© 2020 The Authors. Respirology Case Reports published by John Wiley & Sons Australia, Ltd on behalf of The Asian Pacific Society of Respirology Malignant pleural effusion (MPE) is common with mesothelioma. We report two cases of extraordinarily viscous MPEs associated with mesothelioma. The viscosity prohibited spontaneous gravity-dependent drainage via indwelling pleural catheters. Our ex vivo experiments found very high hyaluronic acid (HA) content within the fluid. Treatment of the fluid with hyaluronidase, but not with deoxyribonucleases, significantly reduced fluid viscosity. The results provide proof that HA can contribute to high viscosity of pleural fluid in mesothelioma. Research into strategies of counteracting HA properties in the management of MPEs may provide further insight

Cheah, Hui Min

Creaney, Jenette

Fitzgerald, Deirdre

Lee, Y. C.Gary

Louw, Amber

English

Research Online @ ECU

Edith Cowan University Research Online ECU Publications Post 2013 2021 Hyaluronic acid in viscous malignant mesothelioma pleural effusion Hui Min Cheah Deirdre Fitzgerald Amber Louw Edith Cowan University Jenette Creaney Y. C.Gary Lee Edith Cowan University Follow this and additional works at: https://ro.ecu.edu.au/ecuworkspost2013  Part of the Diseases Commons 10.1002/rcr2.694 Cheah, H. M., Fitzgerald, D., Louw, A., Creaney, J., & Lee, Y. C. G. (2021). Hyaluronic acid in viscous malignant mesothelioma pleural effusion. Respirology Case Reports, 9(1), article e00694. https://doi.org/10.1002/rcr2.694 This Journal Article is posted at Research Online. https://ro.ecu.edu.au/ecuworkspost2013/9319 Hyaluronic acid in viscous malignant mesothelioma pleuraleffusionHui Min Cheah1,2, Deirdre Fitzgerald1,2,3 , Amber Louw4,5, Jenette Creaney2,6 &Y. C. Gary Lee1,2,3,51Pleural Medicine Unit, Center for Respiratory Health, University of Western Australia, Perth, WA, Australia.2Medical School, University of Western Australia, Perth, WA, Australia.3Department of Respiratory Medicine, Sir Charles Gairdner Hospital, Perth, WA, Australia.4PathWest Laboratory Medicine, QEII Medical Centre, Perth, WA, Australia.5School of Medical and Health Sciences, Edith Cowan University, Joondalup, WA, Australia.6National Centre for Asbestos Related Diseases, Perth, WA, Australia.KeywordsHyaluronic acid, hyaluronidase, malignant pleuraleffusion, mesothelioma, viscosity.CorrespondenceY. C. Gary Lee, Pleural Medicine Unit, Center forRespiratory Health, University of Western Australia,Room 533, Harry Perkins Building, 6 Verdun Street,Nedlands, Perth, WA 6009, Australia. E-mail: gary.lee@uwa.edu.auReceived: 18 September 2020; Revised: 3 November2020; Accepted: 15 November 2020; AssociateEditor: Jasleen Pannu.Respirology Case Reports, 9 (1), 2021, e00694doi: 10.1002/rcr2.694AbstractMalignant pleural effusion (MPE) is common with mesothelioma. Wereport two cases of extraordinarily viscous MPEs associated with mesotheli-oma. The viscosity prohibited spontaneous gravity-dependent drainage viaindwelling pleural catheters. Our ex vivo experiments found very highhyaluronic acid (HA) content within the fluid. Treatment of the fluid withhyaluronidase, but not with deoxyribonucleases, significantly reduced fluidviscosity. The results provide proof that HA can contribute to high viscosityof pleural fluid in mesothelioma. Research into strategies of counteractingHA properties in the management of MPEs may provide further insight.IntroductionPleural effusions affect over 90% of patients with malignantpleural mesothelioma. The malignant pleural effusion(MPE) commonly appears serosanguinous or haemorrhagic,and can be evacuated using a range of devices, such as aspi-ration needles, chest tube, or indwelling pleural catheter(IPC). We report two cases of extraordinarily viscous MPEsin mesothelioma patients. Our ex vivo experiments attrib-uted the viscosity to the high hyaluronic acid (HA) contentwithin the fluid, which could be significantly reduced withhyaluronidase.Case ReportPatient 1A 79-year-old man presented with a left-sided pleural effu-sion which was noted as viscous on the initial therapeuticaspiration. Cytology confirmed malignant mesothelioma.Upon recurrence of the MPE, an IPC (Rocket, UK) wasinserted but the thick fluid prohibited spontaneous drain-age. He was referred to our centre for further management.The fluid was partially evacuated by connecting the IPCvia a connector to a 60-mL syringe through which manualsuction could be applied. The viscosity of the fluid prohibi-ted usual laboratory biochemical analyses of the fluid. Inan attempt to reduce fluid viscosity to clear the remainingfluid, two doses of 5 mg of deoxyribonuclease (DNase) wasinstilled over two days via the IPC, after which ~300 and~200 mL of fluid were drained. The underlying lung re-expanded on chest X-ray (CXR) and talc slurry pleurodesiswas administered via the IPC.Unfortunately, the fluid recurred over the next fourweeks, resulting in a large multiloculated effusion associ-ated with breathlessness and fatigue (Fig. 1A), necessitatinghospitalization. Drainage via the IPC remained difficultbecause of the fluid viscosity. Four doses of intrapleuraltissue plasminogen activator (tPA) 2.5 mg and DNase© 2020 The Authors. Respirology Case Reports published by John Wiley & Sons Australia, Ltdon behalf of The Asian Pacific Society of RespirologyThis is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided theoriginal work is properly cited.2021 | Vol. 9 | Iss. 1 | e00694Page 1Official Case Reports Journal of the Asian Pacific Society of RespirologyRespirology Case Reports5 mg were administered twice daily with good effect. Afluid sample was collected after the first dose (Sample 1)for viscosity and cytological analyses (Tables 1, 2 andFig. 2, respectively). A total of 2.8 L of fluid was drainedwith significant radiographic improvement (Fig. 1B). How-ever, the benefit was short-lived and fluid re-accumulatedwithin two weeks (Fig. 1C), during which time he receivedone cycle of treatment in an immunotherapy trial. A fur-ther attempt with tPA 2.5 mg/DNase 5 mg given in outpa-tient clinic produced only marginal benefit. With strongmanual suction via the IPC, 380 mL of viscous fluid wasaspirated and a further 350 mL was drained the next day.Nonetheless, a massive residual effusion remained.The patient was readmitted two weeks later for ongoingdifficulty with IPC drainage (<100 mL each attempt)(Fig. 1D). Sample 2 was collected for viscosity analyses(Table 1). During the hospitalization, he receivedintrapleural tPA 5 mg/DNase 5 mg and suction via theIPC with little effect (60 and 275 mL after the first twodoses). A further attempt using tPA 10 mg and DNase10 mg yielded 200 mL and minimal radiographic improve-ment. He was discharged and died seven months laterfrom his mesothelioma.Patient 2A 79-year-old female presenting with a right pleural effu-sion underwent video-assisted thoracoscopic pleural biopsy(which showed mesothelioma) and talc poudrage. Thefluid recurred after surgery necessitating a therapeuticFigure 1. The viscous fluid of Patient 1 made drainage via indwelling pleural catheter (IPC) difficult, resulting in large loculated collections of pleuralfluid, requiring hospital admission. The chest radiograph (A) showed almost complete opacification of the left hemi-thorax which cleared withintrapleural tissue plasminogen activator (tPA) and deoxyribonuclease (DNase) therapy (B). However, the benefit was short-lived and significant re-accumulation of fluid was shown on his radiograph two weeks later (C). Further treatment with escalated doses of tPA/DNase failed to produce aresponse (D).Hyaluronan in mesothelioma effusion H.M. Cheah et al.2 © 2020 The Authors. Respirology Case Reports published by John Wiley & Sons Australia, Ltdon behalf of The Asian Pacific Society of Respirologyaspirate which revealed extremely thick exudative pleuralfluid. She underwent regular therapeutic aspiration in anoutside hospital for her MPE over the next four years(during which time she received various systemic therapy)before she was referred to our centre for IPC insertion.The fluid was described as thick and “jelly-like” and failedto drain via the IPC. Manual suction via the IPC (Rocket)was required for complete evacuation of the fluid. Talcslurry was administered after lung re-expansion but thepleurodesis failed.For the following 12 months, drainage was performed(roughly once