Prediction of Pleural Lavage Cytology According to Thin-Section Computed Tomography in Non-Small-Cell Lung Cancer.

BACKGROUND: Although the positive rate of preresection pleural lavage cytology (PLC) is low, it is an important indicator of poor prognosis for non-small-cell lung cancer patients with frequent pleural dissemination (PD) recurrence. Thin-section computed tomography (TSCT) can reveal relationships between a primary tumor and the pleura at 1 to 2 mm intervals, and this is associated with visceral pleural invasion (VPI). However, its association with PLC remains unclear. Therefore, we aimed to improve PLC efficiency and predict PD recurrence by understanding the relationship between PLC and preoperative TSCT findings. PATIENTS AND METHODS: Between January 2014 and December 2018, we reviewed 978 patients with non-small-cell lung cancer who underwent PLC tests during complete resection surgery. Preoperative TSCT findings were evaluated, and factors with the highest specificity (proportion of patients with radiologically to pathologically diagnosed positive PLC) were investigated. We also evaluated their relationships with VPI and PD recurrence. RESULTS: PLC positive was identified in 55 (5.6%) of the 978 patients. The two TSCT findings predicting PLC results, "the absence of pleural findings," ie, tumor not attached to pleura or without pleural tag, and "consolidation-to-tumor ratio ≤0.5", had a specificity of 100% (95% confidence interval: 90.4%-100%); additionally, all cases with these findings were VPI negative and had no PD recurrence. And 24% of the cohort had either of these findings. CONCLUSION: The absence of pleural findings and/or consolidation-to-tumor ratio ≤0.5 of primary tumor on preoperative TSCT can predict PLC negativity with very high probability; therefore, PLC can be omitted for such patients.

Intraoperative rapid diagnosis of pleural lavage cytology in non-small cell lung cancer.

OBJECTIVE: Positive pleural lavage cytology (PLC +) is a poor prognostic factor for non-small cell lung cancer (NSCLC). However, data on the impact of intraoperative rapid diagnosis of PLC (rPLC) are lacking. Therefore, we evaluated the efficacy of rPLC before resection during surgery. METHODS: A total of 1,838 patients who underwent rPLC for NSCLC between September 2002 and December 2014 were studied retrospectively. We assessed the clinicopathological factors between rPLC findings and the impact on survival of patients with curative resection. RESULTS: The rPLC + status was observed in 96 (5.3%) among 1,838 patients. The rPLC + group had more unsuspected N2 (30%) than the rPLC- group (p < 0.001). The 5-year overall survival (OS) of patients who underwent lobectomy or more extensive resection with rPLC + , negative rPLC (rPLC-), and microscopic pleural dissemination (PD) and/or malignant pleural effusion (PE) were 67.3, 81.3, and 11.0%, respectively. In the rPLC + group, the prognosis of patients with pN2 was equal to that of pN0-1 (5-year OS: 77.9% vs. 63.4%, p = 0.263). Undetectable dissemination in the first evaluation immediately after starting surgery was found in 9% of rPLC + patients by additional evaluation of the thoracic cavity. CONCLUSIONS: Patients with rPLC + have more favorable survival than those with microscopic PD/PE after surgery. Curative resection should be performed in patients with rPLC + , even if N2 is detected during surgery. However, the rPLC + group often has N2 upstaging; therefore, systematic nodal dissection should be performed in rPLC + patients for exact staging. rPLC may contribute to preventing oversight PD by re-evaluation during surgery.

Prognostic significance of tumor microenvironment assessed by machine learning algorithm in surgically resected non-small cell lung cancer.

BACKGROUND: A methodology to assess the immune microenvironment (IME) of non-small cell lung cancer (NSCLC) has not been established, and the prognostic impact of IME factors is not yet clear. AIMS: This study aimed to assess the IME factors and evaluate their prognostic values. METHODS AND RESULTS: We assessed CD8+ tumor-infiltrating lymphocyte (TIL) density, forkhead box protein P3+ (Foxp3+ ) TIL density, and programmed death receptor ligand-1 (PD-L1) tumor proportion score (TPS) using a machine-learning algorithm in whole-slide imaging (WSI). We dichotomized patients according to TIL density or TPS and compared their clinical outcomes. Between September 2014 and September 2015, 165 patients with NSCLC were enrolled in the study. We assessed IME factors in the epithelium, stroma, and their combination. An improvement in disease-free survival (DFS) was observed in the high CD8+ TIL density group in the epithelium, stroma, and the combination of both. Moreover, the group with high PD-L1 TPS in the epithelium showed better DFS than that with low PD-L1 TPS. In the multivariate analysis, the CD8+ TIL density in the combination of epithelium and stroma and PD-L1 TPS in the epithelium were independent prognostic factors (hazard ratio [HR] = 0.43; 95% confidence interval [CI] = 0.26-0.72; p = .001, HR = 0.49; 95% CI = 0.30-0.81; p = .005, respectively). CONCLUSION: Our approach demonstrated that the IME factors are related to survival in patients with NSCLC. The quantitative assessment of IME factors enables to discriminate patients with high risk of recurrence, who can be the candidates for adjuvant therapy. Assessing the CD8+ TIL density in the combination of epithelium and stroma might be more useful than their individual assessment because it is a simple and time-saving analysis of TILs in WSI.

