Candidate tumor-specific CD8+ T cell subsets identified in the malignant pleural effusion of advanced lung cancer patients by single-cell analysis.

Isolation of tumor-specific T cells and their antigen receptors (TCRs) from malignant pleural effusions (MPE) may facilitate the development of TCR-transduced adoptive cellular immunotherapy products for advanced lung cancer patients. However, the characteristics and markers of tumor-specific T-cells in MPE are largely undefined. To this end, to establish the phenotypes and antigen specificities of CD8+ T cells, we performed single-cell RNA and TCR sequencing of samples from three advanced lung cancer patients. Dimensionality reduction on a total of 4,983 CD8+ T cells revealed 10 clusters including naïve, memory, and exhausted phenotypes. We focused particularly on exhausted T cell clusters and tested their TCR reactivity against neoantigens predicted from autologous cancer cell lines. Four different TCRs specific for the same neoantigen and one orphan TCR specific for the autologous cell line were identified from one of the patients. Differential gene expression analysis in tumor-specific T cells relative to the other T cells identified CXCL13, as a candidate gene expressed by tumor-specific T cells. In addition to expressing CXCL13, tumor-specific T cells were present in a higher proportion of T cells co-expressing PDCD1(PD-1)/TNFRSF9(4-1BB). Furthermore, flow cytometric analyses in advanced lung cancer patients with MPE documented that those with high PD-1/4-1BB expression have a better prognosis in the subset of 57 adenocarcinoma patients (p = .039). These data suggest that PD-1/4-1BB co-expression might identify tumor-specific CD8+ T cells in MPE, which are associated with patients' prognosis. (233 words).

Single-cell sequencing on CD8+ TILs revealed the nature of exhausted T cells recognizing neoantigen and cancer/testis antigen in non-small cell lung cancer.

BACKGROUND: CD8+tumor infiltrating lymphocytes (TILs) are often observed in non-small cell lung cancers (NSCLC). However, the characteristics of CD8+ TILs, especially T-cell populations specific for tumor antigens, remain poorly understood. METHODS: High throughput single-cell RNA sequencing and single-cell T-cell receptor (TCR) sequencing were performed on CD8+ TILs from three surgically-resected lung cancer specimens. Dimensional reduction for clustering was performed using Uniform Manifold Approximation and Projection. CD8+ TIL TCR specific for the cancer/testis antigen KK-LC-1 and for predicted neoantigens were investigated. Differentially-expressed gene analysis, Gene Set Enrichment Analysis (GSEA) and single sample GSEA was performed to characterize antigen-specific T cells. RESULTS: A total of 6998 CD8+ T cells was analyzed, divided into 10 clusters according to their gene expression profile. An exhausted T-cell (exhausted T (Tex)) cluster characterized by the expression of ENTPD1 (CD39), TOX, PDCD1 (PD1), HAVCR2 (TIM3) and other genes, and by T-cell oligoclonality, was identified. The Tex TCR repertoire (Tex-TCRs) contained nine different TCR clonotypes recognizing five tumor antigens including a KK-LC-1 antigen and four neoantigens. By re-clustering the tumor antigen-specific T cells (n=140), it could be seen that the individual T-cell clonotypes were present on cells at different stages of differentiation and functional states even within the same Tex cluster. Stimulating these T cells with predicted cognate peptide indicated that TCR signal strength and subsequent T-cell proliferation and cytokine production was variable but always higher for neoantigens than KK-LC-1. CONCLUSIONS: Our approach focusing on T cells with an exhausted phenotype among CD8+ TILs may facilitate the identification of tumor antigens and clarify the nature of the antigen-specific T cells to specify the promising immunotherapeutic targets in patients with NSCLC.

Proteomic profiling of HTLV-1 carriers and ATL patients reveals sTNFR2 as a novel diagnostic biomarker for acute ATL.

