The Utility of Rapid On-Site Evaluation during Bronchoscopic Biopsy: A 2-Year Respiratory Endoscopy Central Experience

Background Rapid on-site evaluation (ROSE) is commonly used to evaluate the adequacy of biopsy materials in fine-needle aspiration; however, the diagnostic performance of ROSE during fiber optic bronchoscopy (FOB) biopsy under direct vision is rarely reported. Here, we evaluated the role of ROSE during FOB biopsy of visible lesion in trachea or bronchi. Methods The role of ROSE was prospectively evaluated in consecutive bronchoscopy specimens obtained between January 2016 and January 2018. The agreement and accuracy between ROSE and final histopathological interpretation were assessed. The frequency and possible reasons for discrepancy between ROSE and definitive histopathology results were identified. Histological and cytological classification was performed according to the International Association for the Study of Lung Cancer, the American Thoracic Society, and the European Respiratory Society (IASLC/ATS/ERS) criteria of lung ADCs classification. Results The study enrolled 651 patients, of which 33 were excluded because of insufficient cells. Final diagnosis of malignancy was achieved in 462 cases (74.8%), whereas 156 cases (25.2%) were nonmalignant. ROSE and pathology were well correlated for the diagnosis of squamous cell carcinoma (SCC) (Kappa = 0.718, p < 0.05), adenocarcinoma (AdC) (Kappa = 0.662; p < 0.05) and small cell lung cancer (SCLC) (Kappa = 0.955; p < 0.05). In 24 cases diagnosed as malignant by ROSE and nonmalignant by pathology, the lesion tissues were surgically excised and re-analyzed, and the 24 cases were finally confirmed as malignant by pathology. Conclusions ROSE technique allows bronchoscopists to obtain viable and adequate material for the diagnosis of histopathology, and provides them with an onsite preliminary diagnosis especially in cases with inconclusive macroscopic appearance. ROSE and pathology should be used in combination to increase the accuracy of diagnosis.


Background
In recent years, rapid on-site evaluation (ROSE) has been rapidly popularized in endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) [1][2][3][4][5], or fine needle aspiration biopsy (FNA) [6][7][8] for assessing materials' adequacy and categorizing diagnosis during biopsy procedure. Chandra et al. [9] and Nakajima et al. [2] demonstrated that ROSE is critical for assessing the adequacy of cytological smears and cytology is comparable to histology in the diagnosis of lung lesions. In addition, Fassina et al. [8] reported a satisfactory overall agreement of 71.4% was achieved in differentiating the cancer histological types in fine needle aspiration (FNA) for ROSE, and Nakajima et al. [2] reported a concordance rate of ROSE and final pathologic diagnosis of 94.3% in endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA). e use of ROSE may allow an earlier termination of the procedure based on the confirmation that diagnostic tissue has been retrieved, and may, therefore, improve diagnostic sensitivity by redirecting sampling to adjacent sites in the event of a negative ROSE finding [10,11]. In addition, ROSE can help bronchoscopists determine whether additional specimen needs to be collected for further ancillary studies, such as immunochemistry, molecular detection, and microbiology [8]. Visible lesions are biopsied under direct vision using fiber optic bronchoscopy (FOB); however, the quality and adequacy of biopsy materials are not known. Because ROSE has good intermodality agreement with histopathology and can provide the bronchoscopist with an onsite preliminary diagnosis, it may solve these problems. However, the utility of ROSE during bronchoscopy biopsy under direct vision is rarely reported, and so its role has remained unclear. We conducted this prospective randomized study to further clarify the role of ROSE in assessing materials' adequacy and categorizing diagnosis during biopsy procedure.

