Cells In Effusions Research Articles

Development of an optimal protocol for molecular profiling of tumor cells in pleural effusions at single-cell level.

Liquid biopsy analyzes the current status of primary tumors and their metastatic regions. We aimed to develop an optimized protocol for single‐cell sequencing of floating tumor cells (FTCs) in pleural effusion as a laboratory test. FTCs were enriched using a negative selection of white blood cells by a magnetic‐activated cell sorting system, and CD45‐negative and cytokeratin‐positive selection using a microfluidic cell separation system with a dielectrophoretic array. The enriched tumor cells were subjected to whole‐genome amplification (WGA) followed by genome sequencing. The FTC analysis detected an EGFR exon 19 deletion in Case 1 (12/19 cells, 63.2%), and EML4‐ALK fusion (17/20 cells, 85%) with an alectinib‐resistant mutation of ALK (p.G1202R) in Case 2. To eliminate WGA‐associated errors and increase the uniformity of the WGA product, the protocol was revised to sequence multiple single FTCs individually. An analytical pipeline, accurate single‐cell mutation detector (ASMD), was developed to identify somatic mutations of FTCs. The large numbers of WGA‐associated errors were cleaned up, and the somatic mutations detected in FTCs by ASMD were concordant with those found in tissue specimens. This protocol is applicable to circulating tumor cells analysis of peripheral blood and expands the possibility of utilizing molecular profiling of cancers.

ES07.02 Role of Pleural Fluid Molecular Markers and PD-L1 Testing

Patients with pleural metastases or lymphatic metastases in 50-60% develop pleural effusions (PE). Conventional cytology is negative (CNPE) in up to 40% of malignant PEs. The cells in PE are in a different microenvironment, affecting biology and biomarkers' expression considering the impact of tumor-host cell interactions. Obtaining PE, an early diagnostic step provides important biomarkers; when other tissue samples might not be available. PE represents tumor heterogeneity; tumor cells in pleural effusions might represent more lesions than biopsies. There are studies involving genomic, transcriptomic, proteomic, and metabolomics. Circulating cell-free DNA from PF supernatants may provide relevant information to clinicians that might complement or replace invasive diagnostic procedures. Cytology negative and positive PE is useful in early decision-making and when no additional time is available for later diagnostics steps due to disease aggressiveness. Biomarkers show high specificity, and the low sensitivity limits the diagnostic value that might increase with the PE amount available. Molecular testing on PE specimens is becoming a feasible alternative to tissue biopsy for phenotyping malignant PEs in lung cancer patients. PE might be suitable for assessing PD-L1 expression in malignant cells, and we compare the results of those reported for histological specimens.

The Pediatric Fluid Cytology: A 2-year experience of a tertiary care pediatric center of North India

Background: Body fluid analysis containing exfoliated cells in effusion may reveal information about neoplastic and non-neoplastic etiology. Most important indication of fluid cytology is to look for malignant cells. CSF examination is another investigation performed routinely in all tertiary care centers in addition to other body fluids examined.
 Aims and Objective: The aim of the study was to examine the body fluids for cytological examination in diagnosis, prognostic and therapeutic tool for management of pediatric cases.
 Materials and Methods: Retrospective data analysis of 170 fluid cytology cases taken from departmental data archive of pediatric cases (0-18 years) from Jan 2018-Dec 2019 was tabulated observation was done by 2independent pathologists. The smears were stained with Romanowsky stains and specialized stains of AFB and Rhodamine auramine, MPO, PAS and PAP as per requirement.
 Results: There were higher number of male (n=99, 58.23%) compared to female (n=71, 41.76%). The commonest fluid cytology received was the CSF (n=150, 88.23%) with 77 samples presenting with significantly high TLC for age (using standard age analysis parameters). The diagnosis was grouped into the Neoplastic and Non neoplastic category. The non-neoplastic category was further divided into infective where most common was viral pleocytosis (n=50, 48 Viral lymphocytic pluecytosis, 1case rubella positive CSF, 1 Primary HLH with viral infection) followed by TB(n=10, 6 pleural, 2 CSF, 2 Peritoneal), bacterial (n=07, microbiology culture was done) and TORCH, Rubella with plasmacytosis (n=01). Another non-neoplastic category was of inherited disease where single case of Griscelli’s syndrome was diagnosed showing hemophagocytosis on CSF examination. Neoplastic Cytology was seen in 07 cases where all the cases were hematolymphoid malignancies.
 Conclusion: This study concludes that fluid cytology is a useful diagnosis, prognostic, therapeutic tool in diagnosis and management of pediatric cases.