Adult T-cell leukemia/lymphoma (ATL) is a human T-cell leukemia virus type 1 (HTLV-1)-associated T-cell malignancy with generally poor prognosis. Although only ∼5% of HTLV-1 carriers progress to ATL, early diagnosis is challenging because of the lack of ATL biomarkers. In this study, we analyzed blood plasma profiles of asymptomatic HTLV-1 carriers (ACs); untreated ATL patients, including acute, lymphoma, smoldering, and chronic types; and ATL patients in remission. Through SOMAscan, expression levels of 1305 plasma proteins were analyzed in 85 samples (AC, n = 40; ATL, n = 40; remission, n = 5). Using gene set enrichment analysis and gene ontology, overrepresented pathways in ATL vs AC included angiogenesis, inflammation by cytokines and chemokines, interleukin-6 (IL-6)/JAK/STAT3, and notch signaling. In selecting candidate biomarkers, we focused on soluble tumor necrosis factor 2 (sTNFR2) because of its active role in enriched pathways, extreme significance (Welch's t test P < .00001), high discrimination capacity (area under the curve >0.90), and novelty in ATL research. Quantification of sTNFR2 in 102 plasma samples (AC, n = 30; ATL, n = 68; remission, n = 4) using enzyme-linked immunosorbent assay showed remarkable elevations in acute ATL, at least 10 times those of AC samples, and return of sTNFR2 to AC state levels after achieving remission. Flow cytometry and immunostaining validated the expression of TNFR2 in ATL cells. No correlation between sIL-2 and sTNFR2 levels in acute ATL was found, suggesting the possibility of sTNFR2 as an independent biomarker. Our findings represent the first extensive blood-based proteomic analysis of ATL, suggesting the potential clinical utility of sTNFR2 in diagnosing acute ATL.

Automated histological classification of whole-slide images of gastric biopsy specimens.

BACKGROUND: Automated image analysis has been developed currently in the field of surgical pathology. The aim of the present study was to evaluate the classification accuracy of the e-Pathologist image analysis software. METHODS: A total of 3062 gastric biopsy specimens were consecutively obtained and stained. The specimen slides were anonymized and digitized. At least two experienced gastrointestinal pathologists evaluated each slide for pathological diagnosis. We compared the three-tier (positive for carcinoma or suspicion of carcinoma; caution for adenoma or suspicion of a neoplastic lesion; or negative for a neoplastic lesion) or two-tier (negative or non-negative) classification results of human pathologists and of the e-Pathologist. RESULTS: Of 3062 cases, 33.4% showed an abnormal finding. For the three-tier classification, the overall concordance rate was 55.6% (1702/3062). The kappa coefficient was 0.28 (95% CI, 0.26-0.30; fair agreement). For the negative biopsy specimens, the concordance rate was 90.6% (1033/1140), but for the positive biopsy specimens, the concordance rate was less than 50%. For the two-tier classification, the sensitivity, specificity, positive predictive value, and negative predictive value were 89.5% (95% CI, 87.5-91.4%), 50.7% (95% CI, 48.5-52.9%), 47.7% (95% CI, 45.4-49.9%), and 90.6% (95% CI, 88.8-92.2%), respectively. CONCLUSIONS: Although there are limitations and requirements for applying automated histopathological classification of gastric biopsy specimens in the clinical setting, the results of the present study are promising.

Automated histological classification of whole slide images of colorectal biopsy specimens.

BACKGROUND: An automated image analysis system, e-Pathologist, was developed to improve the quality of colorectal biopsy diagnostics in routine pathology practice. OBJECTIVE: The aim of the study was to evaluate the classification accuracy of the e-Pathologist image analysis software in the setting of routine pathology practice in two institutions. MATERIALS AND METHODS: In total, 1328 colorectal tissue specimens were consecutively obtained from two hospitals (1077 tissues from Tokyo hospital, and 251 tissues from East hospital) and the stained specimen slides were anonymized and digitized. At least two experienced gastrointestinal pathologists evaluated each slide for pathological diagnosis. We compared the 3-tier classification results (carcinoma or suspicion of carcinoma, adenoma, and lastly negative for a neoplastic lesion) between the human pathologists and that of e-Pathologist. RESULTS: For the Tokyo hospital specimens, all carcinoma tissues were correctly classified (n=112), and 9.9% (80/810) of the adenoma tissues were incorrectly classified as negative. For the East hospital specimens, 0 out of the 51 adenoma tissues were incorrectly classified as negative while 9.3% (11/118) of the carcinoma tissues were incorrectly classified as either adenoma, or negative. For the Tokyo and East hospital datasets, the undetected rate of carcinoma, undetected rate of adenoma, and over-detected proportion were 0% and 9.3%, 9.9% and 0%, and 36.1% and 27.1%, respectively. CONCLUSIONS: This image analysis system requires some improvements; however, it has the potential to assist pathologists in quality improvement of routine pathological practice in the not too distant future.

