The Value and Limitations of Cell Blocks in Endobronchial Ultrasound-Guided Fine-Needle Aspiration Cytology: Experience of a Tertiary Care Center in North India.

BACKGROUND: Endobronchial ultrasound (EBUS)-guided fine-needle aspiration cytology (FNAC) is recommended for diagnosing bronchial neoplasms and evaluating mediastinal lymph nodes. However, it may not be possible to subtype or definitely categorize many bronchial neoplasms on FNAC smears alone. Obtaining adequate diagnostic material is often a problem. In such cases, cell blocks made from FNAC material may serve as a useful adjunct. Aim: To study the value and limitations of cell blocks in adding diagnostic information to EBUS guided FNAC smears.
MATERIAL AND METHODS: One hundred and eighty-five cases of EBUS guided FNAC having concomitant cell blocks were reviewed. The cases were evaluated for the extent of adequacy, of definite benign/malignant categorization and of definite subtyping in malignant tumors in these cases. The proportion of cases in which cell blocks added information to FNAC smears alone for the above parameters were calculated.
RESULTS: Cell blocks provided additional information in 31 out of 185 cases. Cell blocks were necessary for subtyping 24/59 malignant tumors, definite categorization into benign and malignant in 10/140 adequate samples, and increasing adequacy in 6/185 total samples. A total of 45 samples were inadequate in spite of adding information from cell blocks to smears.
CONCLUSION: Cell blocks added clinically significant information to EBUS guided FNAC and should be used routinely. To make it more useful, alternative methods of cell block preparation (including proprietary methods) may be evaluated. Copyright:

INTRODUCTION

Lung cancer is the leading cause of cancer-related deaths.[1] Lung cancers are traditionally divided into small cell and non-small cell (adenocarcinoma, squamous cell carcinoma, large cell carcinoma) types. Subtyping non-small cell lung cancers into squamous cell carcinoma and adenocarcinoma is essential for proper treatment and care of the patients. Endobronchial ultrasound (EBUS) guided endobronchial needle aspiration (EBNA) and transbronchial needle aspiration (TBNA) are recommended for diagnosis of central lung lesions and for evaluation of mediastinal nodal enlargements suspicious of metastasis.[2] However, the resulting fine-needle aspiration cytology (FNAC) smears may not always be able to subtype the malignant lesions.[3] Cell block technique done on aspirate material has been found to be a useful adjunct to smears, allowing for subtyping of the cancer by immunohistochemistry (IHC).[45678910111213] Some studies have assessed the additional value of making cell blocks in EBUS TBNA samples, with their results being ambiguous.[1415161718192021]

In view of the promise held by EBUS guided FNAC, we started such services in our hospital, which is a tertiary care center in North India. In this study, we examined the value and limitations of cell block technique when added to conventional smears in diagnosing and subtyping of lung lesions and mediastinal metastasis in our setting. This, to the best of our best knowledge, is the first study in an Indian population to evaluate role of cell blocks in EBUS TBNA samples.

MATERIALS AND METHODS

The research protocol for this study was approved by the institutional ethics committee and was conducted ethically in accordance with the World Medical Association Declaration of Helsinki. Two hundred consecutive cases of EBUS guided EBNA and TBNA from January 2019 to November 2019 were examined for this study. We excluded patients who had already received chemotherapy or radiotherapy for any cancer, patients with markedly vascular lesions and patients who refused to consent for more than one pass.

All cases were subjected to FNAC. Smears and cell blocks were taken from the same site. In our institute, we usually attempt a maximum of three passes for the FNAC smears, with a separate pass attempted for cell block. In some cases, only washings from each pass were taken and a separate pass for cell blocks omitted. Routine air dried and wet-fixed smears for FNAC examination were made. One Air dried smear was stained with toluidine blue and screened for rapid on-site examination (ROSE) by a cytopathologist. In cases where multiple sites were sampled, material for cell block was taken from one site in all cases except for two cases where cell blocks were made from two separate lymph nodes. The smears made were stained with Papanicolaou stain and May-Grunwald-Giemsa stain according to routine laboratory protocols.

Cell block preparation: The aspirate was flushed into a sodium citrate vial. After adding 5 ml formalin to it, it was left for 1-2 hours for fixation and then centrifuged at 2000 rpm for 8 min. The supernatant was discarded and 6-8 ml formalin added to the sediment which was then centrifuged at 2000 rpm for 8 min. The supernatant was again discarded and the cell pellet at bottom of test tube expelled out onto a piece of filter paper, wrapped, fixed in formalin for 1-2 hours and processed as a small biopsy. Then, sections made from the cell block were stained with routine hematoxylin-eosin stain. IHC markers stains were applied when necessary. The IHC stains used included Synaptophysin and chromogranin (for neuro-endocrine tumors and small cell carcinoma), TTF-1 and Napsin (for adenocarcinoma), and p40 (for squamous cell carcinoma).

