of the cells can be assessed by immunocytochemistry	(1) Immediate assessment of the cellularity is difficult, (2) the absence of the whole nuclei, (3) the formation of cross-linking between nucleic acid and proteins	The most common preparation for molecular tests
Direct smear	(1) Immediate cellularity assessment, (2) whole nuclei available, (3) better quality of DNA, (4) malignant cells can be seen in microscope	(1) The slide has to be sacrificed, (2) strict validation needed, (3) cover slip removal takes time	The cells are obtained for molecular tests by scarping, lifting, or transferring
Liquid-based cytology	(1) Intact whole nuclei, (2) the better quality DNA or RNA, (3) multiple slide preparation can be done	CytoRich fixative gives suboptimal DNA	SurePath and ThinPrep preparations are available
Cell pellet	(1) Whole nuclei present, (2) direct transfer of the cells for molecular tests	The slide has to be made for evaluation of cellularity and cell viability	The supernatant fluid can also be used as liquid biopsy

THE ADEQUACY OF THE REPRESENTATIVE TUMOR CELLS IS AFFTECTED BY TWO WAYS

The total cellularity of the sample: The overall cellularity of the sample is directly proportional to the DNA yield for the molecular test. Hence, high cellular usually reassures that the molecular tests will be effective

Tumor fraction: The contamination of reactive mesothelial cells and inflammatory cells may affect the proportion of the tumor cells in the sample. The low tumor fraction is a challenge to the cytologist as it may decrease the sensitivity of the molecular test because the DNA molecules may have come from the nonneoplastic cells. It has been noted that 20% tumor cells is the minimum threshold to avoid the false-negative molecular test.[

THE ROLE OF CYTOPATHOLOGISTS IN MOLECULAR TESTING

The cytologists have several vital responsibilities in the execution of the molecular tests [Figure 1]. The primary duty of the cytologist is to examine the stained cytology smear or section of the cellblock to assess the cytomorphology and to have a provisional diagnosis. It is also essential to find out the slide/smear for an adequate number of viable tumor cells. The area of the slide can be marked for future use. Next step is to decide the exact molecular tests for the diagnosis/typing/primary source of the tumor. In the case of management of the tumor, the decision of the precise molecular analysis should be taken by the combined team consisting of oncologist, molecular pathologist, and cytopathologist.

Figure 1:

Schematic diagram showing the duties and responsibilities of the cytologist in the execution of molecular tests on effusion sample.

MOLECULAR TESTS

Several molecular tests can be done in the effusion fluid.

PCR

PCR can be done in the very tiny amount of DNA or RNA. This technique is mainly target based, and therefore, only selected genetic alterations are detected by PCR. With the help of real-time PCR, we can quantitate DNA or RNA of the sample. The specific gene mutation can be detected in the extremely minute amount of DNA or RNA with the help of the various sophisticated PCR techniques that include droplet digital PCR, allele-specific PCR, amplification refractory mutation system, and single-stranded conformation polymorphism (SSCP).[ With the help of PCR, single gene mutation in the cell or cell-free DNA can be detected.[

Fluorescent in situ hybridization (FISH)

FISH uses DNA probes labeled with fluorescent dyes. FISH can be used in the archival material, and no culture of the cell is needed for this test. It requires a fluorescent microscope for the interpretation of the signals. However, if the chromogen tagged DNA probe is used, then the signal can be interpreted by the light microscope. As the specific DNA probe is used in FISH, so only the selected chromosomal changes can be interpreted by this technique. The various genetic mutational changes have been studied in effusion sample by FISH.[ The signal in FISH can be interpreted along with the cytomorphology of the cells.

Gene sequencing

In this process, the sequential order of nucleotides in the DNA (or RNA) is assessed. The earliest and the classical method of gene sequencing is Sanger’s sequencing. In this method, the nucleotide incorporation is terminated by the addition of 2’, 3’-dideoxynucleotides (dATP, dTTP, dCTP, and dGTP). Each terminated fragment of DNA is recorded at a time, and from that data, ultimately whole DNA sequence is generated. Sanger sequencing platform is, therefore, a slow process as only a fragment of DNA is sequenced in each reaction. However, in the case of NGS, a large number of DNA fragments are simultaneously analyzed. Hence, it is also called as NGS which is also called as high-throughput sequencing. The different types of NGS include pyrosequencing, microelectrophoretic methods, hybridization sequencing, and real-time observation of single molecules. Overall, the basic steps of NGS are (a) to make a library of DNA, (b) simultaneous huge millions of parallel sequence, (c) analysis of data by bioinformatics, and (d) the data interpretation. NGS has been used successfully in effusion samples of various metastatic carcinomas.[

