International audienceIn this contribution, an approach developed previously for mice is used for human biopsy. In the case of patient 1, Pt detection is performed 6 days after the last oxaliplatin infusion, while for patient 2, the biopsy was performed more than 15 days after his first platin infusion and several dialysis. Even for these biological samples, experiments show that synchrotron mediated mXRF is a suitable tool to detect Pt in kidney biopsy, and thus probably for any organ exposed to Pt. Therefore, mXRF could also be of major interest to decipher the mechanism beyond Pt induced neurotoxicity, ototoxicity on human biopsy. Pharmacoavailability of chemotherapies is a major concern because some treatment failures are explained by poor tumor penetration of the active molecule. mXRF could be an elegant way to map the distribution of Pt inside cancerous cells at the micrometer scale. Pt and Zn are only two of the numerous trace elements that mXRF can detect; heavy metal intoxication diagnosis and the toxicity mechanism probably could also benefit from this innovative technique

Alex Kellum

Alexandre Hertig

Aurélien Bataille

Chantal Jouanneau

Christian Mocuta

Dominique Bazin

Emmanuel Esteve

Emmanuel Letavernier

Eric Rondeau

Jean Philippe Haymann

John J. Rehr

Karine Provost

Kris Jorissen

Michel Daudon

Pierre Ronco

Solenn Reguer

Stephan Rouzière

AbstractIn this contribution, an approach developed previously for mice is used for human biopsy. In the case of patient 1, Pt detection is performed 6 days after the last oxaliplatin infusion, while for patient 2, the biopsy was performed more than 15 days after his first platin infusion and several dialysis. Even for these biological samples, experiments show that synchrotron mediated μXRF is a suitable tool to detect Pt in kidney biopsy, and thus probably for any organ exposed to Pt. Therefore, μXRF could also be of major interest to decipher the mechanism beyond Pt induced neurotoxicity, ototoxicity on human biopsy. Pharmacoavailability of chemotherapies is a major concern because some treatment failures are explained by poor tumor penetration of the active molecule. μXRF could be an elegant way to map the distribution of Pt inside cancerous cells at the micrometer scale. Pt and Zn are only two of the numerous trace elements that μXRF can detect; heavy metal intoxication diagnosis and the toxicity mechanism probably could also benefit from this innovative technique

Esteve, Emmanuel

Bazin, Dominique

Jouanneau, Chantal

Rouzière, Stephan

Bataille, Aurélien

Kellum, Alex

Provost, Karine

Mocuta, Christian

Reguer, Solenn

Jorissen, Kris

Rehr, John J.

Hertig, Alexandre

Rondeau, Eric

Letavernier, Emmanuel

Haymann, Jean Philippe

Daudon, Michel

Ronco, Pierre

Elsevier - Publisher Connector 

How to assess the role of Pt and Zn in the nephrotoxicity of Pt anti-cancer drugs?: An investigation combining μXRF and statistical analysis. Part II: Clinical application 

AbstractNephrotoxicity is a specific and serious adverse effect of cisplatin which limits its utilization. Here we present an experimental approach to decipher its mechanisms focusing on the renal distribution of Pt and Zn in cisplatin exposed mice kidneys. We combined Synchrotron Radiation (S.R.) μX-ray fluorescence with statistical treatments. μX-ray fluorescence data were collected on a set of mice biopsies after cisplatin, oxaliplatin and carboplatin injections. Even if this investigation is based on a limited number of samples, these preliminary data seem to reveal a stronger correlation between the spatial repartition of Pt and Zn for cisplatin than for the other Pt containing drugs. The cisplatin injection induced a redistribution of medullary Zn across the corticomedullary junction where histological lesions develop. These results were confirmed by evaluation of Pearson's and Manders' co-localization coefficients. These data suggest that Zn is involved in the nephrotoxicity of cisplatin. This could lead to new diagnosis, physiopathological and even therapeutical approaches

Estève, Emmanuel

Thiaudière, Dominique

Rondeau, Éric

How to assess the role of Pt and Zn in the nephrotoxicity of Pt anti-cancer drugs? An investigation combining μXRF and statistical analysis: Part I: On mice 

Haymann, Jean-Philippe

HAL-Inserm

How to assess the role of Pt and Zn in the nephrotoxicity of Pt anti-cancer drugs?: An investigation combining mXRF and statistical analysis. Part II: Clinical application

