
==== Front
J Cytol
J Cytol
JCytol
J Cytol
Journal of Cytology
0970-9371
0974-5165
Wolters Kluwer - Medknow India

JCytol-41-143
10.4103/joc.joc_4_24
Original Article
Cytomorphological Features as a Subtyping Tool of Non-Small-Cell Lung Cancer in Brushing Bronchoscopic Samples
Gardić Nikola G. 12
Miljković Dejan M. 12
Lovrenski Aleksandra N. 12
1 Department of Pathology and Molecular Diagnostics, Institute for Pulmonary Diseases of Vojvodina, Sremska Kamenica, Serbia
2 Faculty of Medicine, University of Novi Sad, Serbia
Address for correspondence: Dr. Nikola G. Gardić, Put Doktora Goldman 4, 21204, Sremska Kamenica, Serbia. E-mail: nikola.gardic@institut.rs
Jul-Sep 2024
18 7 2024
41 3 143149
10 1 2024
07 5 2024
Copyright: © 2024 Journal of Cytology | Indian Academy of Cytologists
2024
https://creativecommons.org/licenses/by-nc-sa/4.0/ This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms.
Background and Objective:

Nowadays, the separation of adenocarcinomas (ADCs) and squamous cell carcinomas (SCCs) is crucial given that there are new specific targeted therapies. So, the aim of this study was to examine the differences in cytomorphological features between ADC and SCC in bronchoscopic brush samples.

Material and Methods:

The retrospective study was conducted over a 3-year period at Western Balkan University Hospital. All brushing samples were analysed. According to the histopathological report, patients were classified into ADC and SCC groups. The cytomorphological features analysed in 95 samples were presence of necrosis, cell distribution, nuclear atypia, size of nuclei, and visibility of nucleoli. Statistical analysis was performed in JASP, and P values <0.05 were considered significant.

Results:

The necrotic background was more frequent in SCC samples. Small clusters sized ≤200 µm were found in 17.95% of samples from the SCC group and 53.57% in the ADC group. Large clusters sized >400 µm were found in 43.59% in the SCC group, while in the ADC group, it was found in 5.36%. There were no differences in nuclear atypia between groups. Nuclei that were >5x lymphocyte size were found more often in samples from ADC than in the SCC group (37.50 vs 10.25%). In 89.75% of samples from the SCC group, nuclei were ≤5x lymphocyte sizes, while in the ADC group, the percentage was 63.5%. Nucleoli were more often visible in samples from the ADC group compared to the SCC group (92.86% vs 64.10%, P < 0,05).

Conclusions:

Small clusters, large nuclei, and visible nucleoli were more frequent in the ADC group (P < 0.05), while large clusters, small nuclei, and invisible nucleoli were more frequent in the SCC group (P < 0.05).

Adenocarcinoma
bronchoscopy
cytology
morphology
non-small-cell lung carcinoma
squamous cell carcinoma
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pmcINTRODUCTION

Lung cancer represents the main cause of cancer-related death worldwide in both genders. It was estimated that 350 people/per day will die from lung cancer in 2022, which is more than breast, prostate, and pancreatic cancers combined. Lung cancer is the third most commonly diagnosed cancer after breast cancer and prostate cancer.[12]

Traditionally, cytopathology had been doing a good job in distinguishing the two broad categories of lung cancers, including small-cell carcinoma and non-small-cell carcinoma. Back then, further subclassification of non-small-cell carcinomas was not really important because it did not impact the choice of treatment. However, nowadays, the separation of adenocarcinomas (ADCs) and squamous cell carcinomas (SCCs) is important given that there are new targeted therapies effective in a subset of non-small-cell carcinomas, largely ADCs, and there are complications or lack of efficacy if some drugs are used in other types of tumors.[345]