Materials and Methods
All 651 patients who underwent bronchial biopsy combined with ROSE in TaiHe Hospital (Shiyan, Hubei, China) were enrolled in the study. Informed consent was obtained from all patients, and FOB biopsy was performed by a bronchoscopist. Study was approved by the Taihe Hospital Ethics Committees. ROSE was performed in the presence of a cytopathologist in the procedural room of bronchoscopy when possible, cytopathologist immediately interpreted whether the sample was negative (no malignant cells) or positive (definitive cytopathologic evidence of malignancy or special infection evidence). A small portion of each biopsy pass was placed on a slide, smeared, and stained with Diff-Quik for onsite analysis. Slide preparations were performed by the procedural pulmonologist or by an assistant who was a member of the procedural team. A smear was considered adequate if it contained an abundancy of preserved cells or showed sufficient cellularity compatible with the clinical and/or radiological findings. Inadequate material was subjected to re-biopsy instantaneously. e tissue was fixed with formalin and sent to the pathology department for immunohistochemical stains and molecular studies. We categorized the histological and cytological subtypes in accordance with the IASLC/ATS/ ERS [12]. Our diagnoses include definitely adenocarcinoma (AC), favor AC, definitely SqCC, favor SqCC, and NSCLC-NOS, we classified definitely adenocarcinoma (AC) and favor AC into AC, and definitely SqCC, favor SqCC into SqCC in our research. e diagnosis was made on the basis of histologic characteristicsclear-cut evidence of pearl formation, keratinization for SqCC differentiation, and glandular structure for AC differentiation, then IHC evaluation staining intensity was graded as focal and weak, diffuse and strong, negative. Final diagnosis was determined with p63, TTF-1, and neuroendocrine immunostain for all cases. p63(+) was accepted for an SCC, TTF-1(+) was accepted for an ADC. Diagnosis was assigned as adenosquamous cell carcinoma, when both p63 and TTF-1 are positive. If cytology was NSCLC-NOS but histology was SqCC or AC, the criterion for final diagnosis was accepted on the basis of histology. If histology was NSCLC-NOS but cytology was SqCC or AC, the final diagnosis was accepted on the basis of histology also. If IHC was not available, histology and final diagnosis was NSCLC-NOS.
When granulomatous inflammation with necrosis was observed during ROSE, biopsy specimen was considered sufficient and supported for tuberculosis diagnosis by ROSE.
A er that histopathology, Zeihl-Neelson (ZN) stains combined with PCR were performed for final diagnosis of tuberculosis. Trachea and bronchi fungal infections are mainly aspergillus in our study, visible aspergillus hyphae provide evidence for cytology or histopathology diagnosis, microbial cultures of specimen was needed.

Statistical Analysis.
Receiver operator characteristic (ROC) curves were designed to assess sensitivity and specificity for the estimated parameters. Chi-square test was used to compare diagnostic accuracy rates between the disease specific groups. Intermodality agreement between ROSE results and final histopathological diagnosis was assessed by calculating a κ-score. Probability values <5% (푝 < 0.05) were considered statistically significant.

Diagnostic Categories of Malignancy.
Good overall consistency was achieved in categorizing the histological types of malignancy (344/438; 78.5%) (  Table 3. Table 4 shows the cytopathological correlation and diagnostic accuracy in various lung lesions. e diagnostic accuracy of ROSE was 89.4% for SCC, 80.6% for AC, and 95.3% for SCLC, similar to that of histology. all nonmalignant cases diagnosed by histopathology were considered as malignancy by ROSE, which identified 16 cases of SCC, seven cases of AC, and one small cell carcinoma case. ese 24 cases were finally diagnosed as malignant tumors by computed tomography (CT)-guided percutaneous lung biopsy, pleural biopsy, TBNA, or surgical specimen biopsy, which confirmed 16 cases of SCC, seven cases of AC, and one case of small cell carcinoma. (h) e diameter of mycosis is generally 7-10 μm, the division of mucor is right angle bifurcation or hyphae are not separated, aspergillus appears as acute angle bifurcation, with necrotic background and neutrophils (Diff-quick). (i) A er the alveolar phagocytic cells phagocytose the dust, they evolve into dust cells, which show that the cytoplasm contains abundant black carbon particles.

