{"paper_id":"60933d7c-81f6-421a-9041-f800da538542","body_text":"I nt erna ti o nal S oci ety  o f Pl eu ral  D is ea s es  \n© Copyright International Society of Pleural Diseases, 2018                      1   \n                All rights reserved \n \n                                     \n                              \n                          Review     \n   \n                    PD-L1 and Immunotherapy in Patients with   \nNon-Small Cell Lung Cancer and Malignant Pleural Effusion \n  \n \n \n \nCole Liberator, MD1       \nJonas Heymann, MD2     \nAnjali Saqi, MD, MBA2       \nCatherine Shu, MD1   \nJohn Crapanzano, MD2   \nWilliam Bulman, MD1 \n    \n     \n                     \n \nDepartments of 1Medicine and 2Pathology and Cell Biology, New York-Presbyterian Hospital-Columbia University \nIrving Medical Center, New York, NY \nCorresponding author:  Cole Liberator, MD, Division of Pulmonary, Allergy and Critical Care, Columbia Unive r-\nsity Irving Medical Center, 622 West 168th Street, PH-840, New York, NY 10032 \nCdl2142@cumc.columbia.edu \nFunding support:  This research received no specific grant from any funding agency in the public, commercial or \nnot-for-profit sectors. \nConflict of interest disclosures:  Catherine Shu has done an advisory board for Genentech.  Anjali Saqi has a pa-\ntent on a cell block device and has consulted for Boston Scientific. William Bulman is a consultant for Medtro nic. \nAbstract:  Immunotherapy has emerged as a potent tool in the treatment of lung cancer, particularly in \npatients with advanced disease.  Multiple drugs are now available which cause an anti-tumor immune re-\nsponse by blocking the interaction between programmed cell death protein 1 (PD -1) and its ligand, PD -\nL1, which is expressed in some tumors. This review expl ores the role of immunotherapy and the pract i-\ncal implications of testing for PD -L1 in patients with malignant pleural effusion. \nKey Words:  PD-L1, immunotherapy,  malignant pleural effusion, lung cancer \nPD-1 and PD-L1 Complex \nX-ray Diffraction \n \nCourtesy RCSB Protein Data Bank \nLin, D.Y. et al. \nPNAS USA (2008) 105 3011-3016 \ndoi.org/10.1073/pnas.0712278105 \n \n\nInt erna ti o nal  S oci ety  o f  Pl eu ral  D is eas es  \n \nPD-L1 & Immunotherapy in the Pleural Space                     2  \n \nIntroduction \n    Dr. William Coley, fresh out of surgical resi-\ndency in 1890, established a practice at the new-\nly built New York Cancer Hospital on West \n106th Street and Eighth Avenue in Manhattan. \n    One of his first patients, seventeen year old \nElizabeth Dashiell consulted him concerning a \nsarcoma on the dorsum of her \nhand. Her death following a \nfutile arm amputation deeply \naffected the young surgeon.  He \nresearched hospital records and \nfound the case of a German \nman who, after multiple proce-\ndures to remove a sarcoma from \nhis cheek, developed wound \nerysipelas (Streptococcus py-\nogenes).  The sarcoma gradual-\nly decreased in size, finally dis-\nappearing altogether, never to \nrecur. \nDr. Coley theorized that a \nprovoked immune system could \nengage in an anti-tumor im-\nmune response.  He experimented with live and \nkilled bacteria and bacterial toxins in patients \nwith a variety of cancers, and published his work \nas a small case series.1  Interest in “Coley’s Tox-\nins” as a possible treatment modality was mod-\nest and short-lived, supplanted by the exciting \ndevelopment of radiation therapy. \nOver a century later, we are witnessing a \nparadigm shift in cancer treatment that proves \nDr. Coley to be prescient, at the very least.  Can-\ncer immunotherapy has emerged as an exciting \nnew tool for fighting multiple types of cancer, \nparticularly in patients with advanced disease.  \nIn this review, we examine the possible benefits \nand the limits of our knowledge in the realm of \nimmunotherapy for patients with non-small cell \nlung cancer (NSCLC) involving the pleural \nspace. \nMalignant pleural effusion (MPE) is a com-\nplication seen with nearly all types of cancer.  \nLung cancer is the most common, with MPE \nfound in nearly one third of patients.  