Accumulation of TNFR2-expressing regulatory T cells in malignant pleural effusion of lung cancer patients is associated with poor prognosis.

BackgroundRegulatory T cells (Tregs) may represent a major cellular mechanism in immune suppression by dampening the anti-tumor response in malignant pleural effusion (MPE). Tumor necrosis factor receptor type II (TNFR2) has emerged as a novel identification for the maximally suppressive subset of Tregs in the tumor environment. At present, the significance of TNFR2 expression on Tregs in MPE remains unclear.MethodsThe distribution of TNFR2+cells in Tregs and effector T cells (Teffs) in MPE, peripheral blood (PB), and tuberculosis pleural effusion (TPE) were determined. The associations between TNFR2+Tregs frequencies present in MPE and the clinical and laboratorial characteristics of patients with lung cancer were investigated. The immunosuppressive phenotype of TNFR2+Tregs in MPE was analyzed. The effects of the TNF-TNFR2 interaction on the immunosuppressive function of Tregs was explored. The efficacy of targeting TNFR2 for MPE therapy was examined. The source of TNF in MPE was identified.ResultsWe observed that markedly higher levels of TNFR2 were expressed in MPE Tregs compared with the levels expressed in MPE Teffs, PB Tregs, or in TPE Tregs. The frequencies of TNFR2+Tregs were positively correlated with the number of tumor cells in MPE, as well as the volume of MPE. High frequencies of TNFR2+Tregs in MPE indicated short survival time and poor performance status for MPE patients. Compared to TNFR2-Tregs, TNFR2+Tregs expressed higher levels of immunosuppressive molecules cytotoxic T lymphocyte-associated protein 4 (CTLA-4), programmed cell death-ligand 1 (PD-L1), and replicating marker Ki-67. Consequently, the proportions of interferon gamma (IFN-γ)-producing cytotoxic T lymphocytes (CTLs) were significantly increased after TNFR2 blockade. Furthermore, tumor necrosis factor (TNF), through interaction with TNFR2, enhanced the suppressive capacity of Tregs by up-regulating CTLA-4 and PD-L1 expression. Interestingly, T helper 1 (Th1) and T helper 17 (Th17) cells are the major source of TNF in MPE, suggesting that MPE Teffs may paradoxically promote tumor growth by boosting MPE Treg activity via the TNF-TNFR2 pathway.ConclusionsOur data expanded the immunosuppressive mechanism present in MPE induced by Tregs, and provides novel insight for the diagnosis, disease evaluation, and treatment of MPE patients.

Clinical and laboratory characteristics of lymphoid neoplasms in serous effusions: a single centre experience in China.

Lymphoid neoplasms in serous effusions are uncommon, and the details of lymphoid neoplasms in serous effusions from China are still unclear. Between January 2004 and December 2019, all patients with lymphoid neoplasms in pleural effusions, ascites, and pericardial effusions in our hospital, were reviewed. A total of 65 patients with lymphoid neoplasms were collected during this period. The top three neoplasms were diffuse large B-cell lymphoma (DLBCL) (n = 20, 30.7%), myeloma (n = 13, 20.0%), and T-lymphoblastic lymphoma (n = 7, 10.8%). In pleural effusions involving DLBCL, the cytomorphology of DLBCL cells was diverse; most pleural effusions were present during the tumor course (92.9%); bilateral pleural effusions were predominant (57.2%); and the median survival time was only 1.23 months after the effusion. In pleural effusions involving myeloma, 90.9% of cases (10/11) had a high ratio (> 1.0) of immature to mature plasma cells; paraprotein types of IgA (36.4%) and light chain λ (36.4%) were the most frequently found; bilateral pleural effusions were easily found (n = 10, 90.9%); and the median survival time was only 1.4 months after the effusion. In pleural effusions involving DLBCL, most of our patients with effusions are present during the tumor course, and bilateral pleural effusions are predominant. In pleural effusions involving myeloma, the paraprotein types of IgA and light chain λ are the most frequently found, and it has a high ratio of immature to mature plasma cells in pleural effusions.