Discussion
In this study, we demonstrate that ROSE ensures the quality of biopsy specimens during FOB procedure, and the results of ROSE correlated well with the final pathological diagnosis based on hematoxylin and eosin (H&E) staining of biopsy samples of FOB. Although technical advances in histopathology have reduced the processing time for small biopsies to a few hours [13], cytology remains the method of choice for an immediate assessment of sample adequacy, whereas biopsies can be used for different purposes [14]. e proportion of inadequate specimens reportedly ranges from 2.7% to 14.3% during FNA biopsy [6,15,16], consistent with our finding of 5.1% (33/651) inadequacy. Koul et al. [17] reported that only 11% of cases show adequate materials on first FNA, and an average of 3.5 biopsy passes required to obtain adequate material during FNA; however, our data suggested that 46.6% of the cases had adequate materials on first biopsy, 76.6% had adequate materials on second biopsy, and 86.6% satisfactory samples were obtained on third biopsy. A possible reason for this result was that lesions could not be visualized directly during FNA, and ultrasound or imaging was needed for detecting the location and size of lesions. Conversely, lesions were visible during FOB biopsy, and an experienced bronchoscopist detected suspicious lesions directly.
Considering patients' safety and cost effectiveness, studies suggest that ROSE can quickly evaluate the adequacy of TBNA or FNA samples [15,18,19], eliminate unnecessary biopsies, help reduce costs, and avoid or reduce complications [11,20,21]. e present study showed that using FOB combined with ROSE, adequate specimens were obtained in 86.6% of patients in 3 biopsies, suggesting that this regimen is appropriate for FOB biopsy.
Whether lung lesions can be accurately diagnosed using ROSE remains controversial [9]. erefore, in the present study, we combined bronchial FOB biopsy with ROSE, and analyzed the diagnostic accuracy of specimens for benign and malignant lung diseases. Chandra et al. [9] compared diagnosis by bronchial lavage fluid, brush, TBNA, and percutaneous needle aspiration biopsy specimens by ROSE, and showed that ROSE cytology and histology were comparable, and ROSE may be superior to histopathology for the diagnosis of lung tumors. In the present study, evidence of malignancy was not obtained by histology in 24 lung cancer patients, whereas ROSE detected cancer cells in these patients. Further analysis by CT-guided percutaneous lung puncture, thoracoscopic pleural biopsy, and TBNA confirmed the presence of lung cancer in the 24 patients. One possible reason for this discrepancy is that biopsy specimens contain fewer malignant tissue components, whereas they are sufficient for cytological smears. Alternatively, pathological sections may have failed to effectively obtain lesion tissue. Ravaioli et al. [22] showed that TBNA combined with ROSE is useful for the rapid diagnosis of lung cancer, as it not only allows evaluation of the adequacy of TBNA needle biopsy specimens, but also enables the classification of lung cancer [23,24]. Celik et al. reported that ROSE has high diagnostic yield over subclassification of NSCLC-NOS [24], and cytology, with or without cell block, successfully subclassifies NSCLC-NOS cases and can be a substitute for IHC in resource-poor laboratories and in low-income countries [23]. In our present study, there was good agreement between ROSE and final pathology for SCLC. e diagnostic accuracy of 96.8% was in agreement with reports by Fassina et al. [8] and Ravaioli et al. [22], who reported an overall accuracy for small cell carcinoma of 90-97%. In addition, the ROSE results for SCC and AC were consistent with the final pathological diagnosis, but with a low Kappa value. Poorly differentiated SCC or AC, however, displays some features that may be confused with other poorly differentiated neoplasms [25]. It is not always possible based on morphology alone to distinguish these neoplasms from poorly differentiated neoplasms in limited samples [26]. e characteristics of small cell carcinoma cells are more obvious, as cancer cells are arranged in a row and crowded, the cytoplasm is smaller or even absent, and the nucleus has a salt and pepper appearance, with a nuclear model ( Figure 2); therefore, the diagnostic accuracy and pathological consistency are better.
Reactive changes in bronchiolar epithelium might be sometimes pronounced, making it difficult to distinguish these from malignant epithelial cells, but reactive atypia in the bronchiole epithelium is characterized by loose sheets of bronchiolar cells with moderate nuclear enlargement, irregularity, and hyperchromasia, Usually, these atypical reactive cells occur as a few dispersed cells or few small cell clusters, which helps to avoid a diagnosis of malignancy during ROSE [27,28]. In addition, there is a difference between alveolar phagocytes and lung adenocarcinoma, pulmonary macrophages with abundant foamy cytoplasm and frequent intracytoplasmic carbon particles [27,28]. Adenocarcinoma with signet ring, adenocarcinoma is the most common primary lung tumor and displays a variety of morphological features. Most tumors have acinar, papillary, or micropapillary features, although the acinar type is the most common [27,28]. e features of tuberculosis under the microscope include granulomatous inflammation, necrosis, mixed lymphocytes and epithelial-like cells, and Langhans giant cells [29], sarcoidosis characterized by nonnecrotizing epithelioid cell granulomas and different from granulomatous inflammation of tuberculosis [28]. For Aspergillus and Cryptococcus, accurate diagnosis is possible if the fungus is identified. erefore, fungal and tuberculosis infections can be accurately diagnosed onsite by experienced pathological cytologists, thus gaining time for timely clinical treatment. Although ROSE has many advantages, there are also disadvantages such as the need for a professionally trained cytopathologist on site [9].
In conclusion, ROSE can provide guidance for the bronchoscopist to obtain adequate lesion specimens during FOB biopsy and can provide an onsite preliminary diagnosis especially in cases showing an inconclusive macroscopic appearance. Because ROSE and pathology have their own advantages and disadvantages, they can complement each other to improve the accuracy of diagnosis.

Conclusions
In this study, we demonstrated that ROSE technique allows bronchoscopists to obtain viable and adequate material for the diagnosis of histopathology, and provides them with an onsite preliminary diagnosis especially in cases with inconclusive macroscopic appearance. ROSE and pathology should be used in combination to increase the accuracy of diagnosis.
Data Availability e datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Ethical Approval
is study was approved by the Taihe Hospital Ethics Committees (2018KS017) and was performed in accordance with the principles of Good Clinical Practice following the Tri-Council guidelines. All patients or their substitute decision makers provided written informed consent prior to patients.

Consent
Written consent has been obtained for all participants or their substitute decision makers.

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Authors' Contributions
Conception and design: Hansheng Wang, Meifang Wang. Acquisition of data: Na Wei, Yunyun Wang. Analysis and interpretation of data: Xin Qian, Guoshi Luo. Dra ing or revisiting the article: Yijun Tang, Tao Ren, Chang Xiong and Hongbo Li. Hansheng Wang and Na Wei are contributed equally to this work.