Cytology Reporting System for Lung Cancer from the Japan Lung Cancer Society and the Japanese Society of Clinical Cytology: An Extensive Study Containing More Benign Lesions.

INTRODUCTION: The Japan Lung Cancer Society (JLCS) and the Japanese Society of Clinical Cytology (JSCC) have proposed a new four-tiered cytology reporting system for lung carcinoma (JLCS-JSCC system). Prior to the proposal, the Papanicolaou Society of Cytopathology (PSC) had proposed a revised reporting system (PSC system), which comprises the "neoplastic, benign neoplasm, and low-grade carcinoma" category (N-B-LG category), in addition to the 4 categories of the JLCS-JSCC system. This study aimed to evaluate the interobserver agreement of the JLCS-JSCC system with an additional dataset with more benign lesions in comparison with the PSC system. METHODS: We analyzed 167 cytological samples, which included 17 benign lesions, obtained from the respiratory system. Seven observers classified these cases into each category by reviewing one Papanicolaou-stained slide per case according to the JLCS-JSCC system and PSC system. RESULTS: The interobserver agreement was moderate in the JLCS-JSCC (k = 0.499) and PSC (k = 0.485) systems. Of the 167 samples, 17 samples were benign lesions: 7 pulmonary hamartomas, 5 sclerosing pneumocytomas, 2 squamous papillomas, one solitary fibrous tumor, one meningioma, and one lymphocytic proliferation. There were diverse sample types as follows: 11 touch smears, 3 brushing smears, 2 aspirations, and one sputum sample. Fourteen samples (82.3%) were categorized into "negative" or "atypical" by more than half of the observers in the JLCS-JSCC system. Conversely, 3 samples were categorized as "suspicious" or "malignant" by more than half of the observers in the JLCS-JSCC system. On the other hand, 11 samples (64.7%) were categorized into the N-B-LG category by more than half of the observers in the PSC system. CONCLUSIONS: The concordance rate in the JLCS-JSCC system was slightly higher than that in the PSC system; however, the interobserver agreement was moderate in both the JLCS-JSCC and PSC systems. These results indicate that both the JLCS-JSCC and PSC systems are clinically useful. Therefore, both systems are expected to have clinical applications. It may be important to integrate the 2 systems and construct a universal system that can be used more widely in clinical practice.

Cytology Reporting System for Lung Cancer from the Japan Lung Cancer Society and Japanese Society of Clinical Cytology: An Interobserver Reproducibility Study and Risk of Malignancy Evaluation on Cytology Specimens.

INTRODUCTION: The classification of lung carcinoma is based on small biopsies and/or cytology in 80% of patients with non-small cell carcinoma. However, there is no widely accepted classification system for respiratory cytology. The Japan Lung Cancer Society (JLCS) and Japanese Society of Clinical Cytology (JSCC) have proposed a new four-tiered cytology reporting system for lung carcinoma with the following categories: (1) "negative for malignancy," (2) "atypical cells," (3) "suspicious for malignancy," and (4) "malignancy." OBJECTIVE: The aim of this work was to perform an interobserver reproducibility study to confirm the utility of the four-tiered reporting system on respiratory cytological samples. METHODS: We analyzed 90 cytological samples obtained with bronchoscopy. Seven observers classified these cases into each category by reviewing one Papanicolaou-stained slide per case according to the three-, four-, and five-tiered reporting systems. RESULTS: The interobserver agreement was fair in the three- (κ = 0.50), four- (κ = 0.45), and five-tiered (κ = 0.45) reporting systems. However, the four-tiered reporting system provided more precise information than the three-tiered reporting system in patient management. The risk of malignancy in the four-tiered reporting system was also stratified well: 19.3% for "negative for malignancy," 45.6% for "atypical cells," 74.7% for "suspicious for malignancy," and 88.1% for "malignancy." CONCLUSIONS: The reporting system proposed by the JLCS and JSCC was designed to enhance the communication between clinicians and pathologists and among different institutions. It is simple and applicable to cytological diagnosis of any respiratory diseases. We propose establishing an international classification for respiratory cytology, harmonizing the reporting systems proposed by different countries.