Cultivation of cancer cells in malignant pleural effusion and their use in a vascular endothelial growth factor study

Cultivation of cancer cells in malignant pleural effusion and their use in a vascular endothelial growth factor study

Improving performance of recently introduced flow cytometry-based approach of malignant cell screening in serous cavity effusion.

Microscopy has been recognized as the "gold-standard" cellular analysis of serous cavity effusion. However, this method is time-consuming, labor-intensive, and requires accomplished skills. Here, we investigated the efficiency of hematology analyzer in screening malignant cells in serous cavity effusion. A total of 991 serous cavity effusion samples and 370 validation specimens collected from different departments were sent to the clinical laboratory for routine cell count using the automated hematology body fluid (BF) mode and exfoliative cytology simultaneously. High-fluorescent cells (HFCs) were measured as the relative count (HF%) and absolute count (HF#) by BF mode. Receiver operating characteristic curve analysis was combined with scattergram rules to screen malignant cells. HF# and HF% in malignant samples (subgroup) were significantly higher than those in benign samples, and the HF# and HF% levels were different between ascites and pleural effusion (PE). The area under the curve values were also different between ascites and PE. Positive of malignant cells was very high when the ascites or PE sample touching Rule 1 positive and either Rule 2 negative or positive. The cutoff levels of HF# were 5.5 HFC/μL on the basis of Rules 1 and 2 negative, whereas 83.5 HFC/μL on the basis of Rule 1 negative but Rule 2 positive in ascites. By contrast, the cutoff levels of HF% were 0.55 HFC/100 WBC on the basis of Rules 1 and 2 negative, whereas 4.95 HFC/100 WBC on the basis of Rule 1 negative but Rule 2 positive in PE. Serous cavity effusion will be increasingly analyzed using the automated hematology analyzer BF mode in the future because of its rapidness and convenience. The combined application of HFC with scattergram rules is a feasible and useful approach to screen malignant cells in serous cavity effusion.

Diagnosis and categorization of malignant effusions: A 6-year review from a single academic institution.

Cytologic detection of malignant cells in pleural, peritoneal, or pericardial effusion most likely indicates advanced stage of malignant disease. There are a few studies updating the categorization of malignant effusions. The electronic pathology database was searched to identify consecutive cases of malignant effusion during a 6-year period. Patient age and gender, origins of known malignancy, and cytologic diagnoses were recorded and summarized. A total of 1059 specimens included 561 (53%) pleural, 441 (41.6%) peritoneal, and 57 (5.4%) pericardial fluids. Most of the pleural (516, 92.0%), peritoneal (418, 94.8%), and pericardial (53, 93.0%) specimens were derived from patients with a single known malignancy. More common origins involving pleural fluid were lung (152, 27.1%) followed by breast (103, 18.4%) and gastrointestinal tract (76, 13.5%). The most common etiology for women and men was breast (102, 30.8%) and lung (67, 36.2%), respectively. More common origins involving peritoneal fluid were gastrointestinal (158, 35.8%) and gynecologic (156, 35.4%) tracts, and breast (46, 10.4%). The most common etiology for women and men was Mullerian (156, 55.5%) and gastrointestinal tract (94, 68.6%), respectively. Most common origins involving the pericardial fluid were breast (20, 37.7%) and lung (17, 29.8%). Breast and lung were the most common etiology for women (20, 57.1%) and men (8, 44.4%), respectively. Breast and lung remain to be the most common origin of both malignant pleural and pericardial effusion for women and men, respectively. The most common origin involving peritoneal effusion is Mullerian for women and gastrointestinal tract for men.