Clinical significance of immunocytochemical staining for peritoneal lavage cytology in pancreatic cancer.

BACKGROUND: Peritoneal lavage cytology for pancreatic ductal adenocarcinoma is conducted with both an intraoperative rapid diagnosis by Papanicolaou staining (cytology-rapid) and a final diagnosis by immunocytochemical staining at a later date (cytology-final) in our hospital. However, the clinical significance of cytology-final has not yet been elucidated. METHODS: A total of 675 pancreatic ductal adenocarcinoma patients who underwent pancreatectomy and cytology between 2002 and 2018 were retrospectively reviewed. Diagnostic results of cytology-rapid and cytology-final and survival outcomes were analyzed. RESULTS: A total of 43 patients (6.4%) were diagnosed as cytology-rapid (+), and all of them were ultimately diagnosed as cytology-final (+). Among the 632 patients with cytology-rapid (-), 19 (3.0%) were eventually diagnosed as cytology-final (+). The overall survival of patients with cytology-rapid (+) and that of patients with cytology-rapid (-) did not differ to a statistically significant extent (median survival time 26.4 vs 32.9 months; P = .106). In contrast, the overall survival of patients who were diagnosed as a false-negative result by cytology-rapid was significantly worse than that of patients diagnosed as a true negative (18.7 vs 34.8 months; P = .031). The overall survival of patients with cytology-final (+) was significantly worse than that of patients with cytology-final (-) (23.6 vs 34.8 months; P = .012). A multivariate analysis showed that cytology-final (+) was an independent prognostic factor for the OS (hazard ratio = 1.43; P = .049), whereas cytology-rapid (+) was not. CONCLUSION: Immunocytochemical staining may be a useful complement to a diagnosis of cytology by conventional Papanicolaou staining in pancreatic ductal adenocarcinoma patients.

Artificial Intelligence-Powered Prediction of ALK Gene Rearrangement in Patients With Non-Small-Cell Lung Cancer.

PURPOSE: Several studies reported the possibility of predicting genetic abnormalities in non-small-cell lung cancer by deep learning (DL). However, there are no data of predicting ALK gene rearrangement (ALKr) using DL. We evaluated the ALKr predictability using the DL platform. MATERIALS AND METHODS: We selected 66 ALKr-positive cases and 142 ALKr-negative cases, which were diagnosed by ALKr immunohistochemical staining in our institution from January 2009 to March 2019. We generated virtual slide of 300 slides (150 ALKr-positive slides and 150 ALKr-negative slides) using NanoZoomer. HALO-AI was used to analyze the whole-slide imaging data, and the DenseNet network was used to build the learning model. Of the 300 slides, we randomly assigned 172 slides to the training cohort and 128 slides to the test cohort to ensure no duplication of cases. In four resolutions (16.0/4.0/1.0/0.25 µm/pix), ALKr prediction models were built in the training cohort and ALKr prediction performance was evaluated in the test cohort. We evaluated the diagnostic probability of ALKr by receiver operating characteristic analysis in each ALKr probability threshold (50%, 60%, 70%, 80%, 90%, and 95%). We expected the area under the curve to be 0.64-0.85 in the model of a previous study. Furthermore, in the test cohort data, an expert pathologist also evaluated the presence of ALKr by hematoxylin and eosin staining on whole-slide imaging. RESULTS: The maximum area under the curve was 0.73 (50% threshold: 95% CI, 0.65 to 0.82) in the resolution of 1.0 µm/pix. In this resolution, with an ALKr probability of 50% threshold, the sensitivity and specificity were 73% and 73%, respectively. The expert pathologist's sensitivity and specificity in the same test cohort were 13% and 94%. CONCLUSION: The ALKr prediction by DL was feasible. Further study should be addressed to improve accuracy of ALKr prediction.

Application of enhancer of zeste homolog 2 immunocytochemistry to bile cytology.