Detection of Subclinical Peripheral Artery Ischemia in Healthy Male Smokers by an Ankle-Brachial Index After Exercise: Sasayama Study.

We investigated the relationship between smoking and the risk of nonnormal (≤0.99) ankle-brachial index (ABI) at rest and after ankle plantar flexion exercise in healthy male community dwellers. A cross-sectional study was performed including 228 Japanese men aged 40 to 64 years without a history of cardiovascular diseases. Participants were classified as never, ex-, and current smokers. We estimated the multivariate-adjusted odds ratios (ORs) for nonnormal ABI of ex- and current smokers in relation to never smokers after adjusting for age and other confounding factors. At rest, the prevalence of nonnormal ABI was not significantly different by smoking status. After exercise, the prevalence of nonnormal ABI increased from 1.8% to 11.5% in ex-smokers and from 3.8% to 17.0% in current smokers, while the prevalence did not significantly change in never smokers. The multivariate-adjusted OR for nonnormal ABI after ankle plantar flexion exercise, in relation to never smokers, was 3.85 (95% confidence interval [CI]: 0.79-18.9) for ex-smokers and 6.97 (95% CI: 1.32-36.7) for current smokers. Our results suggest that ABI after ankle plantar flexion exercise is useful for early detection of subclinical peripheral artery ischemia in male smokers without typical symptoms.

Does flat epithelial atypia have rounder nuclei than columnar cell change/hyperplasia? A morphometric approach to columnar cell lesions of the breast.

Columnar cell lesions of the breast encompass columnar cell change/hyperplasia (CCC/CCH) and flat epithelial atypia (FEA). These have attracted researchers because emerging data suggest that FEA may represent the earliest histologically detectable non-obligate precursor of breast cancer. However, it is occasionally difficult to distinguish FEA from CCC/CCH because of similar histology. Although the nuclei of FEA are frequently described as relatively round compared with those of CCC/CCH, there are few morphometric studies to support this statement. The aim of this study was to provide objective data as to the nuclear shape in columnar cell lesions. As a shape descriptor, we adopted ellipticity that is defined by the formula 2b/2a, where a is the length of the long axis of the ellipse and b is the length of the short axis. Contrary to circularity, ellipticity reflects the overall configuration of an ellipse irrespective of surface irregularity. Our image analysis included generating whole slide images, extracting glandular cell nuclei, measuring nuclear ellipticity, and superimposing graded colors based on execution of results on the captured images. A total of 7917 nuclei extracted from 22 FEA images and 5010 nuclei extracted from 13 CCC/CCH images were analyzed. There was a significant difference in nuclear roundness between FEA and CCC/CCH with mean ellipticity values of 0.723 and 0.679, respectively (p < 0.001, Welch's t test). Furthermore, FEA with malignancy had significantly rounder nuclei than FEA without malignancy (p < 0.001). Our preliminary results suggest that nuclear ellipticity is a key parameter in reproducibly classifying columnar cell lesions of the breast.

Enhancing automatic classification of hepatocellular carcinoma images through image masking, tissue changes and trabecular features.

BACKGROUND: Recent breakthroughs in computer vision and digital microscopy have prompted the application of such technologies in cancer diagnosis, especially in histopathological image analysis. Earlier, an attempt to classify hepatocellular carcinoma images based on nuclear and structural features has been carried out on a set of surgical resected samples. Here, we proposed methods to enhance the process and improve the classification performance. METHODS: First, we segmented the histological components of the liver tissues and generated several masked images. By utilizing the masked images, some set of new features were introduced, producing three sets of features consisting nuclei, trabecular and tissue changes features. Furthermore, we extended the classification process by using biopsy resected samples in addition to the surgical samples. RESULTS: Experiments by using support vector machine (SVM) classifier with combinations of features and sample types showed that the proposed methods improve the classification rate in HCC detection for about 1-3%. Moreover, detection rate of low-grades cancer increased when the new features were appended in the classification process, although the rate was worsen in the case of undifferentiated tumors. CONCLUSIONS: The masking process increased the reliability of extracted nuclei features. The additional of new features improved the system especially for early HCC detection. Likewise, the combination of surgical and biopsy samples as training data could also improve the classification rates. Therefore, the methods will extend the support for pathologists in the HCC diagnosis.