Human inflammatory dendritic cells in malignant pleural effusions induce Th1 cell differentiation

Dendritic cells are crucial for the initiation and regulation of immune responses against cancer and pathogens. DCs are heterogeneous and highly specialized antigen-presenting cells. Human DCs comprise several subsets with different phenotypes and functional properties. In the steady state, human DC subsets have been well studied. However, the components of DC subsets and their immune functions during the inflamed setting are poorly understood. We identified and characterized DC subsets in the malignant pleural effusions of NSCLC patients. We analyzed the capacity of these DC subsets to induce T-cell differentiation. We observed the presence of inflammatory DCs (infDCs) and macrophages in the malignant pleural effusions of NSCLC patients, as identified by the CD11C+HLA-DR+CD16−BDCA1+ and CD11C+HLA-DR+CD16+BDCA1− phenotypes, respectively. InfDCs represented approximately 1% of the total light-density cells in the pleural effusion and were characterized by the expression of CD206, CD14, CD11b, and CD1α, which were absent on blood DCs. InfDCs also expressed CD80, although at a low level. As infDCs did not express CD40, CD83 and CD275, they remained functionally immature. We found that TLR agonists promoted the maturation of infDCs. Compared with macrophages, infDCs had a weaker capacity to phagocytose necrotic tumor cell lysates. However, only infDCs induced autologous memory CD4+ T-cell differentiation into Th1 cells. For the first time, we found that infDCs were present in the malignant pleural effusions of NSCLC patients. We conclude that infDCs represent a distinct human DC subset and induce Th1 cell differentiation in the presence of TLR agonists.

Rapid purification of lung cancer cells in pleural effusion through spiral microfluidic channels for diagnosis improvement

Lung cancer is one of the leading causes of death worldwide. Fifteen percent of lung cancer patients will present with malignant pleural effusion initially, and up to 50% will have malignant pleural effusion throughout the course of the disease. In this study, we developed a spiral microfluidic device that can rapidly isolate cancer cells and improve their purity through fluid dynamics. This label-free, high-throughput device continuously isolates cancer cells and other unrelated molecules from pleural effusion. Most of the background cells that affect interpretation are flushed to outlets 1 to 3, and cancer cells are hydrodynamically concentrated to outlet 4, with 90% of lung cancer cells flowing to this outlet. After processing, the purity of cancer cells identified in pleural effusion by CD45 and epithelial cell adhesion molecule (EpCAM) antibodies in flow cytometry will be increased by 6 to 24 times. The microfluidic device presented here has the advantages of rapid processing and low cost, which are conducive to rapid and accurate clinical diagnosis.

The Correlation between EGFR Mutation Status and DNA Content of Lung Adenocarcinoma Cells in Pleural Effusion.

Objectives: Lung adenocarcinomas with or without epidermal growth factor receptor (EGFR) mutations have shown different drug effects against EGFR inhibitors. But it is not very clear if EGFR mutation status affects the biological behavior of lung adenocarcinoma, because tumor gene regulation is very complicated and can be affected by many factors. We aimed to explore if EGFR mutation status is related with tumor malignant degree by investigating the relevance of EGFR mutation status with DNA content and aneuploid peaks of lung adenocarcinoma cells in pleural fluids without using EGFR-TKIs.Materials and Methods: 591 cases of lung adenocarcinoma patients in Hebei Tumor Hospital who had undergone EGFR gene detection and DNA quantitative analysis were collected from January 2012 to August 2018.They were divided into two groups: EGFR mutant group and non-mutant group. EGFR mutations were detected by Amplification Refractory Mutation System (ARMS) and ABI 7500 Fluorescence quantitative PCR with pleural effusions. DNA content and aneuploid peaks were detected by LD DNA image cytometry (DNA-ICM). Rank-sum test of SPSS 16 was used for statistical analysis.Results: The maximum DI, the mean DI of the first 20 cells greater than 5C, the percentage of cells greater than 5C and the number of cells greater than 9C of the first 20 cells in the mutant group were all higher than those in the non-mutant group, having statistical significance (p<0.001); the peaks of aneuploid cells in the mutant group occurred more often than those in the non-mutant group, having statistical significance (p<0.001).Conclusions: Our study has shown that advanced lung adenocarcinomas with EGFR-mutations had higher DI values, more aneuploid cells and more frequent aneuploid peaks compared with those without EGFR-mutations, suggesting that advanced lung adenocarcinomas with EGFR mutations are more aggressive than those without EGFR mutations.

