clinical investigation see commentary on page 1226

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1 clinicl investigtion 2 Interntionl Society of Nephrology see commentry on pge 1226 Peripherl nturl killer cell nd llo-stimulted T-cell function in kidney trnsplnt recipients ssocite with cncer risk nd immunosuppression-relted complictions Christopher M. Hope 1,2, Alexnder Troelnikov 1,2, Willim Hnf 1, Shilpnjli Jesudson 1,2, Ptrick T. Cotes 1,2, Peter S. Heeger 3 nd Roert P. Crroll 1,2 1 Centre for Clinicl nd Experimentl Trnsplnttion (CCET), Centrl Northern Adelide Renl nd Trnsplnttion Services (CNARTS), Royl Adelide Hospitl, Adelide, SA, Austrli; 2 Deprtment of Medicine, University of Adelide, Adelide, SA, Austrli nd 3 Deprtment of Medicine, Trnsltionl Trnsplnt Reserch Center, Ichn School of Medicine t Mount Sini, New York, New York, USA Reducing immunosuppression hs een proposed s mens of preventing cncer in kidney trnsplnt recipients ut this cn precipitte grft rejection. Here we tested whether nti-tumor nturl killer (NK) cell nd llo-responsive T-cell function in kidney trnsplnt recipients my predict cncer risk nd define risk of rejection. NK cell function ws mesured y the relese of lctte dehydrogense nd T-cell llo-response y interferon-γ quntifiction using pnel of rective T-cell enzyme-linked immunospot (ELISPOT) in 6 kidney trnsplnt recipients with current or pst cncer nd 26 kidney trnsplnt recipients without cncer. NK function ws significntly impired nd the llo-response ws significntly lower in kidney trnsplnt recipients with cncer. With prospective follow-up, kidney trnsplnt recipients with poor NK cell function hd hzrd rtio of 2.1 (9% confidence intervl.97.) for the comined end point of metsttic cncer, cncer-relted deth, or septic deth. Kidney trnsplnt recipients with low interferonγ relese were lso more likely to rech this comined end point. Thus, posttrnsplnt monitoring of llo-immunity nd NK cell function is useful for ssessing the risk of over immunosuppression for the development of mlignncy nd/or deth from cncer or sepsis. Kidney Interntionl (2) 88, ; doi:1.138/ki.2.237; pulished online 12 August 2 KEYWORDS: cncer; IFN-γ ELISPOT; immunosuppression; kidney trnsplnttion; NK cells Correspondence: Roert P. Crroll, Centre for Clinicl nd Experimentl Trnsplnttion (CCET), Centrl Northern Adelide Renl nd Trnsplnttion Services (CNARTS), Royl Adelide Hospitl, Level 9, Est Wing, Adelide, SA, Austrli. E-mil: roert.crroll@helth.s.gov.u Received 9 Ferury 2; revised June 2; ccepted 12 June 2; pulished online 12 August 2 Kidney trnsplnt recipients (KTR) treted with immune suppressnts hve 3- to -fold incresed risk of developing solid orgn cncers nd 6- to 2-fold incresed risk of developing squmous cell crcinom (SCC) over the generl popultion. 1 6 Mlignncies tht develop in KTR tking immune suppressnts re more ggressive, more likely to recur, nd hve poorer prognoses compred with cncers tht occur in immune-competent sujects. 2,3,7 In ddition, KTRs who develop multiple SCCs re t incresed risk of developing susequent de novo solid orgn cncer compred with KTRs who never develop SCCs. 8 The known ssocition etween the risk of mlignncy nd chronic immunosuppression in KTR hve resulted in ttempts to reduce immunosuppressive drugs, s guidelines suggest. 9 This includes reduction in clcineurin inhiitor (CNI) levels nd/or conversion to drug regimens contining mmmlin trget of rpmycin inhiitors, the ltter hving direct nti-tumor effects Although some studies hve indicted tht immunosuppression reduction is ssocited with reduced incidence of cncer in KTRs, 1 12 findings do not uniformly support protective effect nd immunosuppression withdrwl cn increse the risk of developing cute cellulr nd/or ntiody-medited rejection. 11,12 As consequence, identifying nd vlidting iomrkers cple of ccurtely delineting risk of rejection nd mlignncy in KTRs hs ecome priority. With such iomrkers in hnd, it would e possile to individulize immunosuppression so s to limit nti-donor llo-immunity sufficiently to prevent rejection, while simultneously leving sufficient nti-tumor immunity intct so s to prevent development of posttrnsplnt mlignncy. Mesurement of cellulr llo-immunity in KTRs hs the potentil to inform clinicins sout risks of rejection nd mlignncy. Previous studies hve shown the development of pnel of rective T cell (PRT) interferon-γ (IFN-γ) enzymelinked immunospot (ELISPOT) ssy, which quntifies the frequency of primed/memory T cells in the peripherl lood 1374 Kidney Interntionl (2) 88,