The latest edition of WHO Thoracic tumor classification says that there is insufficient published evidence to allow for a definitive diagnosis of non-mucinous ADC based on single or combined cytomorphological features other than unequivocal acinar or papillary formations. But it also says that 3D clusters are more common in cytological smears than true glandular formations. The typical features of individual ADC cells are columnar cell shape, delicate or vacuolated cytoplasm, nuclear grooves and/or intranuclear cytoplasmic inclusions, and nuclei with open chromatin and large nucleoli. SCC is characterized with Robin’s egg blue cytoplasm as a sign of keratinization. Besides that, SCC shows singly scattered or clusters of atypical cells in the background of necrotic debris. Nuclei are usually small, hyperchromatic, and smudgy, and nucleoli are often inconspicuous.[67]

The purpose of this study is to contribute to the validity of cytological diagnosis of bronchoscopic visible tumor specimens and potentially in the future, along with other studies on this topic, to be the basis for forming a cytological score that will increase the sensitivity of cytological diagnosis of non-small-cell lung cancer.

SUBJECTS AND METHODS

After institutional ethical board approval, the electronic data system of the tertiary care hospital was searched for all bronchoscopic brushing samples for a period of 3 years. Patients’ medical records and histopathological reports were reviewed, and the following were analyzed: basic clinical characteristics, stage of the disease (TNM 8th Classification), diameter of lung lesion, and final histopathological and cytopathological report. According to the histopathological report, patients were classified into two groups: ADC group and SCC group.

Sampling and processing of material for histo- and cytopathological analysis

Material for histopathological and cytopathological analysis was obtained by bronchoscopy performed by a pulmonologist–bronchologist. After visualization of the significant lesion, a brush is introduced through the channel for the instrument, and the sample was obtained, which was directly smeared on the slide by the bronchoscopist.

Routine cytologic preparation included air-dried smears that were stained with the MGG (May-Grunwald-Giemsa) technique, which requires May Grunwald solution, 10% Giemsa solution, and distilled water. Tissue biopsies for histopathological examination were fixed in 10% formalin solution and then dehydrated in increasing concentrations of isopropyl alcohol (70%, 80%, 96%, 100%), embedded in paraffin and cut on a rotary microtome (Leica) into 5 µm sections. Tissue sections were stained with the standard H and E (hematoxylin and eosin) method.

Cytomorphological features

Cytomophrological features are qualitative characteristics of cellular arrangements on cytology smears. The following parameters were analyzed: presence of necrosis [Figure 1], cell distribution [Figure 2a-c], size of nuclei [Figure 2d-f], nuclear atypia [Figure 3], visibility of nucleoli, and sign of keratinization [Figure 4]. Cytological samples were evaluated by an experienced cytologist and pathology resident in the manner shown in Table 1. The presence of partially preserved structures of tumor cells with remains of their outlines in foci of necrotic tissue was considered a sign of tumor necrosis [Figure 1]. Necrosis was assessed as focal in cases where the sample contained mostly viable tumor cells with small foci of tumor necrosis. Necrosis is classified as abundant when the samples contained mostly necrotic foci, while viable tumor cells are present in a small amount, which certainly complicates the interpretation of the sample. The presence of necrotic masses of unrecognizable cells mixed with purulent exudate is considered necrosis of inflammatory/infectious etiology, and such cases are not categorized as tumor necrosis. The size of the nuclei is expressed in relation to the size of the lymphocyte. Nuclear atypia was scored as moderate or severe nuclear atypia. The specimen was categorized as moderate nuclear atypia in cases when nuclei were uniform in size and shape, with mild irregularity of the nuclear membrane and a homogeneous or fine granular chromatin pattern. The specimen was categorized as severe nuclear atypia in cases presented with varied sizes of nuclei with bizarre shapes and coarse chromatin patterns. Considering that keratinization is a specific pattern of SCC, this feature was analyzed only in the SCC group.