Immunohistochemical Differentiation between Reactive and Malignant Mesothelial Proliferations in Pleural Effusion

AbstractBackground: The differentiation between benign and malignant mesothelial cells in pleural effusion in some cases can be a challenge. In the current study, we investigated the value of immunohistochemistry (IHC) in making that differ-entiation.Aim of Study: This study aims to examine the value of IHC expression of desmin, EMA, GLUT-1, p53, Ki67, and BAP1 in discrimination between benign and malignant mes-othelial proliferation in pleural effusions and compare their results regarding sensitivity and specificity.Material and Methods: Pleural fluids from 30 cases diagnosed as malignant meothelioma (MM) epitheloid type and 20 cases diagnosed as reactive mesothelial hyperplasia (RMH) were selected, and stained with immunohistochemical stains included BAP-1, desmin, epithelial membrane antigen (EMA), glucose-transport protein 1 (GLUTA]1), Ki67, and p53.Results: BAP-1 was negative in 5% (1 of 20) cases of reactive MH and in 63.3% (19 of 30) of MM cases (p 40% of mesothelial cells in 10% (2 of 20) of benign and 16.7% (5 of 30) of malignant cases (p=0.40). EMA negativity and desmin positivity were found in 75% (15 of 20) of reactive MH cases and 3.3% (one of 30) of MM cases. EMA positivity and desmin negativity were found in 5% (1 of 20) of reactive MH cases and 90% (27 of 30) of MM cases (p<.001). Positivity for both of Desmin and BAP-1 was detected in 80% (16 of 20) of RMH cases. The combination of Desmin and BAP-1 negativity was detected in 60% (16 of 30) of MM cases (p-value p<.001).Conclusions: Cases showed positivity for EMA and neg-ativity for desmin strongly favors malignant mesothelioma. On the contrary, cases showed negative EMA and positive desmin strongly favors Reactive mesothelial hyperplasia. also the combination of Desmin and Bap-1 postivity favors diagnosis of RMH, and the combination of Desmin and BAP-1 negativity favors the diagnosis of MM. Similarly, cases showed strong membranous expression of GLUT-1 and/or strong nuclear expression of p53 strongly favors diagnosis of malignant mesothelioma. Proliferative index showed detected by Ki67 showed no significant difference between reactive and malignant cases.

Tumor infiltrating T lymphocyte components in malignant pleural effusion of lung adenocarcinoma and their killing activities to autologous tumor cells

To analyze the components of tumor infiltrating T lymphocyte (TIL) cells in malignant pleural effusion of lung adenocarcinoma, and evaluate their killing activities to autologous tumor cells. Methods: Malignant pleural effusions were collected from 17 patients with lung adenocarcinoma. Mononuclear cells were isolated by Ficoll density gradient centrifugation and flow cytometer was used to analyze TIL cell components. TIL and tumor cells were separated through adherent culture. The tumor cells were identified via intramuscular injection of adherent cells into nude mice and the killing effect of cultured lymphocytes on autologous tumor cells was studied. Results: Of the TIL in malignant pleural effusions, T cells accounted for 60.6%-79.3%, while T helper cells were significantly higher than T killer cells (36.63%±1.90% vs 24.64%±2.32%, P<0.001). There were also natural killer (NK) cells and NK T cells in the effusions. Tumor cells were successfully isolated and cultured. The killing activity of cultured TIL to autologous tumor cells was 39.14%±12.04%, and the killing activity of TIL with high proliferation rate to autologous tumor cells was higher than that of low proliferation group (50.51%±3.80% vs 29.04%±5.77%, P<0.001). Conclusion: T lymphocytes are the major components of TIL in malignant pleural effusions derived from lung adenocarcinoma, and T helper cells are more than T killer cells. The killing activity of TIL with strong proliferation ability to autologous tumor cells is higher than that of TIL with weak proliferation ability. Therefore, cells from malignant pleural effusions could be used for cellular immunotherapy against tumor.