Comptes Rendus Chimie

lable at ScienceDirectC. R. Chimie 19 (2016) 1586e1589Contents lists avaiComptes Rendus Chimiewww.sciencedirect.comFull paper/MemoireHow to assess the role of Pt and Zn in the nephrotoxicity of Ptanti-cancer drugs?: An investigation combining mXRF andstatistical analysis. Part II: Clinical applicationEmmanuel Esteve a, Dominique Bazin c, d, *, Chantal Jouanneau a,Stephan Rouziere d, Aurelien Bataille a, Alex Kellum c, Karine Provost e,Christian Mocuta f, Solenn Reguer f, Kris Jorissen g, John J. Rehr g,Alexandre Hertig a, h, Eric Rondeau a, h, Emmanuel Letavernier a, b,Jean Philippe Haymann a, b, Michel Daudon a, b, Pierre Ronco a, ba UMR S1155, INSERM/UPMC, 4, rue de la Chine, 75970 Paris cedex 20, Franceb AP-HP, Hôpital Tenon, Service d’Explorations Fonctionnelles, 4, rue de la Chine, 75970 Paris cedex 20, Francec CNRS, LCMCP-UPMC, College de France, 11, place Marcelin-Berthelot, 75231 Paris cedex 05, Franced Laboratoire de Physique des Solides UMR 8502 CNRS, Bat 510, Universite Paris-11, 91405 Orsay, Francee Institut de Chimie des Materiaux Paris Est, UMR 7182 CNRS UPEC, 2e8 rue Henri-Dunant, 94320 Thiais, Francef Synchrotron SOLEIL, L'Orme des Merisiers, Saint-Aubin, BP 48, 91192 Gif-sur-Yvette, Franceg Department of Physics, University of Washington, Seattle, WA 98195, United Statesh AP-HP, Hôpital Tenon, Service UNTR, 4, rue de la Chine, 75970 Paris cedex 20, Francea r t i c l e i n f oArticle history:Received 30 October 2015Accepted 15 February 2016Available online 1 April 2016Keywords:NephrotoxicityPt-based anti-cancer drugsX-ray fluorescenceSynchrotron* Corresponding author.E-mail address: dominique.bazin@upmc.fr (D. Bahttp://dx.doi.org/10.1016/j.crci.2016.02.0151631-0748/© 2016 Académie des sciences. Publishcreativecommons.org/licenses/by-nc-nd/4.0/).a b s t r a c tIn this contribution, an approach developed previously for mice is used for human biopsy.In the case of patient 1, Pt detection is performed 6 days after the last oxaliplatin infusion,while for patient 2, the biopsy was performed more than 15 days after his first platininfusion and several dialysis. Even for these biological samples, experiments show thatsynchrotron mediated mXRF is a suitable tool to detect Pt in kidney biopsy, and thusprobably for any organ exposed to Pt. Therefore, mXRF could also be of major interest todecipher the mechanism beyond Pt induced neurotoxicity, ototoxicity on human biopsy.Pharmacoavailability of chemotherapies is a major concern because some treatment fail-ures are explained by poor tumor penetration of the active molecule. mXRF could be anelegant way to map the distribution of Pt inside cancerous cells at the micrometer scale. Ptand Zn are only two of the numerous trace elements that mXRF can detect; heavy metalintoxication diagnosis and the toxicity mechanism probably could also benefit from thisinnovative technique.© 2016 Académie des sciences. Published by Elsevier Masson SAS. This is an open accessarticle under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).zin).ed by Elsevier Masson SAS.1. IntroductionCisplatin among platin salt based chemotherapies is ofmajor clinical importance in the treatment of many solidcancers [1e6]. The main toxicity that hampers cisplatinutilisation and therefore patient survival is itsThis is an open access article under the CC BY-NC-ND license (http://E. Esteve et al. / C. R. Chimie 19 (2016) 1586e1589 1587nephrotoxicity. Like most tubulointerstitial acute nephritis,cisplatin induced tubulopathy lacks specific histologicalsigns. This is critical because patients exposed to cisplatinare likely to accumulate several nephrotoxic drugs and topresent multiple risk factors of tubular aggression.In the first part of this investigation, we have demon-strated that mXRF using synchrotron radiation can be usedto assess presence and spatial distribution of trace ele-ments such as Pt and Zn in mice kidneys. Interestingly theZn redistribution pattern as well as the Pt spatial distribu-tion seems to be dependant on the injected Pt salt. Asoxaliplatin and carboplatin are known to cause far lesstubular damages than their cisNa conjugated counterpartswe wondered whether the signal we observed could bespecific to cisplatin induced nephropathy, and whether wecould extrapolate the observations