Figure 1 Tumor necrosis – partly preserved outlines of necrotic cells with few viable tumor cells in periphery of necrotic foci, MGG, x200

Figure 2 a – Small clusters (≤ 200 µm), MGG, x100; b – medium clusters (>200; ≤ 400 µm), MGG, x100; c – large clusters (>400 µm), MGG, x100; d – tumor cells nuclei <3x size of the lymphocyte, MGG, x200; e – tumor cells nuclei 3-5x size of the lymphocyte, MGG, x200; f – tumor cells nuclei >5x size of the lymphocyte, MGG, x200

Figure 3 a – Moderate nuclear atypia, MGG, x200; b – severe nuclear atypia, MGG, x200

Figure 4 Sign of keratinization – dense, glassy, homogeneous and Robin’s egg blue appearance of cytoplasm of SCC cells, MGG, x400

Table 1 Analyzed cytomorphological features

Cyto-feature		Classification	method		
Necrosis	Absent	Focal	Abudant		
Cell distribution	Diffuse	≤200 μm	>200, ≤400 μm	>400 μm	
Nuclear atypia	Moderate	Severe			
Size of nuclei	<3x lymphocyte	3-5x lymphocyte	>5 lymphocyte		
Visibility of nuclei	Not visible	Visible			
Sign of keratinization	Absent	Present			

Statistical analysis

The difference between the frequencies of cytomorphological features in relation to the type of lung cancer was tested using the Chi-square test. In relation to other clinico pathological parameters, cytomorphology was examined using the same statistical method. A value of P < 0.05 was considered statistically significant. The sensitivity, specificity, and positive and negative predictive values were determined according to histopathological findings as the gold standard.

RESULTS

Clinical characteristics

The clinical and pathological characteristics of the patients are shown in Table 2. The age of the patients ranged from 25 to 89 years. Most of the patients were former or active male smokers. Slightly more than 80% of patients were in good general condition.

Table 2 Patients’ characteristics

Characterististic	Value	Percentage	
Age	63.62±8.63		
Gender			
    Male	63	66.32%	
    Female	32	33.68%	
Smoking status			
    Smokers (or former)	86	90.53%	
    Non-smokers	9	9.47%	
ECOG-PS			
    0	6	6.32%	
    1	77	81.05%	
    2	7	7.37%	
    3	5	5.26%	
TNM Stage			
    I	4	4.21%	
    II	8	8.42%	
    IIIa	12	12.63%	
    IIIb	35	36.84%	
    IIIc	9	9.47%	
    IV	27	28.42%	
Tumor size (TNM)			
    T1 (≤3 cm)	20	21.05%	
    T2 (>3 and ≤5 cm)	22	23.16%	
    T3 (>5 and ≤7 cm)	25	26.32%	
    T4 (>7 cm)	28	29.47%	
Histological type			
    Adenocarcinoma	56	58.95%	
    Squamous carcinoma	39	41.05%	

The most patients were in the IIIb stage of the disease. As for stages I, II, and IIIa, which are suitable for surgical treatment, together they made up 25.66%. As for the size of the lesion on CT, it ranged from 11 to 140 mm. The mean value was 55.4 ± 27.29 mm.

The frequency of cytomorphological features depending on the group in which the patient is classified according to his pathohistological findings is shown in Table 3.

Table 3 Frequency of cytomorphological features in relation to NSCLC type

	ADC	SCC	Total	χ 2	P	Cr V	
Necrosis							
    Absent	39 (69,64%)	23 (58,97%)	62 (65,26%)	3,561	0,169	0,194	
    Focal	11 (19,64%)	14 (35,90%)	25 (26,32%)				
    Abudant	6 (10,71%)	2 (5,13%)	8 (8,42%)				
Cell distribution							
    Diffuse	8 (14,29%)	1 (2,56%)	9 (9,47%)	27,412	<0,01	0,537	
    ≤ 200 μm	30 (53,57%)	7 (17,95%)	37 (38,95%)				
    > 200; ≤400 μm	15 (26,79%)	14 (35,90%)	29 (30,53%)				
    > 400 μm	3 (5,36%)	17 (43,59%)	20 (21,05%)				
Nuclear atypia							
    Moderate	47 (83,93%)	28 (71,79%)	75 (78,95%)	2,036	0,202	0,146	
    Severe	9 (16,07%)	11 (28,21%)	20 (21,05%)				
Size of the nuclei							
    <3 × lymphocytes	9 (16,07%)	11 (28,21%)	20 (21,05%)	9,089	<0,05	0,309	
    3-5 × lymphocytes	26 (46,43%)	24 (61,54%)	50 (52,63%)				
    >5 × lymphocytes	21 (37,50%)	4 (10,25%)	25 (26,32%)				
Visiability of the nucleoli							
    Not visible	4 (7,14%)	14 (35,90%)	18 (18,95%)	12,377	<0,01	0,361	
    Visible	52 (92,86%)	25 (64,10%)	77 (81,05%)				
Sign of keratinization							
    Absent	N/A	10 (25.64%)	N/A	N/A	N/A	N/A	
    Present	N/A	29 (74.36%)	N/A				
    Total	56 (100%)	39 (100%)	95 (100%)				