The Papanicolaou system for respiratory cytology was followed for reporting and an initial diagnosis made on routine smears.[22] All cases were categorized into one of the following categories: Non-diagnostic, benign, atypical, neoplasm, suspicious, malignant. In case of a malignant lesion an attempt was made to subtype it into small cell vs non-small and for non-small cell carcinoma cases into adenocarcinoma and squamous cell carcinoma. The atypical and suspicious categories were treated as indeterminate categories for this study.

Descriptive statistics with cross tabulation were used describe the baseline diagnosis and categorization made by smears alone. Then, the additional yield of adequate categorization, definite (benign/malignant) categorization, and final subtyping on adding information from cell blocks to that of smears alone were calculated as proportions. Exact 95% Confidence intervals of these proportions were estimated. P values were calculated by performing the exact McNemar's test for paired data.

RESULTS

The 200 consecutive patients underwent EBUS guided procedure from January 2019 to November 2019 for mediastinal or hilar lesions. Cell block was made in 185 of these cases. Rest of the 15 cases were excluded from further analysis. One hundred and forty-seven aspirates were from mediastinal or hilar lymph nodes and 38 aspirates were from lung lesions. Indications for EBUS in this study are given in Table 1.

Table 1

Clinical indication for Endobronchial ultrasound (EBUS) guided Fine-Needle Aspiration in the study

Indications	Number of cases
Lung cancer with or without metastasis	85
Mediastinal lymphadenopathy in proven/suspected non-lung carcinoma	46
Benign conditions (Tuberculosis/sarcoidosis)	30
Suspected lymphoma	3
Interstitial lung disease with mediastinal lymphadenopathy	5
Clinical details not available	16

The summary of the findings on FNAC smear examination alone and when supplemented by cell blocks is given in Figure 1. Cell blocks provided additional information in 31 out of the 185 cases (16.8%, 95% C.I. =11.7% to 22.9%, P value <0.001). The additional information yield due to cell blocks being added to the diagnostic paradigm was subtyping 24 out of 59 (40.7%, 95% C.I. = 28.1% to 54.2%, P value <0.001) malignant tumors, definite categorization into benign and malignant in 10 out of 140 adequate samples (7.1%, 95% C.I. =3.5% to 12.7%, P value = 0.002), and increasing adequacy in 6 out of the 185 total samples (3.2%, 95% C.I. =1.2% to 6.9%, P value = 0.03). In 10 cases, the tumor could not be definitely subtyped, either due to scant tissue, failure of categorization after addition of the routine IHC markers, or since IHC was not ordered by the clinician. We did not get a single case where a benign smear diagnosis was changed to a malignant diagnosis after addition of cell blocks.

Figure 1

Flowchart showing the performance of EBUS guided FNAC in this study highlighting the additional information provided by Cell blocks over FNAC smears alone. Areas where cell blocks provided additional information have been shaded grey. (NSCC = Non-Small cell carcinoma, SqCC = Squamous cell Carcinoma, ACC = Adenocarcinoma, SmCC = Small cell Carcinoma, AdCC = Adenoid cystic carcinoma, NET = Neuroendocrine tumor)

In a small subset of 17 cases, histological correlation was possible. Nine of these cases were diagnostic on both cytology and histology and showed malignancy. Three malignant tumors were missed by FNAC but caught on biopsy, and three more malignant tumors were missed by biopsy but caught on FNAC. One case was classified as inadequate on both FNAC and biopsy.

DISCUSSION

In our study, the primary advantage of adding cell blocks to the diagnostic flow was a marked increase in the definite subtyping of malignancy which confirms the findings of other studies.[141516] This was primarily an effect of the ease with which IHC could be applied to cell blocks. The extent of increased information due to subtyping is greater than reported by some studies,[1516] possibly because many of the tumors were difficult to classify due to the poor differentiation, but is similar to others.[20] The increase in the proper subtype given is essential for selecting further treatment and investigations, especially for Gemcitabine therapy, radiotherapy and selection of molecular markers for targeted therapy. Cell blocks as an adjunct reduces the need for re-biopsy for additional markers by conveniently providing material for IHC. This is especially useful since the lung cancer patients in our setting usually present at an advanced stage with comorbidities, with concomitant difficulty to do repeat biopsies for additional tests. Hence, we feel that the cell block technique should be an essential adjunct to conventional smear preparation.

Besides the benefit of increasing the type specific diagnosis, we also felt, qualitatively, that cell blocks were beneficial in increasing pathologist confidence, even while corroborating the diagnosis made on conventional smear. This, along with the continuous feedback provided by the cell blocks add a value which cannot be quantified by a statistical analysis.