BACKGROUND: Bile cytology has low diagnostic sensitivity and requires ancillary techniques. This study assessed the utility of enhancer of zeste homolog 2 (EZH2) immunocytochemistry (ICC) in bile cytology. METHODS: A total of 141 bile cytology specimens from 141 patients were evaluated retrospectively. Papanicolaou-stained slides were immunostained with an antibody to EZH2. After calculation of the EZH2 labeling index (LI), the cutoff value was determined via receiver operating characteristic curve analysis. Cytological performance with and without EZH2 ICC was evaluated with reference to the final diagnosis. RESULTS: The area under the curve for the EZH2 LI was 0.955, and the cutoff value for identifying benign bile samples versus malignant ones was 24.0%. The sensitivity and specificity values for malignancy were 53.4% and 100% for routine cytology only, 89.0% and 95.7% for EZH2 ICC only, and 89.8% and 95.7% for a combination of routine cytology and EZH2 ICC. The sensitivities of EZH2 ICC only and a combination of routine cytology and EZH2 ICC were significantly improved in comparison with routine cytology only (P < .001). EZH2 ICC alone had a sensitivity of 68.0% and a specificity of 85.7% in bile samples with atypical cytology, a sensitivity of 87.0% in samples that were suspicious for malignancy, and a sensitivity of 85.7% and a specificity of 100% in samples that were negative for malignancy. CONCLUSIONS: EZH2 ICC improved the diagnostic sensitivity for pancreatobiliary adenocarcinoma in bile cytology. This method is particularly meaningful in samples of indeterminate cytology and may be useful as an initial assessment to ensure that no cancer cells are missed.

Classic Hodgkin lymphoma with osseous involvement mimicking Langerhans cell histiocytosis in a child.

Hodgkin lymphoma (HL) commonly presents superficial lymphadenopathy. In addition, HL cells can arise in various organs including the liver and spleen as an extranodal lymphoma. HL in bone is unusual at the initial diagnosis, although some cases show late-stage localization of lymphoma cells to bone. We report the rare case of a young patient with cranial bone classic HL, presumably originating from the skull without any involvement of lymph nodes. As the main clinical manifestation was only tumor mass in the skull without osteoscopic pain, the tentative diagnosis of Langerhans cell histiocytosis was histologically confirmed by an excisional biopsy. Before the final pathological diagnosis as classic HL, we noticed several small lesions in extranodal regions through systemic surveys, suggesting that the cranial lesion appeared antecedent to those lesions. This is a rare and instructive case of cranial bone HL for which a histological diagnosis has been meticulously made.

A mechanism for evasion of CTL immunity by altered O-glycosylation of HLA class I.

Anti-tumour immunity by cytotoxic T-lymphocytes (CTLs) is essential to suppress tumour progression. Cancer cells that evade CTL immunity proliferate in the host, promoting metastasis, but mechanisms underlying this capacity remain unknown. Here we report that bladder cancer cells metastasized to lymph nodes evade CTL immunity by a new mechanism via altered glycosylation. CTLs normally recognize and kill cancer cells presenting antigenic peptides on human leukocyte antigen (HLA) class I. We show bladder cancer cells expressing the O-glycan processing enzyme, core2 ß-1,6-N-acetylglucosaminyltransferase (C2GnT) exhibit HLA class I O-glycan modified with poly-N-acetyllactosamine and are highly susceptible to CTL. In those cells, poly-N-acetyllactosamine on HLA class I O-glycan binds galectin-3 to form a cell-surface molecular lattice, enabling efficient cell-surface retention of HLA class I. In contrast, bladder cancer cells in which C2GnT is downregulated show decreased levels of poly-N-acetyllactosamine on HLA class I O-glycans, attenuating lattice formation and reducing the cell-surface half-life of HLA class I. These tumour cells present antigenic peptides less efficiently, thereby evading CTL lysis and facilitating metastasis.

An ancillary method in urine cytology: Nucleolar/nuclear volume ratio for discrimination between benign and malignant urothelial cells.

BACKGROUND: The essential of urine cytology for the diagnosis and the follow-up of urothelial neoplasia has been widely recognized. However, there are some cases in which a definitive diagnosis cannot be made due to difficulty in discriminating between benign and malignant. This study evaluated the practicality of nucleolar/nuclear volume ratio (%) for the discrimination. METHODS: Using Papanicolaou-stained slides, 253 benign urothelial cells and 282 malignant urothelial cells were selected and divided into a benign urothelial cell and an urothelial carcinoma (UC) cell groups. Three suspicious cases and four cases in which discrimination between benign and malignant was difficult were prepared for verification test. Subject cells were decolorized and stained with 4',6-diamidino-2-phenylindole for detection of the nuclei and the nucleoli. Z-stack method was performed to analyze. RESULTS: When the cutoff point of 1.514% discriminating benign urothelial cells and UC cells from nucleolar/nuclear volume ratio (%) was utilized, the sensitivity was 56.0%, the specificity was 88.5%, the positive predictive value was 84.5%, and the negative predictive value was 64.4%. Nuclear and nucleolar volume, number of the nucleoli, and nucleolar/nuclear volume ratio (%) were significantly higher in the UC cell group than in the benign urothelial cell group (P <0.001). In the verification test using the nucleolar/nuclear ratio (%), four of the seven cases were concordant with the final diagnosis. CONCLUSION: This study analyzed the nuclear and nucleolar volume to establish an index for discrimination of benign and malignant urothelial cells, providing possible additional information in urine cytology. Diagn. Cytopathol. 2016;44:483-491. © 2016 Wiley Periodicals, Inc.