2 CM Hope et l.: Cncer nd immune function in trnsplnttion clinicl investigtion rective to pnel of llogeneic stimultor cells expressing multiple humn leukocyte ntigen (HLA) molecules. Evidence indictes tht positive PRT ELISPOT results identify pre-trnsplnted KTR t risk for developing cute cellulr rejection.,16 Humorl-medited rejection risk cn e determined y mesuring levels of de novo donor-specific ntiodies (DSAs), 17 which trget donor HLA, nd together these ssys my provide enough evidence to determine how strongly primed the immune system is nd how high rejection risk is, efore drug mnipultions in KTRs with cncer. As ccumulting evidence suggests tht nturl killer (NK) cells confer nti-tumor immunity, we postulted tht one potentilly informtive iomrker for ssessing risk of developing posttrnsplnt mlignncy is mesurement of the immune function of NK cells. Although NK cell numers in KTRs with mlignncy hve een investigted, 18,19 NK cell function hs not een extensively studied in KTRs. 2 The mesurement of lctte dehydrogense relesed from lysed NK-sensitive myelogenous leukemi cell line K62 cn e used to determine NK function 21 nd my provide evidence of KTR s ility to cler mlignncy. As first step towrd developing multifceted monitoring pproch for lncing risks of rejection nd mlignncy, we performed cross-sectionl oservtionl study using cohort of KTR with nd without mlignncies using PRT ELISPOT, DSA, nd functionl NK cell ssys. Our results support the hypothesis tht these ssys hve the potentil to guide drug mnipultion tilored to the trnsplnt recipient s immune iologicl function rther thn sed solely on drug levels. RESULTS In n effort to identify iomrkers tht differentite KTRs with nd without mlignncy, we compred immune prmeters including DSA, NK cell function, nd primed T-cell llo-immunity in 82 KTRs: 31 KTRs with current cncer, 2 KTRs with pst cncer, nd 26 control KTRs (without current or pst cncers). The ge nd durtion of immunosuppression were greter in KTRs with pst cncer thn other KTRs (Tle 1). However, immunosuppressive drug doses nd serum levels did not differ mong the groups nor did gender, serum cretinine, rejection episodes, multiple grfts, or NK cell numer (Tle 1). Fewer KTRs with cncer were dministered mycophenolte- or Tle 1 Cohort demogrphics nd clinicl prmeters Pst cncer P-vlue Numers (N) Age, medin (rnge) 63 (43 7) 6 (44 73) 8 (39 76).44 Mle gender, N (%) 24(77) 19(76) 16(62).37 Yers immunosuppressed, medin (rnge) 9(4 3) 14 (4 3) 7(2 31).6 Serum cretinine, medin (rnge) 117 (66 33) 123 (6 22) 18 (1 324).983 egfr, medin (rnge) ml/min/1.73 m 2 6 (2 6) 44 ( 6) ( 6).63 Presence of proteinuri 2(7) 8(32) 8(31).2 Rejection episode(s), N (%) (16) 3 (12) 9 (3).1 Multiple grfts, N (%) (16) 2 (8) (19).3 DSA 4 MFI, N (%) (16) 3 (12) 7 (27).38 Biopsy-proven trnsplnt glomerulopthy 3 (1) (2) 4 ().46 NK cells/μl of lood, medin (rnge) 97 (1 19) 3 (23 377) 86 (4 242).34 Immunosuppressive drug regimen Azthioprine, N (%) 7 (23) (2) 3 (12).42 Mycophenolte, N (%) 13 (42) 16 (64) 22 (8).4 Clcineurin inhiitors, N (%) (48) 13(2) 2(77).68 mtori, N (%) 8 (26) 4 (16) 3 (12).38 Prednisolone, N (%) 27(87) 2(8) 22(8).7 Triple therpy, N (%) 12(39) 11(44) 18(69).6 Immunosuppressive drug dose Azthioprine, medin (rnge), mg 1 (12. 1) 2 (2 1) 1 ( 1).16 Mycophenolte, medin (rnge), g 1 (. 1.) 1 (1 2) 1.2 (. 2).162 Cyclosporine, medin (rnge), mg (, ) 1 ( 2) 12 (8 2).8 Tcrolimus, medin (rnge), mg 4 (1 14) 4 (3 8) 4 (1. 8).92 mtori, medin (rnge), mg 2 (1. 3) 3 (1. 3) 2 (1. 4).67 Prednisolone, medin (rnge), mg (2. 1) (4 1) ( 1).241 Immunosuppressive drug levels Tcrolimus, medin (rnge), μg/l. (2 11) 4 (3 13) 7 (3 13).813 mtori, medin (rnge), μg/l 7. (3 ) 6 ( 13) 2 (1. 4).69 Arevitions: DSA, donor-specific ntiody; egfr, estimted glomerulr filtrtion rte; MFI, men fluorescence intensity; mtori, mmmlin trget of rpmycin inhiitor; NK, nturl killer. Underlined vlues indicte Po.. Kidney Interntionl (2) 88,