Association between serum long-chain n-3 and n-6 polyunsaturated fatty acid profiles and glomerular filtration rate assessed by serum creatinine and cystatin C levels in Japanese community-dwellers.

BACKGROUND: Plasma concentration of n-3 polyunsaturated fatty acids (PUFAs) has been reported to be associated with renal function in Western populations. However, few studies have investigated the association between serum long-chain n-3 and n-6 PUFA profiles and renal function in a Japanese population with high marine-derived long-chain n-3 PUFA intake. METHODS: A cross-sectional study was performed in 549 Japanese rural community-dwellers aged 40 to 64 years. In adjusted analysis of covariance, we assessed the relationship between estimated glomerular filtration rate (eGFR) and tertiles of serum long-chain n-3 and n-6 PUFA profiles ([eicosapentaenoic acid {EPA} + docosahexaenoic acid {DHA}]:arachidonic acid [AA]). GFR was estimated by Japanese specific equations using serum creatinine and cystatin C (eGFRcre and eGFRcys). Using multivariate-adjusted linear regression models, we also assessed the relationships between eGFRs and several n-3 and n-6 PUFAs, which have been suggested to be associated with renal function. RESULTS: In all participants, higher dietary fish intake as assessed by a semi-quantitative questionnaire was associated with higher serum value of (EPA+DHA):AA. Participants in the higher (EPA+DHA):AA tertiles had non-significantly higher eGFRcre and significantly higher eGFRcys (P = 0.016). In addition, eGFRcys in T2+T3 of (EPA+DHA):AA was significantly higher than that in T1 (adjusted mean eGFRcys, T1: 87 ml/min/1.73 m(2), T2+T3: 91 ml/min/1.73 m(2); P < 0.01). Among the PUFAs, only (EPA+DHA) was significantly associated with eGFRcys. CONCLUSIONS: Serum (EPA+DHA):AA, which reflects an individual's fish intake, might be associated with eGFRcys in Japanese community-dwellers.

High-resolution MRI predicts steroid injection response in carpal tunnel syndrome patients.

OBJECTIVES: To correlate median nerve T2 signal and shape at the carpal tunnel with steroid injection (SI) response in carpal tunnel syndrome (CTS) patients. METHODS: One hundred and sixty-three CTS wrists of 92 consecutive patients who were scheduled to undergo SI were prospectively evaluated with 3-T magnetic resonance imaging (MRI) and a nerve conduction study. All patients underwent axial high-resolution T2-weighted MRI (in-plane resolution of 0.25 × 0.25 mm). The CTS wrists were classified into three groups according to the nerve T2 signal and the flattening ratio at the hook of hamate level: group 1, high and oval; group 2, high and flat; group 3, low and flat. Clinical response to SI was evaluated at 6 months after injection. RESULTS: One hundred and thirteen of the 163 wrists (69.3%) responded well to SI. The percentage of improvement was 81.7% (49/60) in group 1, 69.9% (51/73) in group 2, and 43.3% (13/30) in group 3 (P < 0.01). On stepwise logistic regression analysis high-resolution MRI was the only significant independent factor for SI response in CTS patients (P < 0.01). CONCLUSIONS: High-resolution MRI correlates well with SI response in CTS patients and seems useful for predicting SI response. KEY POINTS: • MRI may help determine appropriate care in carpal tunnel syndrome. • MRI helps in therapeutic decision-making whenever steroid injection is considered. • T2 signal decrease of the median nerve correlates with poor outcome. • T2 signal decrease of median nerve may reflect fibrosis and amyloid deposition.

Iterative decomposition of water and fat with echo asymmetry and least-squares estimation (IDEAL) of the wrist and finger at 3T: comparison with chemical shift selective fat suppression images.