While cell blocks reduced slightly the number of indeterminate cases on cytology smear alone, the numbers in which it did provide increased information was small (10 out of 140 adequate tumors were given a definite benign/malignant categorization after addition of cell blocks). A few examples of cases where cell blocks were useful in adding to a smear diagnosis is given in Figure 2. The effect of cell blocks in reducing the indeterminate category, we feel, is mild; this is probably due to unambiguous categorization in most of the adequate cases on smear examination alone.

Figure 2

Three indeterminate cases on smears aided by cell blocks. (a) Case 1 showed few large cells (black arrow) in a lymphoid background (H and E, X400); (b) Cell block of case 1 showed tumor cells which were p40 positive and TTF1 negative on IHC (squamous cell carcinoma) (H and E, X400); (c) Case 2 showed a single cluster of suspicious cells (MGG, X400); (d) Cell block of case 2 showed squamous cell carcinoma (H and E, X400); (e) Case 3 showed an epithelioid granuloma but few large cells were also seen (MGG, X400); (f) Cell block of case 3 confirmed a granuloma. PanCK negative, CD68 positive (H and E, X400)

We observed a mild rise in the adequacy rate on addition of cell blocks in this study which was entirely due to increased diagnosis of benign cases. This result is counter-intuitive since an additional pass should have increased the yield in malignant cases. In this study, smears were prepared till ROSE revealed adequate cellularity, and only then was an additional pass made for cell block preparation. This may have resulted in a resultant hemorrhagic aspirate in the final attempt for cell block, without any appreciable increase in cellularity in many cases. Such a result may possibly be prevented in the future if a washing of the needle is made for cell block analysis after every attempt at FNB, and then an additional pass taken for cell block alone.

In our study, the primary limitation of cell blocks was the high inadequacy rate. This can be attributed to a variety of preanalytical factors including difficult anatomical location of lesion and expertise of the interventionist. This was a new test in our hospital performed by multiple interventionists of varying experience. The process of making routine cell blocks from FNAC material was also new for our laboratory. We used the basic method of histologically processing the cell pellet left after centrifugation, which may be partially responsible for the low cellularity. Methods using thrombin clot or agar gel to stabilize the cell pellets have been reported.[23242526] We tried standardizing on these latter methods, but they gave unsatisfactory results in our setting. There were problems in wax embedding, resulting in rejection of these methods in favor of the basic method we have continued to use. Proprietary methods like the Cellient™ would possibly help,[27] and the value of such methods will be investigated in our laboratory. In spite of these limitations, we obtained a satisfactory adequacy rate for the entire study combining the results of the smear and the cell block. With increasing experience on the part of the interventionists and in making cell blocks, we expect a greater combined adequacy in the future. In spite of the relatively low adequacy rate, this study provides strong evidence that adding cell blocks provide significantly increased pathological information in a large subset of patients.

The other limitations we encountered in cell blocks were finding crushed tissue (included as inadequate in the analysis), and exhaustion of tissue for IHC (in two cases). In seven cases, we could not come to a definite diagnosis even after IHC. Extended panels of IHC markers or parallel gene expression analysis should be investigated, in our opinion, for these analyses.

The small subset of cases where histopathological correlation was possible suggested that EBUS guided FNAC provides additional information compared to endobronchial biopsy and Trans Bronchial Lung Biopsy, as also reported in other studies.[2829] This suggests that FNAC and histopathology should be seen as complementary and not as competitive techniques.

This study aimed at evaluating the additional information that could be obtained by cell blocks over and above routine FNAC smears and not to compare the two methods. Proper smear interpretation remains essential for the success of EBUS guided FNAC in our setting. Perusal of our results in Figure 1 will show that if taken as a competitive technique, routine FNAC smears are superior to cell blocks alone for both getting adequate smears and for proper categorization of the lesion, but are inferior to cell blocks for carcinoma subtyping once a malignancy is diagnosed. Therefore, routine cell blocks should form a complementary, yet essential, component for fine-needle cytological diagnosis of bronchial lesions.

CONCLUSION

Cell blocks substantially increase the value of EBUS guided FNAC. The proportion of lung cancer cases that can be definitely subtyped is markedly increased, helping further clinical management. This additional information is primarily a result of easily applying IHC markers according to established protocols. Addition of cell blocks may also help in diagnosis of lung or mediastinal lymph node lesions by decreasing the number of cases having indeterminate categorization. Even though limited by a high inadequacy rate when used as a stand-alone technique, it should be an essential adjunct to conventional cytopathology examination.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity.

Financial support and sponsorship

Self-funded.

Conflicts of interest

There are no conflicts of interest.