Presence of necrosis

Although the necrotic background was more frequent in SCC samples, the presence of necrosis was not statistically significantly more common in SCC or in the ADC group. There was no necrosis presented in more than 65% of samples, while abundant necrosis was present in less than 10% of samples.

Cell distribution

The distribution of cells in the form of small clusters sized ≤200 µm was found only in 17.95% of samples from the SCC group, while it is much more common in the ADC group (53.57%). The distribution of cells in the form of large clusters sized >400 µm also was found in almost half of samples from the SCC group, while in the ADC group, it was found in 5.36% [P < 0.05; Figure 5].

Figure 5 Frequency of cell distribution types in brushing samples according to groups formed based on type of NSCLC. * - statistically significant difference in frequency of ≤ 200 µm; ** - statistically significant difference in frequency of >400 µm cell clusters. (P < 0.05)

Nuclear atypia

Although severe nuclear atypia was seen in almost 28% of smears in the SCC group, in the ADC group, it was presented in only about 16% of cases; after statistical analysis, severe nuclear atypia did not prove to be statistically significantly more frequent in the SCC group than in the ADC group (P = 0,202). We analyzed correlation between nuclear atypia and nuclear size. There was a low statistically significant positive correlation in the size of the nucleus and the degree of nuclear atypia (Kendall’s tau-C = 0.224; P = 0.007). Also, there was a low statistically significant positive correlation in mentioned features within groups formed based on the histological type of carcinoma (ADC group – Kendall’s tau-C = 0.282; P = 0.005; SCC group – Kendall’s tau-C = 0.258; P = 0.045).

Size of the nuclei

The size of the nuclei proved to be a statistically significant parameter in the NSCLC subtyping. Nuclei that were >5x lymphocytes were found statistically significanly more often in samples from ADC than in the SCC group (37.5 vs 10.25%). In the 89.75% of samples from the SCC group, the size of tumor cell nuclei was ≤5x lymphocyte sizes, while in the ADC group, the percentage was statistically significantly lower (63.5%) [Figure 6; P = 0,002]. The sensitivity, specificity, and positive and negative predictive values of presence of the nuclei that were <5x lymphocytes were 89.74%, 40.58%, 46.05%, and 87.50% for proving squamous cell carcinoma, respectively.

Figure 6 Size of the nuclei as a cytomorphological features in brushing samples according to groups formed based on type of NSCLC; nuclei that were ≤5x lymphocytes were statistically significantly more often found in samples from SCC group than from ADC group; nuclei that were >5x lymphocytes were statistically significantly more often found in samples from ADC group than in the samples from SCC group

Visibility of the nucleoli

As a matter of nucleoli visibility, statistically significant differences were found. Nucleoli were more often visible in the samples that were in the ADC group compared to the SCC group (92.86% vs 64.10%, P < 0.05).

Sign of keratinization

Sign of keratinization was found in 29 out of 39 samples from the SCC group (74.36%). There was no statistically significant correlation between the frequencies of keratinization signs in samples and other analyzed cytomorphological features.

The association between cytomorphological features and the stage of the disease was also analyzed. From the obtained results, we concluded that there is no statistically significant difference in their frequencies between the samples divided into groups based on the stage of the disease. The exception is the presence of necrosis, for which it was shown that 70% of the samples in which abundant necrosis was found belong to the samples obtained from the patients in IV stage of the disease, while there are no cases with abundant necrosis in samples from patients that were in I or II stage of diseases.