MICROARRAY OF DNA AND RNA

Comparative genomic hybridization (CGH) is a laborious and tedious technique that helps to identify the loss, gain, or amplification of the chromosome. In CGH, a test DNA from the patient and a reference DNA from a healthy female are tagged with red and green fluorescence. The admixture of this DNA (1:1 ratio) is then incubated with the metaphasic plate (along with cot-1-DNA), and the red and green fluorescence is measured to find out the loss (excess red) and gain (excess green) of the chromosome. The array-based CGH (a-CGH) technique uses c-DNA or genomic bacterial artificial chromosome (which is known as “chip”) instead of the metaphasic plate. The test and reference DNA is then hybridized on this “chip.” Both CGH and a-CGH are successfully used in the effusion sample specimen.[

Table 2 highlights the basic principles, merits and demerits of different molecular techniques that are used in the effusion sample.

Table 2:

Comparison of various molecular tests used in effusion sample.

Tests	Basic principle	Advantages	Disadvantages		Comments
Polymerase chain reaction (PCR)	Target DNA is amplified with the help of primer, DNA polymerase, and nucleotides	Very minute quantity of DNA neededIt is highly sensitive and specific	Only specific target DNA can be studied	The different sophisticated PCR techniques include droplet digital PCR, amplification refractory mutation system, and single-stranded conformation polymorphism
Fluorescent in situhybridization	Fluorescent tagged DNA probes are incubated with the test DNA to find out the signal in case of complimentary DNA present in the test	Archival material can be usedIt can be correlated with cytomorphology	Target specific
Next-generation sequencing (NGS)	In a single assay, massive parallel sequencing of DNA occurs	Large number of target DNAs can be studied in a single testExtremely rapid sequencing in comparison to Sanger sequenceWhole-genome can be studied, and it is not target specific	High costSophisticated	The different types of NGS include pyrosequencing, microelectrophoretic methods, hybridization sequencing, and real-time observation of single molecules
Comparative genomic hybridization (CGH)	Here, the test (tagged with red fluorescence) and reference (tagged with green fluorescence) in equal proportion are incubated on the metaphase plate followed by the measurement of red and green fluorescence to find out gene deletion or amplification	It works as virtual karyotypingGenome-wide analysis can be done	Relatively large amount of DNA is needed	Array-based CGH gives better resolution and large number of DNA sequences can be studied in a precise way

APPLICATIONS OF MOLECULAR TECHNIQUES IN EFFUSION FLUID

The molecular tests in the effusion sample can be applied for the diagnosis of malignancy, the detection of types and primary sites, the management of individual cases in personalized therapy, and follow-up of the cases.

DIAGNOSIS

Diagnosis of mesothelioma

The mesothelioma-related genes are BAP1 and CDKN2A. “BRCA-1-associated protein” (BAP1) gene is located at 3p21 chromosome. BAP1 gene is preserved in the reactive mesothelial cells and is lost in majority of the cases of mesothelioma. The demonstration of loss of BAP1 in an effusion sample is highly specific (100%) for mesothelioma. FISH can detect the mutational changes of these genes in mesothelioma[ Walts et al. demonstrated BAP1 loss and mutational changes of CDKN2A in the cell block sections of effusion sample.[ They suggested that these are the specific diagnostic makers of mesothelioma. Illei et al. also demonstrated homozygous deletion of CDKN2A by FISH in ThinPrep preparation of effusion sample.[ They noted that FISH in effusion sample increased the diagnostic sensitivity of malignant mesothelioma cases.

Diagnosis of metastatic carcinomas

Mucin (MUC)

The different mucin genes such as MUCI, MUC2, and MUC5AC are quantitated in effusion sample by quantitative RT-PCR technique.[ It was noted that the expression of MUC1 MUC2 and MUC5AC genes was higher in malignant effusion than that of benign effusion. The sensitivity and specificity to detect malignancy in effusion by quantitative estimation of MUC genes by RT-PCR were 86.1% and 91.5%, respectively.