PURPOSE: To compare fat-suppressed magnetic resonance imaging (MRI) quality using iterative decomposition of water and fat with echo asymmetry and least-squares estimation (IDEAL) with that using chemical shift selective fat-suppressed T1-weighted spin-echo (CHESS) images for evaluating rheumatoid arthritis (RA) lesions of the hand and finger at 3T. MATERIALS AND METHODS: MRI was performed in eight healthy volunteers and eight RA patients with a 3.0T MR system (Signa HDxt GE healthcare) using an eight-channel knee coil. FS-CHESS-T1-SE and IDEAL imaging were acquired in the coronal planes covering the entire structure of the bilateral hands with a slice thickness of 2 mm. In the RA patients both images were obtained after intravenous gadolinium administration. Image quality was evaluated on a five-point scale (1 = excellent to 5 = very poor). Synovitis and bone marrow contrast uptake on MR images were reviewed by two musculoskeletal radiologists using the Rheumatoid Arthritis MRI Scoring System (RAMRIS) of the Outcome Measures in Rheumatoid Arthritis Clinical Trials (OMERACT) group. RESULTS: IDEAL showed uniform FS unaffected by magnetic field inhomogeneity and challenging geometry of hand and fingers, while CHESS-T1-SE often showed FS failure within the first metacarpal joint, tip of the finger, and ulnar aspect of the wrist joint. Overall image quality was significantly better with IDEAL than CHESS-T1-SE images (4.43 vs. 3.43, P < 0.01). Interobserver agreement (κ value) for synovitis and bone marrow contrast uptake was good to excellent with IDEAL (0.74-0.91, 0.62-0.89, respectively). CONCLUSION: IDEAL could compensate for the effects of field inhomogeneities, providing uniform FS of the hand and finger than did the CHESS-T1-SE sequence.

Adenocarcinomas with predominant ground-glass opacity: correlation of morphology and molecular biomarkers.

PURPOSE: To retrospectively identify successive changes in peripheral lung adenocarcinoma that feature dominant ground-glass opacity (GGO) at computed tomography (CT) and correlate with biomolecular markers. MATERIALS AND METHODS: The institutional review board approved this retrospective study, and all 25 patients provided informed consent. Patients with lung adenocarcinomas smaller than 3 cm in diameter in whom tumor growth could be evaluated with CT before surgery were included. Two thoracic radiologists evaluated tumor growth by analyzing GGO type (pure or mixed) and size increases. Immunohistochemistry of the p53 protein and molecular analysis of the epidermal growth factor receptor (EGFR) and K-ras genes were performed. The Fisher exact test was used to assess statistical significance. RESULTS: Tumor size increased in 19 of 25 patients (76%) during the observation period. The CT changes in 19 patients were classified into four patterns: persistent pure GGO (n = 8), change from pure to mixed GGO (n = 3), mixed GGO with growth of solid component (n = 4), and mixed GGO with growth of GGO component (n = 4). The remaining six patients (24%) had pure GGO without any interval changes. Staining for p53 was negative in all 14 patients with pure GGO and positive in six of 11 patients (55%) with mixed GGO (P < .01). In these six patients appearance or growth of the solid component was seen. EGFR mutations were found in both pure (36%) and mixed (45%) GGO lesions (P = .70). CONCLUSION: Lung adenocarcinomas with a dominant GGO often possess EGFR mutations. Interval changes in the solid component may be related to p53 inactivation.

Thin-section chest CT findings in systemic lupus erythematosus with antiphospholipid syndrome: a comparison with systemic lupus erythematosus without antiphospholipid syndrome.

PURPOSE: To assess thin-section chest CT findings in systemic lupus erythematosus (SLE) with antiphospholipid syndrome (APS), in comparison with SLE without APS. MATERIALS AND METHODS: We retrospectively reviewed the medical records and thin-section CT findings of 17 consecutive patients with an established diagnosis of SLE with APS, comparing with 37 consecutive SLE patients without APS, between 2004 and 2008, and patients who had other autoimmune disease, such as Sjögren syndrome, were excluded. No significant differences were seen between the two groups in age, gender, smoking habits, or history of steroid pulse and biological therapy. CT images of 2mm thickness obtained with a 16- or 64-detector row CT were retrospectively evaluated by two radiologists in consensus on ultra high-resolution gray-scale monitors. RESULTS: The frequency of thin-section CT abnormalities was higher in SLE with APS group (82%) than in SLE without APS group (43%). Ground-glass opacity (59%), architectural distortion (47%), reticulation (41%), enlarged peripheral pulmonary artery (29%), and mosaic attenuation (29%) were significantly more common in the SLE with APS group than in the SLE without APS group (Fisher's exact test, p<0.01). CONCLUSION: SLE patients with APS have increased prevalence of thin-section chest CT abnormalities than those without APS.