weekly) via IPC. Sample 3 showed a typi-cal specimen. Standard IPC suction bottles were ineffec-tive to clear the thick fluid. Hence, the patient’s carerswere taught to perform IPC drainage using manualsuction.After one year, IPC drainage failed despite the above-mentioned measures causing significant breathlessnessnecessitating a hospital admission. A trial ofintrapleural tPA 2.5 mg/DNase 5 mg was successful anddrained 1.5 L with significant radiographic clearance.Regular IPC drainage was re-established. Her mesothe-lioma continued to progress and she died 10 monthslater.Fluid Viscosity and HA AnalysesPleural fluid viscosity was determined as described bySimpson et al. [1]. HA concentrations were determined inthe MPE supernatant using a commercially available HAbinding assay (Corgenix, USA). To test the contribution ofHA to the viscosity of the effusion, samples were incubatedat room temperature for 10 min with hyaluronidase typeV (0–100 U/mL) diluted in phosphate-buffered saline(both Sigma-Aldrich, USA). To model the effects ofrecombinant human DNase (rhDNase) treatment, theoriginal pleural effusion sample was incubated in the labo-ratory with rhDNase (Pulmozyme®; Roche, USA) dilutedin normal saline (Baxter, Australia) for 4 h before viscositymeasurement.We found that MPE from both patients had very highpleural fluid HA concentrations. Hyaluronidase, but notrhDNase, was effective in reducing the viscosity of thefluid in a dose-dependent manner (Tables 1, 2, Video S1).DiscussionWe described two mesothelioma patients with HA-richand extremely viscous MPE, limiting fluid drainage. OurTable 1. Pleural fluid HA concentrations and viscosity levels (mPas) of pleural fluid after incubation withhyaluronidase (U/mL).HA concentration in pleural fluidFluid viscosity (mPas) after treatment with different concentrationsof hyaluronidase (U/mL)0 1 10 100Patient 1(Sample 1)2.66×106 ng/mL 800.21 54.62 21.91 2.94Patient 1(Sample 2)5.39×106 ng/mL Too viscous Too viscous 153.67 18.61Patient 2(Sample 3)2.31×106 ng/mL 303.03 168.01 6.59 2.87The two samples from Patient 1 were taken about 38 days apart.HA, hyaluronic acid.Table 2. Pleural fluid viscosity levels (mPas) of pleural fluid after incubation with rhDNase.rhDNase (μg/mL)0 2.5 5 10Patient 1 (Sample 1) 780.56 690.93 627.61 567.38Sample 1 was incubated with rhDNase at 2.5, 5, and 10 μg/mL at room temperature for 4 h before viscosity was measured. The range of concen-tration selected was to approximate the concentration of enzyme in a patient with a 1.5-L pleural effusion treated with 5 mg of rhDNase.rhDNase, recombinant human deoxyribonuclease.H.M. Cheah et al. Hyaluronan in mesothelioma effusion© 2020 The Authors. Respirology Case Reports published by John Wiley & Sons Australia, Ltdon behalf of The Asian Pacific Society of Respirology3laboratory work confirmed that HA, present in high con-centrations in these fluids, likely contributed to the highviscosity which could be significantly reduced usinghyaluronidase.MPE from mesothelioma contains a vast number ofimportant mediators. Mesothelioma has been known toexpress HA for over 80 years and HA pleural fluid concen-trations are a valuable ancillary test for diagnosis. In ourprevious study including 96 mesothelioma patients, thepleural fluid HA level has an area under the curve of 0.89in its diagnostic value of mesothelioma, highly comparablewith mesothelin. Pleural fluid HA has also prognosticvalues [2]. Our experiments provide proof to the long sus-picion that HA is involved in generating high viscosityin MPE.HA is a long polymer of disaccharides up to 20,000 kDain size produced by hyaluronan synthases, present inabundance in extracellular matrix. It can promote inflam-mation and angiogenesis—processes involved in MPE for-mation. HA is approved as intra-articular treatment forosteoarthritis and for use in ophthalmologic conditions. Itis also a common ingredient in skin care products [3].HA can be degraded by hyaluronidases, a family ofenzymes, and by various non-enzymatic reactions(e.g. acidic/alkaline hydrolysis) [4]. Hyaluronidases havebeen used in low doses in various conditions, mainly tobreakdown extracellular matrix and enhance drug distribu-tion [5,6]. Whether it can be safely applied intrapleurallyand in sufficient therapeutic quantity to lyse HA in humanMPE will need to be investigated.At present, clinically available options for viscous MPEare limited. Use of tPA/DNase enhanced the evacuation ofthick infected pleural fluid in clinical trials. The regimeprovided some short-term benefits in Patient 1, but subse-quent administration did not prove effective. Our ex vivoexperiments did not find a major role of rhDNase inreducing the viscosity. As tPA is known to stimulate signif-icant production of pleural fluid in healthy and diseasedpleura, it is possible that the dilutional effect of this fluidformation helped with initial drainages. Whether tPA itselfaffects viscosity of MPE is unknown.The composition of pleural fluid may be important toprovide an informed clinical decision to ascertain an effec-tive therapy for pleural effusion management. In cases ofviscous fluid and high HA levels, strategies to counteractHA production or enhance its breakdown warrantexploration.Disclosure StatementAppropriate written informed consent was obtained forpublication of this case report and accompanying images.AcknowledgmentsY. C. Gary Lee is a recipient of the National Health andMedical Research Council (NHMRC)/Medical ResearchFuture Fund Next Generation Practitioner Fellowship.Y. C. Gary Lee and J. Creaney have received project grantfunding from NHMRC, iCare New South Wales Dust Dis-eases Authority, Sir Charles Gairdner Research AdvisoryCommittee, Institute for Respiratory Health, Cancer Coun-cil of Western Australia, and Cancer Australia.Figure 2. The cytology sample from Patient 1 demonstrated materialconsistent with hyaluronic acid (HA) both in intracellular and extracellu-lar locations. The smear of the viscous pleural fluid (A) shows promi-nent extracellular granular material within the background consistentwith HA (Diff-Quick stain, original magnification, 10×). The cell block(B) of the same pleural fluid demonstrates malignant mesotheliomacells with prominent intracytoplasmic vacuoles containing eosinophilicmaterial either with a globular appearance or an appearance similar tothat of crinkled paper, both of which suggest the presence ofhyaluronan (haematoxylin and eosin, original magnification, 40×).Hyaluronan in mesothelioma effusion H.M. Cheah et al.4 © 2020 The Authors. Respirology Case Reports published by John Wiley & Sons Australia, Ltdon behalf of The Asian Pacific Society of RespirologyReferences1. Simpson G, Roomes D, and Heron M. 2000. Effects of strep-tokinase and deoxyribonuclease on viscosity of human surgi-cal and empyema pus. Chest 117(6):1728–1733.2. Creaney J, Dick IM, Segal A, et al. 2013. Pleural effusionhyaluronic acid as a prognostic marker in pleural malignantmesothelioma. Lung Cancer 82(3):491–498.3. Abatangelo G, Vindigni V, Avruscio G, et al. 2020.Hyaluronic acid: redefining its role. Cells 9(7):1743.4. Stern R, and Jedrzejas MJ. 2006. Hyaluronidases: their geno-mics, structures, and mechanisms of action. Chem. Rev. 106(3):818–839.5. Weber GC, Buhren BA, Schrumpf H, et al. 2019. Clinical appli-cations of hyaluronidase. Adv. Exp. Med. Biol. 1148:255–277.6. Buhren BA, Schrumpf H, Hoff NP, et al. 2016. Hyaluroni-dase: from clinical applications to molecular and cellularmechanisms. Eur. J. Med. Res. 21:5.Supporting informationAdditional Supporting Information may be found in theonline version of this article at the publisher’s web-site:http://onlinelibrary.wiley.com/doi/10.1002/rcr2.694/suppinfo.Video S1. Highly viscous pleural fluid from Patient1 (Sample 2) in the tube labelled untreated is shown.Treatment of the same sample with hyaluronidase(100 U/mL for 10 min) significantly reduced the fluidviscosity as shown in the tube labelled treated.H.M. Cheah et al. Hyaluronan in mesothelioma effusion© 2020 The Authors. Respirology Case Reports published by John Wiley & Sons Australia, Ltdon behalf of The Asian Pacific Society of Respirology5

Hyaluronic acid in viscous malignant mesothelioma pleural effusion

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Hyaluronic acid in viscous malignant mesothelioma pleural effusion

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