Characterizing the Effect of Automated Cell Sorting Solutions on Cytomorphological Changes.

INTRODUCTION: Liquid-based cytology has become a widely adopted, automated screening system for gynecologic and nongynecologic cytology. Automated screening systems function by distinguishing atypical cells based on their cytoplasmic and nuclear areas, densitometric measurement, and so on. However, the morphological influence of the washing solution has not been fully considered. Here, we examined the morphological effect and temporal change resulting from saving the cytologic samples in various solutions. METHODS: Cytologic specimens were obtained from the ascites (AS) of patients with peritoneal cancer. Various solutions of a physiological saline, a Ringer's solution, a low-molecular dextran L injection, VOLUVEN 6% solution, MIXID L injection (ML), RPMI-1640 medium, and horse serum (HS) were added to aliquot sediments. All samples were refrigerated at 4°C, and aliquots were subsequently processed at specific time points (0, 1, 2, 4, 7, and 14 days). For all samples, cytoplasmic and nuclear size of the Papanicolaou-stained specimens were measured. RESULTS: In terms of cytoplasmic and nuclear areas, samples stored in ML and HS showed no significant difference compared to the AS sample; in contrast, the other samples were significantly larger in both cytoplasmic and nuclear areas than the AS sample. In examining the temporal change among the solutions, we found that the cytoplasms and nuclei became small over the time course for all of the tested solutions. CONCLUSION: We showed that cells swell in the solution after 1 h of storage and contract as time progresses. Together, our findings have important implications for how mathematical analysis is applied during the automated screening process.

Effects of the Storage Solution Type and Prolonged Storage on the Immunoreactivity of Cells.

INTRODUCTION: In effusion cytology, immunocytochemistry is a useful staining approach to provide important information for diagnosis. Effusion cytology is performed not only for pleural effusions and ascites but also for peritoneal and needle washing from fine needle aspirations or instruments. Although various solutions are used for washing cytology, the effect of the solution type on immunocytochemical reactivity is not fully understood. In this study, we examined the immunocytochemical reactivity of cytological samples after storage in various solutions. METHODS: Cell block specimens were obtained from ascites of patients with peritoneal cancer and pleural effusions of patients with diffuse malignant mesothelioma. Various solutions, including physiological saline (PS), Ringer solution, a low-molecular-weight dextran L injection, Voluven 6% solution, Mixid L injection, RPMI-1640 medium, and horse serum were added to the sediment layers of aliquots. All samples were kept at 4°C, and aliquots were subsequently processed at specific time points (0, 1, 2, 4, 7, and 14 days). Formalin-fixed, paraffin-embedded, cell block samples were prepared for immunocytochemical staining. Immunocytochemical results were analyzed for differences in the percentages of positive cells, using the effusion sample stored for 1 h as standard (100%). RESULTS: For all solutions other than PS, the median and central 50% of values were <100% (with respect to the effusion sample as a standard) after 1 h of storage. Immunoreactivity decreased for most solutions as time progressed. CONCLUSION: Of note, immunocytochemistry results obtained using a washing solution are different from those using an effusion sample. For cytology, when a washing solution was used or when a sample was stored for a long time, the accuracy of the immunocytochemical results was low.

Primary pulmonary meningioma: A rare case report of aspiration cytological features and immunohistochemical assessment.