Utility of intraoperative cytology of resection margins in biliary tract and pancreas tumors.

BACKGROUND: Intraoperative diagnosis of resection margins in the biliary tract and pancreas tumors is important in deciding extent of resection; however, diagnosis based solely on frozen sections is sometimes difficult. Therefore, we investigated the usefulness of intraoperative cytology (IC) combined with frozen section (FS) histology. We present the results with a discussion of the value of this combination and its associated problems. METHODS: We examined 80 bile duct resection margin specimens from 42 patients, and 34 pancreatic resection margin specimens from 29 patients, who underwent intraoperative diagnosis of resection margins during surgery for biliary tract or pancreatic tumors between October 2012 and January 2014. IC was performed on imprint specimens prepared from surfaces of margins being examined. The results were compared with FS and final histology of operative materials. RESULTS: In IC, excluding cases with insufficient material, the results for bile duct margins; sensitivity was 96.7%, specificity 100% and accuracy 98.7%. The results for pancreatic margins; sensitivity was 100%, specificity 92.9%, and accuracy 93.3%. In FS, the results for bile duct margins; sensitivity was 96.8%, specificity 100%, and accuracy 98.8%. The results for pancreatic margins; sensitivity was 66.7%, specificity 100%, and accuracy 97.1%. CONCLUSION: IC is quick, highly accurate and very easy to perform. This study even included a specimen for which only IC led to an accurate diagnosis. IC used in combination with FS can achieve intraoperative diagnosis with high overall accuracy.

Clinical application of immunocytochemical detection of ALK rearrangement on cytology slides for detection or screening of lung adenocarcinoma.

UNLABELLED: Immunohistochemical screening of Anaplastic lymphoma kinase (ALK) rearrangement has been regarded essential and routinely carried out to select treatment for lung adenocarcinoma. However, difficulty to approach a tumor by transbronchial lung biopsy (TBLB), it often fails to obtain tumor tissues whereas tumor cells are contained in cytology specimens simultaneously obtained when the bronchoscopy is done. Therefore we evaluated the expression of ALK protein by using immunohistochemistry (IHC) on TBLB specimens and immunocytochemistry (ICC) on brushing smear cytology slides in the same cases, and compared the concordance rate of IHC and ICC results. ICC was carried out on routine Papanicolau-stained slides after cytology diagnosis and decolorization. RESULTS: Eighteen patients with adenocarcinoma were extracted in the Hirosaki University Hospital and the Hirosaki National Hospital. IHC and ICC results showed a very high concordance rate: sensitivity of ICC in comparison with IHC was 85.7% (6/7), specificity was 100% (11/11), positive predictive value was 100% (6/6), and negative predictive value was 91.6% (11/12). Detection of ALK rearrangement using ICC on routine Papanicolau cytology slides is considered to be advantageous for lung cancer treatments.

Three-dimensional nuclear luminance analysis in well-differentiated adenocarcinoma of the lung.

OBJECTIVE: Well-differentiated papillary adenocarcinoma of the lung (G1 cancer cells) is difficult to distinguish from benign bronchial columnar epithelial cells with reactive atypia (benign cells) in many cases because nuclear atypia is mild. We focused on the 3-dimensional presence of nuclei in cell smears. Several images focused on the nucleus were acquired, and the nuclear luminance was measured and analyzed. STUDY DESIGN: One hundred G1 cancer cells and benign cells (nuclei), respectively, were selected from those on a bronchial brushing preparation for cytology. Images of 41 layers were acquired at 0.25-µm intervals in each cell, and the nuclear luminance was measured (a total of 8,400 images). RESULTS: There were more focus positions in the G1 cancer cell nuclei, showing a 3-dimensional nucleus, compared to benign cells, and the 3-dimensional variation in the coefficient of variation (CV) of nuclear luminance at the focus position was smaller in G1 cancer than in benign cells, showing a significant difference. CONCLUSION: The G1 cancer cells' nuclear structure was more 3-dimensional, and the chromatin distribution was homogeneous. The three-dimensional variation in the nuclear luminance CV could be numerically presented, which might be an objective index for cancer diagnosis.