Malignant \neffusion is also commonly seen in breast cancer, \nHodgkin’s and Non-Hodgkin’s \nlymphoma, and melanoma.2   \nMalignancy in an effusion re-\nsults from hematologic spread, \nlymphatic spread, or direct in-\nvasion from peri-pleural dis-\nease.3-5  For solid tumors other \nthan mesothelioma, malignant \ncells in the pleural space repre-\nsent Stage 4 disease and poor \nsurvival.6 For patients with \nNSCLC, the most common \ncause of cancer-related death \nworldwide,7,8 the one-year \nsurvival rate for patients with \nStage 4 disease on the basis of \na malignant pleural effusion is \n39%.9 \nIn these patients, thoracentesis for pleural \nfluid cytological analysis is a safe, high yield \nbedside procedure to establish diagnosis, histo-\nlogic subtype and disease stage in a single step.  \nIt has been shown to reliably identify targetable \nmutations and gene rearrangements.10-13  Simi-\nlarly high yields are seen with thoracentesis in \nmalignant pleural effusions from other solid \ncancers, including breast carcinoma (70%).14  \nThe yield for liquid tumors including lymphoma, \nleukemia and multiple myeloma ranges from 31-\n55%, with Hodgkin’s lymphoma generally hav-\ning the lowest yield.15,16  \nWilliam Coley MD,  Surgical Resident \n     New York Hospital.  Circa 1890 \n \n\nInt erna ti o nal  S oci ety  o f  Pl eu ral  D is eas es  \n \nPD-L1 & Immunotherapy in the Pleural Space                                 3 \n \nSystemic Treatment Options for Patients \nwith Stage IV NSCLC and MPE \nA decade ago, treatment options for NSCLC \nStage 4 on the basis of pleural involvement con-\nsisted of either palliative systemic chemotherapy \nor pleural interventions including thoracentesis, \ntunneled indwelling catheters, and pleurodesis. \nToday, several new treatment modalities have \nshown efficacy in selected patients. One is muta-\ntion-targeted therapy. Multiple genetic altera-\ntions that drive tumor formation and growth \nhave been identified in a variety of tumors. Cor-\nresponding targeted therapies have been devel-\noped to prolong progression-free survival in pa-\ntients with the corresponding genetic alterations.    \nAdenocarcinoma (ADC), the most common \nNSCLC subtype, contains activated epidermal \ngrowth factor receptor (EGFR) mutations and \nre-arrangement of the anaplastic lymphoma ki-\nnase (ALK). Drugs that target these and other \nmutations have been approved by the Food and \nDrug Administration (FDA) as first-line therapy \nfor patients with advanced disease and are in \nwide clinical use. The treatment of metastatic \ncancer with these drugs therefore often hinges \nupon the detection of these genetic alterations. \nDespite the challenge frequently posed by the \navailability of only limited tissue, clinical tumor \ngenotyping has been extensively validated on \ncytologic and small histologic specimens, in-\ncluding pleural fluid cytology.17-23 Accordingly, \njoint guidelines from the College of American \nPathologists, International Association for the \nStudy of Lung Cancer, and Association for Mo-\nlecular Pathology,24  later endorsed by the Amer-\nican Society of Clinical Oncology(ASCO),25  \nsupport EGFR, ALK, ROS1 and BRAF (by AS-\nCO) testing on cytologic samples, including cell \nblocks prepared from pleural and pericardial \neffusion samples. \nThe newest option available for patients \nwith locally advanced or metastatic disease, in-\ncluding those without a targetable genetic altera-\ntion in the tumor is immunotherapy. This latest \napproach shows that Dr. Coley was correct in \nhis assertion that the immune sy stem plays a \nrole in cancer, but it is a complex one. Tumor \ncells express antigens that are targets for the \nimmune system’s cytotoxic anti-tumor response, \na reaction conducted primarily by tumor specific \ncytotoxic T cells. An anti-tumor immune re-\nsponse can suppress tumor growth by elimina t-\ning cancer cells, but cancer cells, by evading \nimmune surveillance, become free to prolife r-\nate.26   \nTreatment Options in NSCLC Stage 4 \nMechanical Intervention \nChemotherapy \nTargeted therapy against tumor mutations \nImmunotherapy to activate host defenses \n \n At the beginning of this decade, it was \nshown that evasion of the immune system occurs \nby expression of ligands * on tumor cells that \nbind to and activate T cell inhibitory receptors.27  \nMultiple mechanisms for this evasion have \nbeen elucidated, including “immune chec k-\npoints”: biological pathways in the inte raction \nbetween tumor cells and inflammatory cells in \nthe tumor microenviro nment. The most studied \nof these checkpoints is the pathway i nvolving \nprogrammed cell death protein 1 (PD -1) and its \nligands PD-L1 and PD-L2.   \n PD-1 is a receptor protein expressed \nprimarily by T lymphocytes, including tumor-\ninfiltrating CD4 + T cells, B cells, natural killer \nT cells, as well as by monocytes and den dritic \ncells. Binding of the PD -1 by tumor ligands \ninhibits the production (CHECK) o f kinases \ninvolved in T cell activation. 28  Thus, a tumor \ncell expressing PD -L1 can down-regulate the \nactivity of tumor atta cking T cells. 26  Converse-\n\nInt erna ti o nal  S oci ety  o f  Pl eu ral  D is eas es  \n \nPD-L1 & Immunotherapy in the Pleural Space                                 4 \n \nly, prevention of this binding may enable infi l-\ntrating T cells to mount an immune attack on a \ncancer.  \n PD-1 has two ligands, PD-L1 and PD-L2, \nwhich are expressed by various tumors, includ-\ning NSCLC, melanoma, breast carcinoma, renal \ncell carcinoma, ovarian carcinoma and gastroin-\ntestinal carcinoma.29-31  Upregulation PD-L1 on \ntumor cells has been shown to inhibit cytokine \nand T cell activation.29,32 \n In a landmark paper published in 2012, \nBrahmer and colleagues showed that a human \nmonoclonal antibody specific for tumor PD-L1 \ngiven every 2 weeks for \n12 weeks resulted in du-\nrable clinical responses in \npatients with a variety of \ncancers including 75 pa-\ntients with advanced \nNSCLC who had failed or \nprogressed on platinum-\nbased therapy and/or tyro-\nsine kinase inhibitors \n(TKIs). Benefit was seen \nin both adenocarcinoma \n(ADC) and squamous cell \ncarcinoma (SCC). The drug was well \ntolerated. Complete or partial response \nwas seen in up to 17% of patients. Prolonged \nstabilization of disease (at 24 weeks) was seen in \nup to 41%.33   \nSubsequent studies have focused specifically \non patients with NSCLC. Several PD-1-\ninhibitory monoclonal antibodies are now avail-\nable to patients with metastatic NSCLC.34  Pem-\nbrolizumab was initially approved as a second-\nline treatment for both metastatic NSCLC with \nexpression of PD-L1 in at least 50% of tumor \ncells, and for metastatic NSCLC that had pro-\ngressed after platinum-based chemotherapy if \nPD-L1 expression was present in at least 1% of \ntumor cells.35-38  Pembrolizumab later gained \nFDA accelerated approval as a first-line therapy \nin combination with chemotherapy for previous-\nly untreated patients with metastatic NSCLC.39  \nThis approval is contingent on future survival \ndata.  Nivolumab was approved in 2015 for \ntreatment of metastatic NSCLC in patients who \nhad failed chemotherapy, and in patients with \nEGFR or ALK mutations who had failed target-\ned therapy.   \nTaken together, the efficacy of immunother-\napy targeting the PD -1/PD-L1 axis in patients \nwith metastatic lung  cancer has been promi s-\ning, with the potential to increase overall su r-\nvival by several \nmonths compared to \nstandard chemother a-\npy, including patients \nwith SCC and ADC.  \nWhile important que s-\ntions remain, inclu d-\ning which patients are \nmost likely to benefit, \nthe clinical trials ta r-\ngeting PD-1 and PD-\nL1 have shown a te n-\ndency towards i n-\ncreasing efficacy with increasing \nexpression of PD-L1.37,39-41    \nPD-L1 Immunotherapy for Patients with \nNSCLC and MPE \nAll of the large clinical trials of these agents \nin NSCLC required pathologic specimens. The \nPD-L1 tests were developed and validated on \nsurgical specimens. The KEYNOTE-024 trial, \nfor example, which led to first-line pembroli-\nzumab approval, enrolled patients with tumors in \nwhich at least 50% of cells expressed PD-L1 in \nan immunohistochemistry (IHC)-based compan-\nion test (22C3 pharmDx - Dako North America, \nInc., Carpinteria, CA).39  Although patients in \nthe phase III trial of pembrolizumab as first-line \ntherapy had unresectable, stage IV NSCLC, cy-\nCourtesy of Terese Winslow \n©Terese Winslow LLC \nTereseWinslow.com \n\n\nInt erna ti o nal  S oci ety  o f  Pl eu ral  D is eas es  \n \nPD-L1 & Immunotherapy in the Pleural Space                                 5 \n \ntology specimens, including fine needle aspi-\nrates and effusion samples, were not permitted \nfor quantification of PD-L1 expression under the \ntrial protocol.  \nThe stringent requirement for histology samples \nover cytology, along with the paucity of litera-\nture on PD-L1 in cytology samples, may lead \nclinicians and researchers to doubt the suitability \nof effusion samples for PD-L1 testing. The prac-\ntical realities of clinical care, however, differ \nfrom those in a clinical trial. Roughly a third to \nhalf of all patients with NSCLC are diagnosed \non the basis of a cytology specimen.42  For many \npatients with NSCLC staged on the basis of \nMPE, the pleural fluid tumor cells are the only \ncells easily available for PD-L1 testing.   \nNow that immunotherapy is available out-\nside clinical trials, PD-L1 detection and quanti-\nfication is routinely being done on tumor cells \nobtained from pleural and pericardial fluid sam-\nples in clinical practice. The information ob-\ntained is being used to guide clinical decision \nmaking, but there is a lack of literature validat-\ning this approach. Many questions remain open, \nchiefly whether the yield for PD -L1 testing in \npleural fluid is the same as in histologic spec i-\nmens, and whether the PD-L1 status of the \npleural tumor cells is representative of the e n-\ntire tumor burden. An anal ogous concern arises \nwhen testing small bio psy histologic samples \nfrom a large tumor.  It is known that there is \nheterogeneity of PD -L1 expression in different \nregions of the sam e tumor. Therefore, a small \npiece may not represent the status of the \nwhole.26,43  \n   \n   \n   \n   \n   \n   \n   \n Pleural fluid for cytological analysis is pre-\npared by spinning cells down received in either \nan alcohol-based fixative or 10% neutral buff-\nered formalin (NBF) into a pellet known as a \ncell block. It is then processed like a histology \nsample ─ formalin fixed and paraffin embedded \n(FFPE). The suitability of cytologic specimens \nfor other advanced testing, such as tumor muta-\ntion testing, has been well validated. Joint guide-\nlines from the College of American Pathologists, \nthe International Association for the Study of \nLung Cancer and the Association for Molecular \npathology all endorse the use of cytology for \ntesting for EGFR and ALK.24 Theoretically, PD-\nL1 testing should be easy and accurate in cyto-\nlogic preparations.  As currently employed, PD-\nL1 analysis requires evaluation of a minimum of \n100 viable tumor cells to be considered adequate \nfor quantification of PD-L1 expression.  \n    Three tests of PD-L1 expression by IHC are \ncurrently approved by the FDA, each with dif-\nferent grading scales and cut-offs for positivity.  \nVariability between the assays may prove to be a \nlarge barrier to interpretation of biomarker data \nbetween trials.44  However, recent studies show-\ning reasonable concordance between the assays \nare reassuring.40,45 Following correlation with a \nhematoxylin & eosin-stained slide, a pathologist \ntrained in scoring PD-L1 expression scores any \nperceptible membranous staining (≥1+) of tumor \ncells and quantifies the proportion of viable, PD-\nL1-expressing tumor cells in cytology and his-\ntology samples. Staining identified in necrotic \ncells or pulmonary alveolar macrophages is dis-\nregarded.   \n  Clinical Considerations  \nPleural fluid cells may be the only ones easily available \nPD-L1 testing of pleural fluid is done routinely \nPD-L1 data used with paucity of literature support \nObtaining other tissue can be a significant burden  \n\nInt erna ti o nal  S oci ety  o f  Pl eu ral  D is eas es  \n \nPD-L1 & Immunotherapy in the Pleural Space                                 6 \n \n     There is limited literature on the performance \nof PD-L1 testing in pleural fluid cytology.  