we made in the murinemodel to humans.To further investigate this hypothesis we chose toexplore two pathological kidney biopsies gathered in theNephrology department of Tenon’s Hospital. Both patientspresented acute renal failure attributed to acute tubulardysfunction after platin exposure.2. Material and methodsAll samples were investigated on the DiffAbs beamlineat Synchrotron SOLEIL (France). Details regarding the ex-periments have been extensively given in different previ-ous publications [7e12] and also in part I of thisinvestigation.Patient 1 was a 55 year old patient, his main comor-bidities were: liver transplantation in 2005 for chronicalcoholic steatosis and sleep apnea syndrome. In July 2011CT4aN2cM1 epidermoid carcinoma in the postcricoïd areawas diagnosed. A treatment according to the EXTREMEprotocol (Erbitux, Cisplatin, 5Flurouracil) was initiated on04/08/2011. Acute renal failure occurred few days after thefirst infusion, the serum creatinin increased from 96 to607 mmol/l in 6 days. His usual medications by the time ofhis admission in the nephrology intensive care unitincluded: salicylate, valgancyclovir, gabapentine, tacroli-mus, calciparin and pantoprazole. There was no clinicalevidence of infection, hemodynamic constants werenormal; the renal sonogram was normal, and prerenaletiology was ruled out. There was neither hematuria norleucocyturia. There was non-selective proteinuriameasured at 898 mg/mmol creatinine for only 388 mg/mmol creatinine of albuminuria. Immunological in-vestigations were negative. A kidney biopsy was performedon 19/08/2014 (Supplementary data 1), 15 days after thelast cisplatin infusion. Biopsy was cortical, 15 glomeruliwere analyzable, 4 of them were sclerotic. Two glomeruliwere ischemic. There was mild mesangial expansion in theothers. Few tubular sections showed microvacuols as seenin anti-calcineurin toxicity. Less than 5% of the tubules wereatrophic. The tubular epithelium was a bit flattened. Therewere almost no necrotic casts in tubules lumens. Interstitialfibrosis interested 10e15% of the parenchyma. There wasno interstitial inflammation. Some arterioles showed se-vere chronic lesions. Immunofluorescence for IgG, IgA, IgM,C3, C1q, kappa, lambda was negative. This biopsy showedonly mild tubular injuries regarding the severity of therenal failure. The retained diagnosis was acute renal failuresecondary to cisplatin nephrotoxicity. Evolution was pejo-rative; hemodialysis was initiated on 30/08/2014 and wasnot disrupted until the patient’s death four months later.Patient 2 was 57 at the time of the biopsy in 2011. Hewas diagnosed with T4N2M1 colon liberkhunian adeno-carcinoma in January 2008. He was treated surgically andreceived multiple chemotherapies including: FOLFOX(folinic acid, oxaliplatin and 5Fluorouracil), FOLFIRI (FolinicAcid, 5Fluorouracile, Irinotecan), bevacizumab and capeci-tabin. In July 2011, Folfox (including 85 mg/m2 oxalipatin)and bevacizumab were reinitiated. On 16/9/11, during the4th cure of FOLFOX, the patient presented shivering asso-ciated with hyperthermia and nausea. An empiric anti-biotherapy including tazocillin and vancomycin was initi-ated. Subsequently, he presented acute renal failure (serumcreatinin raised from 90 mmol/l to 325 mmol/l and804 mmol/l) with severe hyperkaliemia (6.6 mmol/l). Thepatient was transferred to the intensive care unit andextrarenal epuration had to be initiated. The renal sono-gram was considered normal, and the blood and urinaryionogram couldn’t distinguish prerenal participation. Bloodcell count in urine was negative, but there were hemoglo-binuria and 3þ proteinuria on the urine dipstick. Clinicallythe patient presented rapidly progressing oligoanuria. Thepractician noticed acute hemolysis, thrombopenia andcoagulation factor consumption. No schizocytes werenoticed. Hyperthermia resolved within 24 h; microbiolog-ical investigations were negative and anti-biotherapy wasdisrupted. The Coombs test was 3þ positive for IgG and C3.A kidney biopsy was performed on September 22(Supplementary data 2). Twenty-one glomeruli wereanalyzable on this cortico-medullar biopsy. Four glomeruliwere sclerotic, two were ischemic, whereas the othersdidn’t show any sign of pathology. There was evidence ofacute tubular injury with many necrotic debris in tubuleslumen. Some