TNF Receptor Type II Is Critical to the Expansion and Suppressive Function of Regulatory T Cells in Malignant Pleural Effusion from Lung Cancer Patients

Background: Tumor-infiltrating Tregs have been found to express high levels of tumor necrosis factor receptor type II (TNFR2), which renders their suppressive function more pronounced. However, the importance of TNFR2 expression on human Tregs from malignant pleural effusion (MPE) and the efficacy of targeting TNFR2 for the treatment of MPE remain unclear. Methods: The characteristics of TNFR2 expression by Tregs in MPE and peripheral blood were analyzed. The effects of TNF/TNFR2 pathway on Treg proliferation and suppressive function were explored. The relationships between effector T cells and Tregs via the TNF/TNFR2 pathway were evaluated. The effects on the percentage of CTL cells and the concentration of IFN-γ were measured after TNFR2 blockade. Findings: The percentages of Tregs in MPE were much higher than in peripheral blood. TNFR2 appears to be a specific proliferative and functional marker for Tregs in MPE. TNF promotes TNFR2+Treg proliferation and the expression of CTLA-4 and PD-L1. There is also a strong positive correlation between the level of TNF in Th17 cells and Foxp3 in CD4+CD25hi T cells, and IL-17 up-regulates the expression of TNFR2 on Tregs in vitro, suggesting Th17 cells may promote Tregs in the proliferation and function via the TNF/TNFR2 pathway. Moreover, the proportions of CTL cells and IFN-γ concentration were significantly increased after blocking TNFR2. Interpretation: Our results provide laboratorial evidence that TNFR2 might be a novel and effective target in the further clinical treatment for lung cancer patients with MPE. Funding Statement: This work was supported by grants from National Natural Science Foundation of China (NNSFC; No. 81973990, No. 81770090 and No. 81470274) and The Graduates' Innovation Fund, Huazhong University of Science and Technology (TGI, HUST; No. 2019YGSCXCY 067). Declaration of Interests: The authors declare no potential conflicts of interests. Ethics Approval Statement: The study protocol was approved by our institutional review board for human studies, and informed consent was obtained from all subjects.

1226P - A phase I clinical trial of malignant pleural mesothelioma treated with locally delivered autologous anti-FAP-targeted CAR T-cells