Ectopic meningioma is a generally rare type of benign tumor that very rarely occurs in the lung. Here, we report the cytological findings of a primary pulmonary meningioma with a particular focus on immunohistochemical (IHC) assessment. A healthy 60-year-old woman visited our hospital with an asymptomatic nodule in the right lower lung lobe. She had no particular past-history and no other tumors in the central nervous system or elsewhere according to an imaging examination. Transbronchial fine-needle aspiration cytology revealed clusters of spindle cells in a whorled formation and psammoma bodies. The tumor cells exhibited spindle-shaped cytoplasm, small fusiform or round nuclei and numerous intranuclear cytoplasmic inclusions. IHC staining of the cytological specimen revealed that the tumor cells were positive for epithelial membrane antigen, negative for thyroid transcription factor-1 and p40, and equivocal for claudin-1. Progesterone receptor immunoreactivity of cytology specimen resulted negative at first by manual method but retrieved positive by an autostainer. Following segmentectomy, the pathological diagnosis was a meningothelial meningioma. The patient has remained well without recurrence for 36 months postoperatively. Because the cytological preparation exhibited characteristic findings of meningioma, a correct diagnosis based on pre-operative cytological findings with appropriate IHC would be possible. Here, we report the cytological and IHC features of this case and highlight the importance of IHC-quality assurance.

[Feasibility Study for Storage and Re-Utilization of Circulating Tumor Cells (CTCs)].

BACKGROUND: In this era of precision medicine, monitoring patients requires not only real time but also longitudinal sequence of samples at various time points. Based on this background, we focused on conditioned circumstances on fixation and storage for re-utilization of CTCs. MATERIALS: Instead of actual CTCs, Cell line (H1975) derived from lung cancer was used because of their scarceness of CTCs. METHODS: These cells were put on a slide by using an auto-smear device. The slides were evaluated under various centrifuge forces, fixations for the following storages. RESULTS AND DISCUSSION: The study indicated that 800 rpm for 1 min centrifuge and fixation by 95% ETOH was excellent. Further at least 5 cells per 1 mL cell solution were required for the following procedures including Fluorescence in situ hybridization (FISH) analysis. This study provides insights of new platform for evaluation of CTCs not only real time but also longitudinal sequence at various time points.

Desmoplastic small round cell tumor with sphere-like clusters mimicking adenocarcinoma.

Desmoplastic small round cell tumor (DSRCT) is a rare and aggressive neoplasm that predominantly affects young men. DSRCT often presents as multiple nodules on the serosal surface and is histologically categorized as a small round cell tumor. However, the cytological spectrum of DSRCT is not fully understood because of its rarity. Here, we report an unusual case of DSRCT that showed spheres of cells without stromal cores in pleural fluid cytology material, a finding that is typically associated with metastatic adenocarcinoma and mesothelioma. The specimen from a simultaneous needle biopsy showed the classic histology of DSRCT, comprising nests of small round cells set in desmoplasia. The diagnosis of DSRCT was further supported by immunohistochemical coexpression of cytokeratin and desmin, as well as Ewing sarcoma breakpoint region 1 gene rearrangement, which was determined by fluorescence in situ hybridization. The unusual cytological finding in this case illustrates a potential pitfall of the cytological diagnosis of pleural fluid or ascites. DSRCT should not be excluded from the differential diagnosis when sphere-like round cell clusters are observed in pleural or abdominal effusion, particularly in young male patients.

Recycling and long-term storage of fluorescence in situ hybridization slides.

OBJECTIVES: Although fluorescence in situ hybridization (FISH) technology is adequate, demand exists for additional recycling and long-term storage of FISH slides. METHODS: Formalin-fixed paraffin-embedded slides derived from breast cancer cases were used for this study. Each slide was probed, and then procedures for removing probes were performed, such as removing the fluorescent probe and diamidino-2-phenylindole signals. Formamide was used for removing probes, and then slides were stored dry at room temperature (22°C), 4°C, -20°C, or -80°C for 101 days. Following storage, each slide was probed in a similar manner to the initial probing. Evaluation was performed using automatic signal count software. Tiles and spots were counted immediately after the initial probing. Reprobed spots for each slide were then compared with the initial probing. RESULTS: Slides stored at -20°C and -80°C for 101 days showed the best recovery of probing. CONCLUSIONS: Our approach for probe removal and recycling allows repeated examination of even a limited number of slides.

Pulmonary neuroendocrine tumors with nuclear inclusion.