of those debris showed a macrophagicinflammation response. There were mild non-specificinterstitial inflammations. They were neither acutevascular injuries nor sign of acute microangiopathy.Immunofluorescence was negative for IgA, IgG, IgM, C3,C1q, fibrin, Kappa and Lambda. The final diagnosis wasacute tubular necrosis secondary to oxaliplatin mediatedhemolysis. Evolution was favorable and dialysis was dis-rupted on October 3rd.Renal function slowly normalized.Oxaliplatin was not readministrated, and the patient diedfrom generalized cancer on October 1, 2012.Although these two patients both required dialysisinitially due to acute tubular injury few days after platinsalt exposure, the mechanism underneath these acutekidney injuries is very different. As far as we can say,cisplatin exerted direct toxicity toward the tubular cells ofpatient 1, leading to their definitive destruction and to endstage renal failure. Patient 2 suffered acute intravascularhemolysis due to an immunoallergic mechanism. Renalfailure is secondary to the haemoglobin precipitation in thetubules. Thereforewewonderedwhether we could identifythe presence of Pt in these two kidney biopsies andwhether it’s spatial distribution (among with Zn’s one)would be different.E. Esteve et al. / C. R. Chimie 19 (2016) 1586e158915883. Results and discussionAs described in Part 1 of this project concerning ananimal model of platin induced nephritis, mXRF spectrawere acquired in large areas of the two studied biopsy withmicrometer scale resolution. They were digitally processedto isolate ZnKa and PtLa [13]. The intensity of the respectivepeaks was then used to generate maps representing spatialdistribution of these elements in the two biopsies.The striking result of this investigation comes from thefact that we were able to point out the presence of Pt in thehuman biopsy. To the best of our knowledge, this is the firsttime that suchmeasurements were performed. Note that inthe case of patient 1(Fig.1aeb), Pt detectionwas performed6 days after the last oxaliplatin infusion while for patient 2,the biopsy was performed more than 15 days after his firstoxaliplatin infusion, and several dialysis. We were also ableto detect Zn in the two patients’ biopsies (Fig. 1ced).Pt and Zn seemed to be equally distributed in the biopsyof patient 1. Pt and Zn seemed to be concentrated in themedulla of patient 2. In our experience in mice, Pt and ZnFig. 1. Zn and Pt XRF maps collected for patient 1 (a and b) and for pare found in the cortex after cisplatin infusion only, and webelieve that this is somehow related to cisplatin inducedtubulopathy. This observation likely explains the asym-metry observed in the patient 2 sample. Unfortunately inabsence of analyzable medulla in patient 1 tissue, wecannot definitely confirm our hypothesis.Following the approach defined for mice in part I of thisstudy, the fluorescencemaps for the different samples wereanalyzed using Intensity Correlation Analysis (ICA)methods [14,15] in order to highlight possible spatial cor-relations between Zn and Pt (Fig. 2). Although this provesthe feasibility of this measurement, the absence of medullain the patient 1 biopsy is clearly a limitation of such anapproach.4. DiscussionFor the first time to the best of our knowledge, we haveshown that mXRF can detect Pt and Zn at amicrometer scalenondestructively in human kidney biopsy. From a clinicalpoint of view, routine renal anatomopathology is quiteatient 2 (c and d). Cortico medullar junction is shown in black.Fig. 2. Pearson correlation coefficients between Zn and Pt obtained throughstatistical analysis for the patients 1 and 2.E. Esteve et al. / C. R. Chimie 19 (2016) 1586e1589 1589unequipped to assess the participation of a specific drug inacute tubular necrosis. mXRF allowed us to physically detectthe potential nephrotoxic agent in the tissue of interest.Although the tubular lesions of patient 1 were very discreteafter standard staining, which contrasted with the severityof the renal failure, mXRF could identify traces of remanentPt in the cortex. Such measurements may thus be of pri-mary importance for the clinician.Moreover, the spatial correlation between Pt and Znmay also be discussed using such an approach.Whether thezinc we measured is linked to zinc renal