BackgroundMalignant pleural mesothelioma (MPM) is a cancer with a worldwide increasing incidence due to the use of asbestos. MPM is incurable despite the use of multimodality treatment. Tumor spread confined to the thoracic cavity is a hallmark of MPM, providing a rational for local treatment. We developed a chimeric antigen receptor (CAR) targeting FAP (fibroblast activating protein), a cell-surface antigen that we have shown to be highly expressed in epithelial cancers. MethodsUsing a Δ-CD28-costimulated CAR, we initiated a phase I clinical trial (NCT01722149) to determine the safety and persistence of intra-pleural administered anti-FAP-CAR T cells in the periphery. A single dose of 1x 106 re-directed T cells was administered through a pleural catheter. Patients were monitored on an intensive care unit for 48h. Clinical evaluations of on-target and off-tumor toxicity were assessed for 3 months. Laboratory analyses included cytokines, CRP and the detection of CAR-T cells in the pleural effusion and blood over time. Radiological evaluation with PET-CT scans was performed as standard of care. ResultsThree patients with metastatic MPM were treated (all patients received at least two cycles of chemotherapy previous to CAR T-cell administration). No CAR T-cell-related toxicities were observed. Intense monitoring for cytokine release syndrome showed significant changes on a subset of cytokines. CAR T-cells were detected in the peripheral blood after treatment. Activity of the patient’s redirected T cells was confirmed in vitro. Furthermore, one patient received an anti-PD1 checkpoint blockade antibody 8 months after CAR T-cell instillation with no toxicity. With a median follow-up of 18 months, 2 out of 3 patients are alive. ConclusionsIn this phase I clinical trial, intra-pleural administration of anti-FAP CAR T-cells was well tolerated without any evidence of treatment related toxicity. Persistence of CAR T-cells was documented in the periphery. Clinical trial identificationNCT01722149. Legal entity responsible for the studyUnviersity Hospital Zurich. FundingSwiss Cancer League. DisclosureA. Curioni: Advisory / Consultancy, Speaker Bureau / Expert testimony: AstraZeneca; Advisory / Consultancy: Boehringer Ingelheim; Advisory / Consultancy, Speaker Bureau / Expert testimony: Bristol-Myers Squibb; Advisory / Consultancy, Speaker Bureau / Expert testimony, Travel / Accommodation / Expenses: F. Hoffmann-La Roche; Advisory / Consultancy, Speaker Bureau / Expert testimony, Travel / Accommodation / Expenses: Merck Sharp and Dohme; Advisory / Consultancy: Novartis; Advisory / Consultancy, Speaker Bureau / Expert testimony: Pfizer ; Advisory / Consultancy, Speaker Bureau / Expert testimony, Travel / Accommodation / Expenses: Takeda . C. Britschgi: Honoraria (self), Advisory / Consultancy: AstraZeneca; Honoraria (self), Advisory / Consultancy: Pfizer; Honoraria (self), Advisory / Consultancy: Roche; Honoraria (self), Advisory / Consultancy: Takeda. W. Weder: Advisory / Consultancy, Speaker Bureau / Expert testimony: AstraZeneca; Advisory / Consultancy, Speaker Bureau / Expert testimony: Medtronic. R.A. Stahel: Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): AstraZeneca; Honoraria (self), Advisory / Consultancy: AbbVie; Speaker Bureau / Expert testimony, Research grant / Funding (institution): Bristol-Myers Squibb; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution), Officer / Board of Directors: F. Hoffmann-La Roche; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Merck Sharp and Dohme; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Boehringer Ingelheim; Research grant / Funding (institution): Pfizer; Honoraria (self), Advisory / Consultancy, Officer / Board of Directors: Takeda; Research grant / Funding (institution): Genentech. All other authors have declared no conflicts of interest.

LI-cadherin and CDX2: Useful Markers in Metastatic Gastrointestinal Adenocarcinoma Cells in Serous Cavity Effusion.

The aim of this study was to investigate the expression and significance of liver-intestine cadherin (LI-cadherin) and CDX2 (caudal-type homeobox transcription factor 2) in cytology specimens of metastatic gastrointestinal adenocarcinoma in serous cavity effusion. The serous cavity effusion samples that were suspected of tumor metastasis in the last 5 years were made into paraffin-imbedded cell blocks, a total of 180 cases, including 97 cases of pleural effusion, 78 cases of ascites, and 5 cases of pericardial effusion. The expressions of LI-cadherin and CDX2 were detected by immunohistochemical staining in cell blocks and primary tumor tissues; thereafter, the specificity and sensitivity were analyzed. The positive expressions of LI-cadherin and CDX2 in 63 cases of gastrointestinal adenocarcinoma cells were 65.1% (41/63), 61.9% (39/63), respectively. Of 11 cases of pancreaticobiliary duct cancers, only one case (9.09%) was weakly positive for LI-cadherin and CDX2 expressions, and 107 cases of non-digestive tract cancers were all negative. In 57 matched-pair samples of cell blocks and primary gastrointestinal adenocarcinoma tissues, the positive expressions of LI-cadherin and CDX2 were 64.9% (37/57), 61.4% (35/57), respectively, in cells, and 82.5% (47/57), and 77.2% (44/57), respectively, in tissues. Positive correlation was observed between the expressions of LI-cadherin and CDX2 in metastatic gastrointestinal adenocarcinoma cells (P<0.05). Both LI-cadherin and CDX2 expressions demonstrated positive correlation in cells with paired cancer tissues (P<0.05). The positive expression reached 80.7% with at least one positive marker in the total cell samples through combined detection of LI-cadherin and CDX2, increased by 15.8% and 19.3% compared with using either of the markers alone. Combined use of LI-cadherin and CDX2 can improve the accuracy in the diagnosis of metastatic adenocarcinoma cells in serous cavity effusion and provide some bases of histologic origin because of their high sensitivity and specificity.