3 clinicl investigtion CM Hope et l.: Cncer nd immune function in trnsplnttion CNI-sed regimens compred with KTRs with no cncer (Tle 1). DSA levels in KTRs with cncer KTRs with nd without cncer hd similr frequencies of DSA (positively defined s men fluorescence intensity 4 (refs 17, 22)) s seen in Tle 1. Altering the threshold for defining positive DSA (men fluorescence intensity of 3) did not lter the results (dt not shown). NK cell function in KTRs with cncer To ssess whether the presence of cncer is ssocited with differences in NK cell function, sufficient peripherl lood mononucler cells (PBMCs) were ville in 62/82 (76%) KTRs nd these were tested for their ility to lyse K62 cells. Ptients were dichotomized s either high or low NK-medited lysis y using the medin lysis vlue for the ssy. KTRs with current cncer hd lrger proportion of KTRs with low (omedin lysis) NK cell function compred with KTRs with pst or no cncer (Figure 1, P =.14). When ge nd durtion of immunosuppression were divided into qurtiles, there ws no ssocition with NK lysis high or low ctegoriztion (P =.84 nd P = 1., respectively). NK cells numers in peripherl lood were no different etween KTR groups (Tle 1). Predictive vlue of NK cells in KTRs with cncer Over the prospective period of medin (rnge) = 311 ( ) dys, there were 22 KTRs who developed recurrent (42) SCC skin, 4 who hd progressive solid orgn cncer, 1 who died of metsttic cncer, nd who died of sepsis. A Kpln Meier survivl curve for the development of metsttic cncer or cncer/septic deth ws generted from 41 KTRs with current nd pst cncer (Figure 1). Low NK cell function incresed the risk of these immunosuppressiverelted complictions with hzrd rtio (9% confidence intervl) = 2.1 (.97.), P =.63. Primed T-cell llo-immunity in KTRs with cncer Sufficient T cells were ville to mesure nd compre cellulr llo-immunity in 77/82 (94%) KTRs with nd without mlignncy. Recipient PBMCs were stimulted with pnel of llogeneic B cells in n IFN-γ PRT ELISPOT pltform s performed y the Clinicl Trils In Orgn Trnsplnttion consortium. 23 Stimultors comprise 4 individul B-cell lines tht expressed disprte clss I nd II HLA ntigens. The ssys showed lower frequencies of llo-rective IFN-γ producers in KTRs with current nd pst mlignncy compred with KTRs without cncer (Figure 2). The difference etween groups ws significntly greter thn the intr-ssy coefficient of vrition (31%), indicting true differences etween groups. When ge nd durtion of immunosuppression were divided into qurtiles, there ws no correltion with PRT result (P =.39 nd.6, respectively). Predictive vlue of llo-immunity in KTRs with cncer To exmine whether the PRT ELISPOT predicted immunosuppression-relted clinicl events (metsttic cncer, nd cncer or septic deth), we ssessed whether these events occurred in the KTRs with current nd pst cncer (n = 3) for medin (rnge) 33 (8 1712) dys of event censored follow-up (the very short follow-up (8 dys) ws ptient who died of metsttic cncer). A receiver opertor chrcteristic curve ws constructed to determine whether the PRT ELISPOT ssy could identify KTRs who developed recurrent cncer, mlignnt, or septic deth (Figure 2) in those KTRs who hd current or pst cncer on the smple dte. The receiver opertor chrcteristic curve hd n ccurcy of 69% (re under the curve =.69, P =.2) with sensitivity of 3% nd specificity of 79% t trde-off vlue of 28 spots/3 1 PBMCs (spots, Figure 2). A Kpln Meier survivl curve for