Nuclear inclusion or pseudoinclusion is a peculiar cytological feature, and its recognition in appropriate clinicopathological settings can aid in the diagnosis of several disease entities. To the best of our knowledge, only 1 case of pulmonary neuroendocrine tumor (NET) with nuclear pseudoinclusion has been reported. A review of 227 patients who had undergone surgical resection for pulmonary NETs revealed 2 tumors with different mechanisms of nuclear inclusion. To explore the cause of nuclear inclusion, NET with nuclear inclusion was characterized immunohistochemically and ultrastructurally. Nuclear inclusions were observed in 2 of the 227 (0.9%) patients with pulmonary NETs. The first patient was a 46-year-old woman with small cell carcinoma. Tumor cells with nuclear inclusions were distributed focally. Ultrastructural analysis showed that these inclusions were pseudoinclusions. The second patient was a 62-year-old man with large-cell neuroendocrine carcinoma. Nuclear inclusions were observed in the focal area of the tumor. Immunohistochemical analysis revealed that the intra-nuclear materials consisted of biotin and aberrant cytoplasmic and nuclear accumulation of ß-catenin. Mutational analysis revealed a CTNNB1 gene mutation. Although very rare, diagnostic errors may be observed in cases of pulmonary NETs with nuclear inclusions. The mechanisms of nuclear inclusion differed, with one due to herniation of the cytoplasm into the nucleus (pseudoinclusion) and the other due to accumulation of biotin resulting from a CTNNB1 gene mutation.

Malignant pulmonary epithelioid hemangioendothelioma with hilar lymph node metastasis.

Few cases each of malignant pulmonary epithelioid hemangioendothelioma (PEH) and PEH with lymph node metastasis have been reported. Here we report a case of PEH with lymph node metastasis. A Japanese woman was found to have a 2-cm-diameter mass with small satellite nodules in the right upper lobe of the lung. Microscopic examination revealed solid destructive growth of the main tumor, with epithelioid cells showing cytologic atypia and 3 mitotic figures per 10 high-power fields. Some of the tumor cells had intracytoplasmic lumina that appeared as vacuoles. These lumina were negative for alcian blue and periodic acid Schiff, and contained erythrocytes. However, erythrocytes were seen more frequently within small but distinct vascular channels that were arranged diffusely in the periphery of the main tumor. Other satellite nodules showed conventional PEH morphology. In hilar lymph nodes, the tumor cells resembled those of the main tumor. The vascular origin of the main tumor and satellite nodules was demonstrated by positive immunoreactivity for some endothelial markers. Although the diagnostic features of malignant PEH are not clear, those for PEH in other organs have included nuclear atypia, many mitoses, presence of necrosis, large tumor size, and spindle cell proliferation. The present case met these criteria, except for large tumor size and spindle cell proliferation. In conclusion, atypical cytologic features, the presence of necrosis, a high Ki-67 labeling index, and accompanying nodules of conventional PEH in the same pulmonary lobe suggest that this case was a malignant PEH with hilar lymph node metastasis.

Cytological features of signet-ring cell carcinoma of the lung: comparison with the goblet-cell-type adenocarcinoma of the lung.

Signet-ring cell carcinoma (SRCC) and goblet-cell-type adenocarcinoma (GCA) are mucin-producing lung adenocarcinomas. Primary SRCC shows an aggressive clinical course, whereas GCA shows infrequent distant metastasis, but more frequent intrapulmonary metastases resembling lobar pneumonia. To distinguish SRCC from GCA, this study investigated the respective cytological features of these lesions. We selected 10 cases each of SRCC and GCA from the archival imprint smears. We assessed them for the following 10 cytological features. Necrosis/debris was observed in 60% of the SRCC and 90% of the GCA. A mucinous background was observed in 10% of the SRCC and 90% of the GCA. Significant inflammation was observed in none of the SRCC and 80% of the GCA. Stromal cluster was observed in 30% of the SRCC and 70% of the GCA. Nuclear overlapping was observed in 50% of the SRCC and in all of the GCA. Single tumor cells were observed in 80% of the SRCC and 10% of the GCA. Honeycomb-like cluster was observed in none of the SRCC and 80% of the GCA. Prominent nucleolus was observed in 50% of the SRCC and 40% of the GCA. Nuclear membrane irregularity was observed in 70% of SRCC and 60% of the GCA. Nuclear pleomorphism was observed in all of the SRCC and none of the GCA. The cytological features of SRCC were the presence of single tumor cells and nuclear pleomorphism, whereas that of GCA were the presence of abundant mucin and significant inflammation in the background, and a honeycomb-like cluster.