In \nthe absence of clear data supporting PD-L1 test-\ning in cytologic specimens, some have suggested \nthat it may be necessary to perform more inva-\nsive sampling by core needle biopsy or surgical \nresection in patients \nwith MPE. This ap-\nproach, however, may \nbe burdensome for \npatients with easily \naccessible tumor cells \nand advanced disease. \nAs noted above, test-\ning malignant cells \nprocessed from a pleu-\nral fluid sample for \nPD-L1 with IHC is \ntechnically quite fea-\nsible;46 the question re-\nmains whether these re-\nsults are accurate and \nrepresentative of (or con-\ncordant with) the malig-\nnant disease elsewhere. \nThere is some limited retrospective data to sug-\ngest that this is in fact the case, and little data to \nsuggest that it is not. \nFollowing FDA approval of immunotherapy \nfor NSCLC, multiple groups have published ret-\nrospective analyses of PD-L1 testing in clinical \npractice  These studies serve to inform in a lim-\nited way as to the suitability of MPE specimens \nfor this purpose  Heymann and colleagues pub-\nlished a series of consecutive cytologic speci-\nmens that were diagnostic of lung ADC and \nSCC and compared the results of PD-L1 quanti-\nfication performed in a consecutive series of \nhistologic specimens (both small biopsies and \nresections) collected over the same time \nperiod.47   \nIn the Heymann study, a total of 214 lung \ncarcinoma specimens were collected from 188 \npatients of median age 71.  Pleural and pericar-\ndial effusion specimens from twelve of the pa-\ntients were included. Specimens were deemed \n“positive” if ≥ 50% the viable tumor cells ex-\npressed PD-L1. Twen-\nty-three patients had \ntwo or more specimens \ncollected. PD-L1 ex-\npression was concord-\nant among paired or \ntriplicate samples from \n21 (91%) of these pa-\ntients. Heymann and \ncolleagues concluded \noverall that cytologic \nspecimens of NSCLC \nprovide sufficient cel-\nlularity for quantifica-\ntion of PD-L1 expres-\nsion in a majority of \ncases, and that results \nof PD-L1 expression \ntesting are comparable \namong resection, cytology, and other small bi-\nopsy specimens of NSCLC.  \nA case series published by Skov and Skov in \n2017 had similar findings comparing NSCLC \ncytology to histology, although their series had \nonly a small number of cytologic specimens \nfrom pleural fluid. Their retrospective review of \n86 patients with paired histology and cytology \nspecimens, mostly NSCLC and a few other tho-\nracic neoplasms known to express PD-L1, \nshowed similar rates of positivity across multi-\nple cutoffs for “positive”, from ≥ 1% to ≥ 50%.  \nUsing the ≥ 50% cutoff, the paired specimens \nhad 100% agreement on positive cases and 93% \nagreement on negative cases. Only three patients \nhad a pleural fluid sample, making extrapolation \nFigure:  Pleural fluid collected by thoracentesis from a \nmale smoker aged 60 years with suspected Stage 4 \nM1a primary lung adenocarcinoma.           \n(A) Pleural fluid cell block, H & E; original magnifica-\ntion × 400. (B) Immunohistochemical analysis of PD‐L1 \nexpression demonstrating  1+ membranous staining in \napproximately 75% of tumor cells (“PD‐L1 positive”). \n \nB \nA \n\nInt erna ti o nal  S oci ety  o f  Pl eu ral  D is eas es  \n \nPD-L1 & Immunotherapy in the Pleural Space                                 7 \n \nof the findings overall to this small group diffi-\ncult.48 \nThe largest cohort of pleural samples was \nrecently presented in a case series by Ilie and \ncolleagues. They performed a retrospective re-\nview of 70 paired biopsy and cytology samples \nfrom patients with NSCLC comparing PD-L1 \nexpression across multiple assays  Of those 70 \ncytological samples, 30 were from pleural effu-\nsions while the other 40 were from bronchial \nwashings. Comparisons of PD-L1 tumor expres-\nsion between biopsy and cytology showed \nstrong correlation across multiple different as-\nsays and at both 1% and 50% PD-L1 expression \ncut offs (> 95% positive and negative agree-\nment). Furthermore, intra-class correlation coef-\nficients between isolated pleural cytology and \nbiopsy were greater than 0.8 across various as-\nsays. These findings again support the viability \nof using PD-L1 expression in cytology samples \nto guide treatment eligibility for PD-L1 immu-\nnotherapy.49 \nIssues in PD -L1 testing  in MPE  \nCorrelation of pleural fluid with histological specimens \nRelationship between the pleural fluid yield and the \nwhole tumor character \nComparison of three different PD-L1 tests. \nBest cutoff for “PD-L1 positive“ \nClinical application of limited data on pleural PD-L1 \ntesting \n \nPD-L1 Expression in Other Malignancies \nwith Pleural Invasion \nPD-L1 expression in the pleural fluid of pa-\ntients with other primary malignancies has also \nbeen studied, most notably malignant pleural \nmesothelioma (MPM). Histological expression \nof PD-L1 in MPM is varied, ranging