EpCAM, CEA, Cadherin, MUC1, and mammaglobin

The expression of a panel of genes consisting of EpCAM, CEA, cadherin, MUC1, and mammaglobin was studied in the effusion sample with the help of RT-PCR.[ It was noted that RT-PCR analysis of CEA and Ep-CAM significantly increased diagnostic sensitivity in effusion sample.

The panel of genes

Leichsenring et al. studied a large panel of genes from the cell block of 20 effusion samples with the help of NGS. Somatic mutations were detected all the samples, and it was suggested that NGS in the effusion sample might help in the diagnosis.[

Microsatellite marker

Woenckhaus et al. studied microsatellite markers in the 20 effusion samples by PCR and noted genetic alterations of microsatellite genes in the tumor samples only. The combination of cytological and microsatellite analysis raised the sensitivity of the detection of malignancy from 57% to 79%. It was suggested that in challenging cases, microsatellite markers might help in the diagnosis of malignancy.[

K-ras and p53

Dai et al. performed PCR -based single-stranded conformation polymorphism (PCR-SSCP) in 16 cases of pleural effusion to detect K-ras and p53 gene mutation.[ Interestingly, they used the stained cytology slides for these tests. At first, the cellular areas of the slide were identified and then those areas were marked. The microdissection of the cell was done with the help of micromanipulator system. The PCR-SSCP was done in the microdissected cells to find out mutational changes. There were identical mutational changes in the cells of the effusion sample and resected specimen. The authors suggested that p-53 and K-ras mutation patterns are useful markers to diagnose lung carcinoma in pleural effusion sample.

Claudin 4

Mohamed et al. did RT-PCR from the cell pellet of 75 effusion sample and noted that mRNA of claudin 4 is high in the malignant effusion compared to the benign sample. They suggested that the detection of claudin 4 gene upregulation may help in the diagnosis of malignancy.[

Human telomerase reverse transcriptase (h-TERT)

Shu et al. studied mRNA of h-TERT in 96 effusion samples and noted that mRNA of h-TERT is frequently present in the malignant pleural effusion.[ The sensitivity and specificity of mRNA of telomerase reverse transcriptase are 90 and 95%, respectively. The detection of mRNA of hTERT may help to diagnose malignancy in effusion sample.

Micro-RNAs (mi-RNA) as a diagnostic marker

mi-RNAs are the non-coding RNA of single-stranded RNA of short-length nucleotides. They regulate the expression of oncogenes or suppressor genes. Therefore, mi-RNAs are indirectly linked with carcinogenesis.[ The study of miRNA is easy to do, economically feasible with high sensitivity and specificity. The studies on miRNA as a diagnostic marker in the identification of metastatic carcinoma are promising. Shin et al. noted reduced expression of miRNA-134, miRNA-185, and miRNA-22 in malignant effusion compared to that of benign effusion.[ Wang et al. showed that nine miRNAs (miRNA-200c-3p, miRNA-200b-3p, miRNA-200a-3p, miRNA-429 and miRNA-141-3p, miRNA-205-5p, miRNA-483-5p, miRNA-375, and miRNA-203a-3p) were most frequently associated with metastatic carcinoma in effusion.[ Altered profile of mi-RNA may be a helpful in distinguishing mesothelioma from the metastatic carcinoma in effusion sample. The expression of mi-RNA-200 is reduced in mesothelioma than that of metastatic adenocarcinoma of lung.[ Table 3 highlights the applications of molecular tests in effusion in the diagnosis.

Table 3:

Diagnostic applications of molecular tests.