Analysis of expression patterns of breast cancer-specific markers (mammaglobin and gross cystic disease fluid protein 15) in lung and pleural tumors.

CONTEXT: The lung is the most common site of metastasis during the natural history of malignant tumors. Breast carcinoma has a propensity for distant metastasis, and the lung and pleura are among the most common metastatic sites. Although it is often difficult to make a clear-cut differential diagnosis between the two, distinguishing primary lung carcinoma from breast carcinoma metastatic to the lung is important because the treatment modalities are different. OBJECTIVE: To elucidate the utility of mammaglobin and gross cystic disease fluid protein 15 (GCDFP-15), which are known to be breast-specific antigens, in distinguishing various primary lung and pleural tumors from breast carcinoma metastasizing to the lung. DESIGN: A total of 20 cases of breast carcinoma metastatic to the lung and 263 tumors of nonbreast origin located in the lung and pleura were analyzed. RESULTS: Of the 20 cases of breast carcinoma metastatic to the lung, 10 (50.0%) were immunoreactive for mammaglobin and 9 (45.0%) for GCDFP-15, the frequency of positivity being slightly higher for the former than for the latter. The area immunopositive for mammaglobin showed more diffuse staining than the area immunopositive for GCDFP-15. Furthermore, the specificity of mammaglobin for breast carcinoma metastatic to the lung was superior (98.9%) to that of GCDFP-15 (91.8%). CONCLUSION: The sensitivity of mammaglobin is equal or superior to that of GCDFP-15 for investigation of breast carcinoma. Immunopositivity for mammaglobin is more diffuse than that for GCDFP-15. In terms of practical diagnosis, mammaglobin immunohistochemistry can serve as a differential marker of breast carcinoma and should be added to the immunohistochemical panel.

Cytopathologic factors can predict invasion in small-sized peripheral lung adenocarcinoma with a bronchioloalveolar carcinoma component.

BACKGROUND: Patients with noninvasive, small-sized primary adenocarcinomas of the lung have excellent prognosis after lobectomy. Several researchers have suggested that limited resection could be an acceptable alternative for these patients. Therefore, a preoperative or intraoperative judgment of invasiveness would be one of the critical determinants of the surgical procedure in each case. Cytopathologic findings that can distinguish invasive from noninvasive adenocarcinomas remain to be elucidated. METHODS: Imprint smears were obtained from 60 resected adenocarcinomas with nonmucinous bronchioloalveolar features. Thirteen cytologic factors were evaluated: the presence of necrosis, fibrovascular tissue, proportion of macrophages, the presence of large tumor cell clusters, nuclear grooves, nuclear overlapping, variation in nuclear size, chromatin pattern, presence of a nucleolus, intranuclear inclusions, multinucleated cells, spindle cells, and mitosis. Each factor was examined by univariate analysis for correlation with the presence of histopathologic invasion. RESULTS: In the univariate analysis, 5 cytologic factors--presence of tumor cell clusters consisting of more than 50 tumor cells (P < .001), nuclear overlapping in more than 3 layers (P < .001), presence of nuclear grooves (P = .007), more than 3-fold variation in nuclear size (P < .001), and 1 mitotic cell per 1000 tumor cells (P = .035)--were associated significantly with invasion. Among these, nuclear overlapping in more than 3 layers (P = .003) and more than 3-fold variation in nuclear size (P = .005) were found to be independent predictive factors for invasion by multivariate analysis. CONCLUSIONS: Using imprint smears, the presence of invasion in small-sized primary adenocarcinomas of the lung is predictable by the 2 above-mentioned cytologic findings. Imprint smear cytology may effectively aid intraoperative judgement of invasion in cases where frozen section histology is difficult to interpret.