metabolismmodification, protective mechanisms such as heavy metaldetoxification metallothionein induction, a witness ofplatin induced damaged DNA linked protein recruitmentsuch as ZNF143 or to deleterious mechanisms such asactivation and accumulation of profibrotic metal-loproteases such as MeprinA is unclear. However, cisplatinseems to be the only Pt salt able to induce a redistributionof renal Zn to the cortex, and this is probably an interestingnew pathway to explore.We are aware that this study is preliminary and containsseveral limitations. Renal biopsies grant access to a verysmall sample of kidney tissue and the representativity ofthe sample is a constant concern. Nevertheless, an advan-tage of such an approach is that we are able to investigatethe paraffin bloc through a non-destructive technique.Therefore it does not prohibit further exploitation of thesample by the anatomopathologist. In this experiment thelack of medullar tissue in the patient 1 biopsy hampersgreatly our ability to reproduce the cisplatin specific Pt andZn spatial distribution pattern we identified in mice.Nevertheless, we believe that the unique ability of mXRF todissect matter could be of great utility in the understandingand diagnosis, and therefore the prevention of Pt relatednephrotoxicity.5. ConclusionThis set of experiments shows that synchrotron medi-ated mXRF is a suitable tool to detect Pt in kidney biopsy andconsequently probably so on any organ exposed to Pt.Therefore, mXRF could also be of major interest to decipherthe mechanism beyond Pt induced neurotoxicity, ototox-icity. Pharmacoavailability of chemotherapies is a majorconcern because some treatment failures are explained bypoor tumor penetration of the active molecule. Thus mXRFcould be an elegant way to map the distribution of Pt insidecancerous cells at the micrometer scale. Pt and Zn are onlytwo of the numerous trace elements mXRF can detect.Heavy metal intoxication, diagnosis and toxicity mecha-nisms could probably also benefit from this innovativetechnique.Appendix A. Supplementary dataSupplementary data related to this article can be foundat http://dx.doi.org/10.1016/j.crci.2016.02.015.References[1] J. Ferlay, E. Steliarova-Foucher, J. Lortet-Tieulent, S. Rosso,J.W. Coebergh, H. Comber, D. Forman, F. Bray, Eur. J. Cancer 49(2013) 1374.[2] E. Wong, C.M. Giandomenico, Chem. Rev. 99 (1999) 2451.[3] B. Desoize, C. Madoulet, Crit. Rev. Oncol. Hematol. 42 (2002) 317.[4] L. Kelland, Nat. Rev. Cancer 7 (2007) 573.[5] D. Wang, S.J. Lippard, Nat. Rev. Drug Discov. 4 (2005) 307.[6] A.V. Klein, T.W. Hambley, Chem. Rev. 109 (2009) 4911.[7] D. Bazin, X. Carpentier, O. Traxer, D. Thiaudiere, A. Somogyi,S. Reguer, G. Waychunas, P. Jungers, M. Daudon, J. Synchrotron Rad15 (2008) 506.[8] D. Bazin, X. Carpentier, I. Brocheriou, P. Dorfmuller, S. Aubert,C. Chappard, Biochimie 91 (2009) 1294.[9] C. Nguyen, H.K. Ea, Thiaudiere, S. Reguer, D. Hannouche, M. Daudon,F. Liote, D. Bazin, J. Synchrotron Rad 18 (2010) 475.[10] X. Carpentier, D. Bazin, P. Jungers, S. Reguer, D. Thiaudiere,M. Daudon, J. Synchrotron Rad 17 (2010) 374.[11] D. Bazin, M. Daudon, C. Chappard, J.-J. Rehr, D. Thiaudiere, S. Reguer,J. Synchrotron Rad 18 (2011) 912.[12] D. Bazin, A. Dessombz, C. Nguyen, H.-K. Ea, F. Liote, J. Rehr,C. Chappard, S. Rouziere, D. Thiaudiere, S. Reguer, M. Daudon, J.Synchrotron Rad 21 (2014) 136.[13] Pymca: http://pymca.sourceforge.net/.[14] O. Hignette, P. Cloetens, G. Rostaing, P. Bernard, C. Morawe, Rev. Sci.Instrum 76 (2005) 063709.[15] O. Hignette, P. Cloetens, C. Morawe, C. Borel, W. Ludwig, P. Bernard,A. Rommeveaux, S. Bohic, AIP Conf. Proc. 879 (2007) 792.

How to assess the role of Pt and Zn in the nephrotoxicity of Pt anti-cancer drugs?: An investigation combining μXRF and statistical analysis. Part II: Clinical application

Emmanuel Estève

Dominique Thiaudière

Éric Rondeau

How to assess the role of Pt and Zn in the nephrotoxicity of Pt anti-cancer drugs? An investigation combining μXRF and statistical analysis: Part I: On mice

HAL - UPEC / UPEM

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How to assess the role of Pt and Zn in the nephrotoxicity of Pt anti-cancer drugs?: An investigation combining μXRF and statistical analysis. Part II: Clinical application

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