from 20-\n63% in limited studies.50,51 This wide range is \nattributed to different thresholds for PD-L1 posi-\ntivity (>1% to >10%) as well as different per-\ncentages of MPM tumor subtypes between stud-\nies. Mansour and colleagues published a case \nseries of PD-L1 expression in pleural effusions \nfrom 74 patients with MPM  Of the analyzed \ncases, 10% had greater than 50% PD-L1 expres-\nsion while 38% of the samples had greater than \n1% expression. Of note, 13 of the 74 cases had \nto be excluded due to insufficient cell block ma-\nterial or malignant cell number. \nConclusion \n     The demonstration of a survival benefit in \nadvanced NSCLC from mutation-targeted thera-\npies and with PD-L1 immunotherapy has \nchanged the way we approach the treatment of \nlung cancer. Many questions regarding PD-L1 \ntesting remain under debate, including which \ntest to use, whether to incorporate staining re-\nsults of tumor-associated inflammatory cells, \nwhich cutoff to use for “positive”, and what to \ndo with negative tumors, given that some pa-\ntients with PD-L1 negative tumors respond to \ntherapy. A true measure of the utility of PD-L1 \ntesting in pleural effusion samples would be a \nprospective trial with paired testing of malignant \ncells from pleural fluid and matched surgical \nspecimens, with an analysis of clinical response \nin immunotherapy-treated patients. In light of \nthe emerging data from case series, however \nlimited, and given the increase in indications \nand popularity of PD-L1 inhibitors, the clin-\nical equipoise for such a trial is evaporat-\ning.  In the absence of such a trial, more data \nshould be collected from patients who have \npaired samples collected in the course of \nroutine clinical care. \nIn light of what we know now, quantifica-\ntion of PD-L1 expression in pleural fluid speci-\nmens of NSCLC is feasible.  The results are \ncomparable to histologic specimens in the ma-\njority of patients.  For patients with stage IV dis-\n\nInt erna ti o nal  S oci ety  o f  Pl eu ral  D is eas es  \n \nPD-L1 & Immunotherapy in the Pleural Space                                 8 \n \nease on the basis of a malignant effusion, PD-L1 \ntesting on tumor cells from the pleural space is a \nlow-risk, minimally invasive way to determine \nPD-L1 status. Existing data justify using this \ninformation to guide treatment decisions.  For \npatients, the ultimate question is not one of PD-\nL1 status, but of therapeutic benefit and prolon-\ngation of survival. Discordance in PD-L1 testing \ndoes occur, and testing can be suboptimal or fail \nfor technical reasons. For patients with MPE and                                                            \nfailed testing or equivocal results, or for those  \nPD-L1 negative patients with limited therapeutic \nalternatives, it is appropriate to consider \nresampling or testing other sites of disease.  \nA Final Thought \nWe find ourselves now at the beginning of a \nnew era of cancer treatment. We have learned \nmuch about the immune system’s role in cancer \nprevention. And we have learned much about its \nlimitations. We have shown that we can alter the \ninteraction between tumor cells and normal \ncells. Yet, there is much to be learned and the \nwork goes on. The final question remains to be \nanswered. Can we enable the immune system to \neradicate an established cancer? \n \n      \n                                                                               \n \n \n \n \n \n \n \n \n \n \n \n \n \nThe authors express their gratitude to \nJerome L. Slate MD, FCCP for his assistance                                                        \nin the preparation of this manuscript. \n\n\nInt erna ti o nal  S oci ety  o f  Pl eu ral  D is eas es  \n \nPD-L1 & Immunotherapy in the Pleural Space                                 9 \n \nREFERENCES \n1. Coley WB, MD. The Classic: The Treatment of Malignant Tumors by Repeated Inoculations of Erysipelas: \nWith a Report of Ten Original Cases. Clinical Orthopaedics & Related Research January. 1991;262:3-11. \n2. Management of Malignant Pleural Effusions. 2000;162(5):1987-2001. \n3. Rodriguez-Panadero F, Naranjo FB, Mejias JL. Pleural metastatic tumours and effusions. Frequency and \npathogenic mechanisms in a post-mortem series. 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