Liquid biopsy-based single-cell metabolic phenotyping of lung cancer patients for informative diagnostics

Accurate prediction of chemo- or targeted therapy responses for patients with similar driver oncogenes through a simple and least-invasive assay represents an unmet need in the clinical diagnosis of non-small cell lung cancer. Using a single-cell on-chip metabolic cytometry and fluorescent metabolic probes, we show metabolic phenotyping on the rare disseminated tumor cells in pleural effusions across a panel of 32 lung adenocarcinoma patients. Our results reveal extensive metabolic heterogeneity of tumor cells that differentially engage in glycolysis and mitochondrial oxidation. The cell number ratio of the two metabolic phenotypes is found to be predictive for patient therapy response, physiological performance, and survival. Transcriptome analysis reveals that the glycolytic phenotype is associated with mesenchymal-like cell state with elevated expression of the resistant-leading receptor tyrosine kinase AXL and immune checkpoint ligands. Drug targeting AXL induces a significant cell killing in the glycolytic cells without affecting the cells with active mitochondrial oxidation.

γδT Cells in Lung Cancer Malignant Pleural Effusion: Friend? Foe?

γδT Cells in Lung Cancer Malignant Pleural Effusion: Friend? Foe?

Pathologic Considerations and Standardization in Mesothelioma Clinical Trials

The accurate diagnosis of mesothelioma is critical for the appropriate clinical management of this cancer. Many issues complicate making the diagnosis of mesothelioma including the presence of reactive mesothelial cells in benign pleural effusions, the heterogeneity of mesothelioma histopathology, the relatively high incidence of other epithelial malignancies that metastasize to the pleura, and primary sarcomas that arise within the pleura. Given the rapidly evolving field of molecular profiling and the need for translational correlates in mesothelioma clinical trials, the National Cancer Institute (NCI)–International Association for the Study of Lung Cancer–Mesothelioma Applied Research Foundation Clinical Trials Planning Meeting was convened in March 2017 to develop a consensus on standard pathology guidelines for future NCI-sponsored clinical trials in mesothelioma. This consensus statement covers recommendations for specimen handling, pathologic classification and diagnosis, biobanking, and tissue correlative studies.

Implementation of functional precision medicine for anaplastic lymphoma kinase-rearranged non-small lung cancer

Anaplastic lymphoma kinase (ALK)-rearranged kinase drives non-small cell lung cancer (NSCLC) in 3–7% of patients and treatment consists of a broad range of approved inhibitors which are administered sequentially in case of resistance developing invariably after approximately 1 year of therapy. Besides the former standard tyrosine kinase inhibitor (TKI) crizotinib, the second-line ALK inhibitors alectinib, ceritinib, brigatinib, as well as the third-line lorlatinib are approved for the treatment of ALK-positive NSCLC patients. The main challenge is to find individual schemes of ALK inhibitors therapy which provide the best benefit for the patients. A host of ALK fusion partners, rearrangement variants and ALK mutations prevent a direct correlation of ALK alterations and sensitivity to specific TKIs within the framework of genomic precision medicine. However, recurrent ALK-positive NSCLC is an aggressive disease resulting in accumulation of tumor cells in pleural effusions which may be collected for in vitro sensitivity testing in a manner designated as functional precision medicine. Provided that these cells are present in sufficient numbers, they can be exposed to all possible drug candidates and their chemosensitivity tested in short-term proliferation assays. In addition to ALK-directed TKIs, cytotoxic drugs and inhibitors of non-target bypassing pathways may be included in the tests. In contrast to other trials in genome-guided precision medicine, a range of suitable therapeutics is available for modified ALK proteins. In summary, ALK-positive NSCLC with pleural effusions offer a unique model to develop, test and validate concepts of functional precision medicine.