As for other clinical parameters, no statistically significant differences in the frequency of cytomorphological characteristics were found.

DISCUSSION

Flexible bronchoscopy is routinely used for obtaining specimens for diagnosing different lung diseases. Although the histopathological analysis is the gold standard for the diagnosis of lung cancer, the guidelines prefer different combinations of sampling techniques in bronchoscopy.[8]

Visually identified abnormalities within the airways concerning malignancy, infection, or inflammatory disorders are typically evaluated with endobronchial brushings, biopsies, and/or bronchial washings. In order to reduce the influence of the type of procedure on morphology of cells, we chose only one type of samples to analyze for the study. Bronchial brushing is an effective tool for obtaining exfoliative cytologic specimens, especially in infiltrative mucosal disease.[9] The sensitivity, specificity, and positive and negative predictive values of the brushing cytology sample were 75%, 100%, 100%, and 88%, respectively.[8] We could not perform analysis of predictive values because the practice in our institution is that cytological and histological bronchoscopic samples are correlated with each other during analysis and a common conclusion is drawn based on them.

Nadjafi and colleagues claim that in their institution, the initial diagnostic samples were used for EGFR and ALK testing in 83% of cases and that samples were in 44.5% of cases cytological samples.[10] The small quantity of samples used for biopsies or cytology must be carefully managed in order for them to provide precise information regarding diagnosis and predictive tests required for the treatment of lung cancer.[11]

MGG stain has been used for respiratory and lymph node cytology samples in our institution for decades. The literature suggests that MGG is the standard method in hematology, but it has become a routine stain in diagnostic cytopathology for a variety of air-dried preparations. After all, the choice of stain is a matter of personal and institutional criteria.[12] Most of the authors with whom we compared the results used Romanowsky’s stain alone or in combination with Papanicolaou’s stain.[1314151617]

Rekhtman et al.[18] claim that classification based on cytomorphological parameters is sufficient for lung cancer subtyping in 88% of cases. When the characteristic cytomorphologic features are present, this subclassification of NSCLC often can be made without difficulty. Yet, it may be a real challenge in poorly differentiated tumors, especially when the only material for evaluations is smear.[19] For this reason, we believe that it is of crucial importance to investigate all procedures for obtaining samples in order to direct the immunophenotyping of tumors as precisely as possible and thereby reduce the consumption of material and the repetition of invasive procedures to obtain the material and reduce the costs of all analyzes required for immunophenotyping.

Zakowski and colleagues examined the concordance of the diagnoses in 93 cases of nine cytologists from nine different institutions in the differentiation of the two most common types from the NSCLC group. Of 818 chances to correctly identify all the cases, 753 correct diagnoses were made, resulting in 91.7% overall group accuracy or accuracy of all the cytologists calculated together. But if we considered the results by the cases alone, the results were not so good. Of the 93 analyzed cases, in 36% of ADCs and 29% of SCCs, at least one or even more cytologists had disagreement in the report.[20]

Many studies suggest the ability of ROSE to inform intraprocedural decision-making contributes to shorter procedure times as the reliable positive results preclude the need for further sampling, particularly transbronchial lung biopsy.[21222324] Samples obtained during ROSE were not used in our study. In our institution, ROSE is performing usually for the evaluation of intraoperative imprint of undiagnosed lung nodules and for the evaluation of bronchoscopic fine needle aspirations of lymph nodes and centrally located tumors. Unlike the mentioned authors who investigate the ROSE in order to determinate the malignancy, that is, diagnostic sample, Ravaioli et al.[25] demonstrated that it was possible to establish the histological type of the most frequent lung tumors during ROSE using a few easily identifiable cytomorphological features. In their results, the ADC was characterized by the presence of one or more nucleoli and small/medium cell clusters, both of which showed high specificity and sensitivity. In particular, the presence of nucleoli and small/medium cell clusters were observed in 81% and 90% ADC, respectively. As a matter of this point, our results agree. In our sample of ADCs, visible nucleoli and small/medium clusters (≤200 µm and >200; ≤400 µm clusters) were represented in 92.86% and 80.36%, respectively. But we must point out that the mentioned authors did not explain in detail the method for cluster classification. Also, Ravioli state necrosis is a valid parameter in the typing of lung carcinoma. In their results, as many as 75% of the samples that had necrosis present in over 50% of the sample were SCC, while ADCs were only in about 10% of such samples. In our results, focal or abundant necrosis was more common in SCC samples compared to ADC, but this difference was not statistically significant. Regarding the presence of necrosis, we agree with Japanese authors that distinct necrosis is rarely present in ADC samples (4.48%). However, in our samples, although relatively rare, it was found much more often (abundant necrosis in 10.71% and focal necrosis in 19.64%). The reason for this is that the aforementioned study analyzed ADC with a diameter of 3 cm or less, and our results suggested that the presence of necrosis is more common in tumors of patients that were in a higher stage of the disease.[26]