Genetic marker/s	Number of samples	Sample preparation	Molecular tests	Results	Comments
BAP1 and CDKN2A (p16)
BAP1 and CDKN2A (p16)[26]	67 samples;32 MM, 35 atypical reactive mesothelial cells	Cell block section of effusion	Immunocytochemistry for BAP1 and FISH for CDKN2A	Homozygous deletion of CDKN2A (p16) by FISH is more sensitive than loss of BAP1 immunostaining.	Good diagnostic markers of malignant mesothelioma
CDKN2A[27]	32 total samples; 19 benign and 7 MM and 6 cytological suspicious	ThinPrep	Dual color FISH	Homozygous CDKN2A deletion was noted in 6/7 MM and 6/6 specimens with suspicious cytology	FISH increased diagnostic sensitivity of MM
MUC1 MUC2, and MUC5AC[28]	89 total samples; 54 malignant, 35 benign	Cell pellet	QT-RT-PCR	The expression of MUC1 MUC2, and MUC5AC genes are higher in malignant effusion	The sensitivity and specificity of to detect malignancy in effusion are 86.1% and 91.5%, respectively
CEA, Ep-CAM, E-cadherin, mammaglobin, MUC1[29]	114 total cases; 71 malignancy, 43 benign	Cell pellet	RT-PCR	The combined application of cytology and RT-PCR of CEA and Ep-CAM had high sensitivity and specificity (90.1%)	RT-PCR analysis of CEA and Ep-CAM significantly increased diagnostic sensitivity
A large panel of genes (161 genes)[22]	20 samples	Cell block	NGS	Somatic mutations were detected all these cases	The genetic markers assessment may have diagnostic importance
Microsatellite markers[30]	20 samples; 14 malignant and 6 benign	Cell Pellet	PCR	Genetic alterations of microsatellites were noted in tumor patients only	A combination of cytological and microsatellite analysis may help in the diagnosis of difficult cases
P53 and k-RAS[31]	16 cases	Microdissected cells from the glass slide	PCR-single-stranded conformation polymorphism	The same mutational changes were noted in the cytology sample and resected specimen	Mutational changes of p53 and k-RAS may help in the diagnosis of malignancy in effusion sample
Claudin 4[32]	75 total cases; 56 malignant and 19 benign	Cell pellet	RT-PCR	mRNA of claudin 4 detected in cells of effusion samples in malignant cases	The detection of claudin 4 gene upregulation may help in the diagnosis of malignancy
Telomerase reverse transcriptase[33]	96 total cases; 41 malignant and 55 benign	Cell pellet	RT-PCR	mRNA of telomerase reverse transcriptase is frequently present in malignant effusion sample than the benign sample	The sensitivity and specificity of mRNA of telomerase reverse transcriptase are 90% and 95%, respectively, and the detection of TERT may help in the diagnosis of malignancy

MM: Malignant mesothelioma, CEA: Carcinoembryonic antigen, EpCAM: Epithelial cell adhesion molecule, MUC1: Mucin 1, RT-PCR: Real-time reverse transcriptase-polymerase chain reaction

MANAGEMENT AND PROGNOSIS

In the era of personalized medicine, molecular tests can be used extensively in the effusion samples.

EGFR and ALK

Introduction of targeted therapy for EGFR and ALK has changed the scenario of the management of lung adenocarcinoma.[

Several studies have demonstrated EGFR and ALK mutation in pleural fluid samples.[ PCR and NGS have been applied from either cell block or cell pellet material of effusion fluid for the demonstration of EFGR mutation.[ Less than 5% of tumor cells were sufficient for the detection of EGFR mutation by NGS.[ There was a high concordance between the result of the resected specimen and the effusion sample.[ EGFR mutational analysis from effusion sample was successful in more than 90% of cases, and various studies concluded that EGFR mutational status from the effusion sample could be used for targeted therapy.[

EML4-ALK gene fusion was demonstrated in the effusion sample by FISH or RT-PCR, and the majority of the studies were done successfully on the cell block material of effusion sample and concluded that the demonstration of ALK gene rearrangement in effusion could be used for targeted therapy instead of a tissue section from the biopsy material.[

Her2/neu

Her2/neu gene amplification is successfully demonstrated by FISH technique in effusion sample by various researchers.[ Nizzoli et al. successfully showed Her2/ neu gene amplification in the archival May-GrünwaldGiemsa (MGG) stained slide.[ FISH in the archival MGG stained slide is particularly useful in cases that need pre-operative chemotherapy, but tissue section or cell block is not available.

BRCA1 and BRCA2

Mutational changes of BRCA1 and BRCA2 have been demonstrated by NGS in effusion samples.[ There was complete concordance of mutation results between histopathology sections and effusion samples. Fumagalli et al. performed NGS on archival MGG slides by scrapping the representative cytology smear. They concluded that BRCA test is reproducible in effusion fluid sample of ovarian cancer and a useful tool for clinical decision-making.[

Cancer stem cell markers

Sherman-Samis et al. performed quantitative RT-PCR of various cancer stem cell markers (NANOG, OCT4, SOX2, SOX4, SOX9, LIN28A, and LIN28B) in a large number of effusion samples.[ They noted that higher SOX2 and SOX9 protein expressions were associated with overall shorter survival. Table 4 highlights the applications of molecular tests in effusion in the management and prognostic assessments.