Frequency and significance of pulmonary nodules on thin-section CT in patients with extrapulmonary malignant neoplasms.

PURPOSE: To determine the frequency and significance of pulmonary nodules detected on thin-section CT in patients with extrapulmonary malignant neoplasms. MATERIALS AND METHODS: The institutional review board approved this study. This study retrospectively evaluated 308 patients with extrapulmonary carcinomas or sarcomas and had undergone thin-section chest CT (2mm slice thickness) for staging. Three radiologists identified non-calcified nodules and evaluated the size, the growth and the distance from the nearest pleural surface. The characteristics of the nodules were defined based on the results of either a diagnostic biopsy or nodule growth. RESULTS: One or more non-calcified pulmonary nodules were detected in 75% of the patients (233/308). One hundred and thirty-seven of these patients had nodules that met the criteria of either benign or malignant nodules. Nodules smaller than 10mm were more likely to be benign, whereas those 10mm or greater were more likely to be malignant (22/26, 85%; P<.0001). Most nodules less than 10mm from the pleura were benign (91%), whereas approximately half of the nodules 10mm or more away from the pleura were malignant (20/43, 47%; P<.0001). Patients with melanoma, sarcoma, or testicular carcinoma were more likely to have malignant nodules. A multivariable analysis demonstrated the nodule size (P<.0001) and distance from the pleura were predictive of malignancy. CONCLUSION: The nodule detection rate on thin-section CT in patients with extrapulmonary malignancy is high. Most of the nodules smaller than 10mm or less than 10mm from the pleura are benign.

Osteosclerotic lesions in patients treated with gefitinib for lung adenocarcinomas: a sign of favorable therapeutic response.

OBJECTIVE: To assess the frequency of osteosclerotic changes on CT that appeared after treatment with gefitinib in patients with lung adenocarcinoma and the relationship between the osteosclerotic changes and the response to the therapy. MATERIALS AND METHODS: Our study included 41 patients with lung adenocarcinoma who underwent chest CT both before (CTpre) and after (CTpost) starting treatment with gefitinib. The presence or absence of bone metastases was assessed on the CTpre, and the interval bony change after the therapy was classified as lytic, sclerotic, or no changes on the CTpost. The relationship between treatment results of primary lung cancer and interval bony changes was evaluated. RESULTS: Osteosclerotic lesions were identified in 11 patients (27%) on CTpost; in 6 of 11 patients osteosclerotic lesions newly appeared where the CTpre showed no bone metastasis before the gefitinib therapy. There were significant differences in the therapeutic response of the primary cancers (P < 0.001) and in the survival rate (P < 0.01) in patients with osteosclerotic changes versus those without osteosclerotic changes. CONCLUSION: Osteosclerotic changes on CT, observed after gefitinib treatment in patients with lung adenocarcinomas, may be an indicator of a good therapeutic response.

Usefulness of computerized method for lung nodule detection in digital chest radiographs using temporal subtraction images.

RATIONALE AND OBJECTIVES: The aim of this study was to evaluate the usefulness of a novel computerized method for lung nodule detection on digital chest radiographs using temporal subtraction images. MATERIALS AND METHODS: To significantly reduce the number of false-positive results while maintaining high sensitivity, temporal subtraction images, which can enhance interval changes on sequential chest radiographs, were used. Fifty-one cases with lung nodules <3 cm and 51 cases without lung nodules were selected for an observer performance test. Twelve radiologists participated in this observer performance test. The radiologists' performance was evaluated using receiver-operating characteristic analysis, on a continuous rating scale. To estimate the numbers of cases affected beneficially and those affected detrimentally using this computerized method, the computer output was assumed to have an effect on an observer's diagnosis when there was a difference in rating score of ≥30% between the first and second ratings. RESULTS: The average area under the curve for all radiologists increased significantly from 0.849 to 0.950 with the computerized method (P < .001). The mean number of cases affected beneficially was significantly higher than that of cases affected detrimentally (8.92 vs 1.25, P < .001). CONCLUSIONS: The novel computerized method using temporal subtraction images would be useful in detecting lung nodules on digital chest radiographs.