SCC is characterized by the presence of keratinizing tumor cells with markedly hyperchromatic nuclei with irregular edges. Keratinization is best seen on samples stained with Papanicolaou’s staining and has a dense, glassy appearance, distinctly orange in color, while on samples stained with Romanowski’s method, it is blue, glassy, and homogeneous in appearance and can be extensive and form extracellular keratin pearls. In poorly differentiated SCC, keratinization is much less prevalent, so the diagnosis must also be made on the basis of other cytomorphological characteristics.[62728] Indian authors divided samples of SCC into groups of well, medium, and poorly differentiated; the different representations of patterns of SCC between the groups are discussed. However, what stands out in all three groups as unchanged characteristics is the presence of necrosis.[1528] As we pointed out in the results, necrosis was more common in SCC than in ADC, but this difference was not statistically significant.[15] Regarding cluster size, the literature agrees with our results that large clusters are characteristic of SCC.[29] However, there is a discrepancy in relation to our results when we talk about the nucleolus. In our results, nucleoli were less commonly visible in squamous carcinomas compared to the others, but it was visible in 64.10% of SCC, which makes this characteristic inapplicable in distinguishing types of NSCLC.[162829]

Tumor necrosis is a result of chronic ischemic injury due to rapid tumor growth. The literature suggests that mechanical and compressive effects of a high cancer cell proliferation index cause obstruction of micro- and macrovasculature, leading to ischemic intratumoral infarcts.[30] Morphology of the tumor cells are still partially preserved in tumor necrosis, with swelling of tumor cells, which are accompanied by chromatin flocculation, plasma membrane rupture, and eventual shedding of the cytoplasmic contents into the extracellular space. Unlike tumor necrosis, in the inflammatory necrosis, we more commonly see the morphological pattern of liquefactive necrosis, in which we observed viscous liquid mass as the dead cells are being digested with masses of neutrophil granulocytes and macrophages.[31] The limitation of our study is that there is qualitative description on quantification of necrosis and there is no precise parameter on distinguishing tumor and inflammatory necrosis. Furthermore, previous published data investigating necrosis in cytological or histological specimens have used similar, subjective techniques to value the necrosis.[252628]

Our results suggest that some cytomorphological parameters are more often presented in SCCs or ADCs, but our opinion is that it is still not enough to become a standard in NSCLC subtyping. Small clusters (≤200 µm), nuclei sized >5x size of lymphocyte, and visible nucleoli were statistically significantly more frequent in samples from the ADC group, compared to the SCC group. Large clusters (>400 µm), nuclei sized <5x size of lymphocytes, and invisible nucleoli were statistically significantly more frequent in samples from the SCC group, compared to the ADC group. Consequently, it is necessary to develop more research on this topic in order to provide more information or even a score system that will help cytopathologists to differentiate the two most common types of NSCLC, especially in poorly differentiated tumors.

Key messages

The authors compared the frequencies of cytomorphological features between subtypes of non-small-cell lung cancers and concluded that some of new cytomorphological features were statistically significantly more often present in adenocarcinoma or squamous cell carcinoma.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

Acknowledgement

The article was read and approved by all authors and received approval from the head of the department.
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