Table 4:

Applications of molecular tests for the management and prognosis assessment.

Molecular markers	Specimen preparation	Tests	Results and comments
EGFR
EGFR[40]	Cell pellet from fresh sample	NGS	Less than 5% tumor cells can also be processed. There was high concordance of the result of pleural effusion and biopsy sample from tumor. The molecular analysis by NGS can be used for targeted therapy
EGFR[41]	Cell block	NGS	Clinically relevant genetic alterations can be detected in effusion sample for the targeted therapy
EGFR, ALK, and ROS1[42]	Cell block	PCR for EGFR and K-ras; FISH for ALK	Molecular tests were successful in 91% samples. Molecular tests in effusion sample can be used for targeted therapy
ALK
ALK[43]	Cell block	RT-PCR and FISH	EML4-ALK fusionGene can be demonstrated successfully in pleural effusion sample. Both RT-PCR and FISH are specific and can be used for targeted therapy
ALK[44]	Cell block	FISH	ALK rearrangement can be successfully demonstrated in cell block of pleural effusion
ALK[45]	Cell block	FISH and RT-PCR	Cell block of effusion had good performance for ALK detection and can be used instead of tumor tissue.
Her2/neu
Her2/neu[46]	Cytospin	FISH	Her2 gene amplification can be detected in effusion sample even in the small amount of cells.
Her2/neu[47]	Archival May-Grünwald-Giemsa stained smear	FISH	HER-2/neu gene amplification can be demonstrated by FISH from May-Grünwald-Giemsa stain
BRCA1 and BRCA2
BRCA1 and BRCA2[48]	Cell pellet	NGS	There was complete concordance (100%) of mutation result between effusion cytology and histology samples.
BRCA1 and BRCA2[49]	Archival May-Grünwald-Giemsa stained smear	NGS	BRCA test was reproducible in effusion fluid sample of ovarian cancer and a useful tool for clinical decision-making
Cancer stem cell markers
NANOG, OCT4, SOX2, SOX4, SOX9, LIN28A, and LIN28B[50]	Cell pellet	QRT-PCR	Higher SOX2 and SOX9 protein expression were associated with overall shorter survival

CONCLUSION AND FUTURE PERSPECTIVES

Serous effusion provides enough material for all the tests that can be done on tissue biopsy. All the molecular analyses, including a-CGH, FISH, PCR, and NGS, can be done successfully from the serous fluid sample. For the diagnostic purpose, immunocytochemistry on the effusion sample is highly useful in the majority of the cases. The molecular tests are costly and time consuming. Therefore, it is not recommended for diagnostic purposes in a routine situation. However, the molecular tests can be done in undiagnosed effusion cases when the combined clinical information, radiological investigations, and immunocytochemistry are not helpful. The selection of molecular tests largely depends on the clinical setup. In the case of diagnosis of malignancy, the expression of EpCAM, CEA, telomerase reverse transcriptase, or microsatellite gene mutation may be helpful. The molecular tests should be selectively chosen to identify the source of metastasis.

mi-RNA is a promising biomarker in effusion sample because of relative more stability in the biological sample, high sensitivity and specificity, and economically feasible to evaluate. However, it is associated with certain limitation. The one specific mRNA is often regulated by several miRNA. Hence, it is often difficult to specify the target molecule. Hence, the derangement of a particular mi-RNA may be linked with two different malignancies. More extensive study in this area is required to find out the exact panel of miRNA in the diagnosis and even prognosis of the metastatic diseases in the effusion sample.

The information on selected gene mutational changes in effusion sample may be immensely helpful to introduce targeted therapy. The array-based CGH can give information on molecular changes of a large number of genes and may be very helpful for therapeutic purpose. Another less explored area is cell-free DNA of the supernatant fluid of the effusion sample. NGS of the supernatant fluid of the effusion may work as a liquid biopsy and may provide us with a plethora of information.

In the era of molecular technology, the cytopathologist plays many vital roles. The cytologist may provide the provisional cytological diagnosis to guide the clinicians for ordering the molecular tests. He can also assess the cellularity of the samples for the selection of the slide for the molecular analyses. Finally, the cytologist may take part the central role of integrating the molecular data, clinical history, and cytological features for the ultimate conclusion.

Data sharing

Only the published data were used in this paper.