recurrent cncer, mlignnt, or septic deth in KTRs who hd current or pst cncer ws generted sed on this cutoff vlue (Figure 2c). KTRs who 2 1 (n =22) High NK function Low NK function Pst cncer (n =19) (n =21) Ptient event-free survivl High NK function Low NK function Time (dys) Figure 1 Dichotomized nturl killer (NK) cell function in kidney trnsplnt recipients (KTRs). The depicted NK cell function is dichotomized on the medin mount of lctte dehydrogense (LDH) relesed when KTR peripherl lood mononucler cells (PBMCs) re co-cultured with K62 t rtio of 2:1, high NK function (lck)/low NK function (gry). () Br grph of 62 KTRs. The 22 KTRs with current cncer hve greter proportion of KTRs with low NK cell function (68%) compred with the 19 KTRs with pst cncer (26%) nd 21 KTRs with no cncer (33%) with χ 2 -test P-vlue of.14. () A Kpln Meier survivl curve depicting the dichotomized dt of the 41 KTRs with current nd pst cncer, developing metsttic cncer, or dying of immunosuppressive-relted diseses (i.e., cncer or sepsis) over dys. KTRs with low NK cell function (gry line) hve lower survivl proportions t 36 dys (32% vs. 71%) nd lower overll survivl with hzrd rtio (9% confidence intervl) = 2.1 (.97.), log-rnk P =.63, when compred with KTRs with high NK cell function (lck line) Kidney Interntionl (2) 88,

4 CM Hope et l.: Cncer nd immune function in trnsplnttion clinicl investigtion Spots per 3 1 PBMCs P<.1 Sensitivity% (n = 29) Pst cncer (n = 24) (n = 24) % Specificity% c 1 Ptient event-free survivl 7 2 >28 spots <28 spots Time (dys) Figure 2 Allo-stimulted interferon-γ (IFN-γ) relese from kidney trnsplnt recipients (KTRs). Allo-response ws mesured y co-culturing 1 1 llogeneic B cells with 3 1 peripherl lood mononucler cells (PBMCs) from KTRs nd quntitting IFN-γ relesed from cells (spots) vi enzyme-linked immunospot (ELISPOT). The intr-ssy coefficient of vrition (C.V.) ws determined to e 31%. () Box plot representing medin (rnge) numers of spots from 77 KTRs. The KTRs with current (n = 29) nd pst (n = 24) cncer, oth hve decresed IFN-γ llo-responses when compred with KTRs with no cncer (n = 24, Po.1) using Kruskl Wllis non-prmetric test. () A receiver opertor chrcteristic (ROC) curve for the 3 KTRs who hd current or pst cncer, who went on to developed metsttic cncer or died of immunosuppressive drug-relted diseses (i.e., cncer or sepsis) over the medin (rnge) of dys followed = 33 (8 1712). The ROC hs n ccurcy of 69% (P =.2) with trde-off of 28 spots per 3 1 PBMC (spots) leding to sensitivity of 3% nd specificity of 79%. The 28 spots trde-off ws identified using single vlue tht hd oth the highest likelihood rtio nd Youden s index. (c) A Kpln Meier survivl curve for metsttic cncer or immunosuppressive-relted deth (cncer or sepsis)-free dys dichotomized on the trde-off vlue of 28 spots, over dys. KTRs with o28 spots (gry line) hve lower survivl rtes (23% vs. 61%, t dys), log-rnk test P =., when compred to KTRs with 428 spots (lck line). hd current or pst cncer nd with low llo-rectivity reched this comined end point two times quicker thn those with high llo-rectivity (Figure 2), i.e. in posttrnsplnt setting, low llo-response is ssocited with significntly higher risk of recurrent cncer, mlignnt, or septic deth in KTRs with ny history of cncer. Determining ssocition of immunosuppressive regimen to either immunologicl ssy Irrespective of the comintion of drugs used in drug regimen, there were no effects on NK function or PRT results (Figures 3 nd 4). The determining fctor in these ssys ws the presence of cncer in the recipient t the time of the ssy. As KTRs with pst cncer hd similr NK function profiles to those with no cncer (Figure 2), these two groups were comined for the nlysis of the effect of immunosuppressive regimens. With regrd to NK function (Figure 3), current cncer ptients were more likely to hve low NK function irrespective of numers of drugs in the regimen, the presence or the sence of mycophenolte mofetil (MMF), or the presence or the sence of CNI. Conversely, ptients with no current cncer were more likely to hve high NK function irrespective of the comintion of the drugs regimen. The proportion of KTR with impired NK function ws higher in those with current cncer compred with no current cncer for KTRs on triple therpy or MMF-sed regimens, P =.73 nd P =.29, respectively, χ 2 -test (Figure 3). When compring MMF presence or sence in the regimen, there ws only sttisticlly significnt difference in proportions in those on MMF y cncer sttus (P =.21). When compring the effect of CNI on NK function, there were no sttisticlly significnt differences etween the groups, ut KTRs with cncer were more likely to hve impired NK function (Figure 3c). With regrds to llo-response (Figure 4), current cncer ptients hd lower PRT responses irrespective of the drug regimen compred with KTRs with no current cncer. The differences were more pronounced in those on triple therpy (P =.1), those tking MMF (P =.14), or those tking CNI (P =.14). Kidney Interntionl (2) 88,

5 clinicl investigtion CM Hope et l.: Cncer nd immune function in trnsplnttion 2 High NK function Low NK function drugs (n =6) drugs (n =16) + 3 drugs (n =24) drugs (n =16) + MMF (n =7) MMF (n =) + MMF (n =29) MMF (n =11) c CNI (n =1) CNI (n =12) + CNI (n =27) CNI (n =13) Figure 3 Immunosuppressive drug regimen nd cncer ssocitions to nturl killer (NK) cell function in kidney trnsplnt recipients (KTRs). A pnel of r plots depicting NK cell function dichotomized on the medin NK cell function of 62 KTRs, with vrious drug regimens nd with either current cncer (Cncer) or pst/no cncer (No Cncer). () KTRs on triple therpy (three drugs) do not differ from their one to two drug counterprts. However, KTRs with cncer hve greter proportion of KTRs with low NK cell function thn the KTR with no cncer when on 1-2 drugs (63 vs. 19%, P =.29) ut does not rech sttisticl significnce when on three drugs (83% vs. 3%, P =.73). () KTRs on mycophenolte (MMF)-sed regimens do not differ from their non-mmf-sed counterprts. However, there ws greter proportion of KTRs with cncer tht hve low NK function compred with KTRs with no cncer, which is only sttisticlly significnt when KTRs were not on MMFsed regimens (67% vs. 18%, P =.21). (c) KTRs on clcineurin inhiitor (CNI)-sed regimens do not differ from their non-cni counterprts. Although KTR with cncer on CNI nd not on CNI hve greter proportion of KTR with low NK function (7% nd 67%, respectively), these do not sttisticlly differ from their non-cncer counterprts (3% nd 36%, respectively) with (P =.6 nd P =.116, respectively). All sttisticl nlysis performed using Fisher s exct tests. Determining ssocitions tht grft function hs with oth immunologicl ssys There were three grft filures nd two iopsy dignoses of trnsplnt glomerulopthy. These ptients lso developed proteinuri nd were found to hve HLA DSAs. All hd PRT428 spots, medin (rnge) = 7 (3 11), nd 4/ (8%) hd high NK function. Proteinuri ws lso ssocited with higher NK function (Figure ), ut we did not find n ssocition with estimted glomerulr filtrtion rte (Figure ). Serum cretinine, estimted glomerulr filtrtion rte (Modifiction of Diet in Renl Disese formul), previous rejection episodes, nd multiple trnsplnts were not different etween the KTR groups. However, the presence of proteinuri 4 mg/dy ws less common in those with current cncer compred with pst cncer nd no cncer groups (P =.29). Biopsy-proven trnsplnt glomerulopthy ws t similr prevlence of 1% 2% etween groups (Tle 1). Ctegorized estimted glomerulr filtrtion rte did not ssocite to NK cell function (P =.276, Figure ) or lloresponse (P =.86, Figure c). The presence of proteinuri ws ssocited with higher NK cell function (P =.26, Figure ) ut not llo-responses (P =.473, Figure d). DISCUSSION This study provides evidence tht posttrnsplnt monitoring of llo-immunity nd NK cell function is useful for ssessing risk of over immunosuppression s mnifested y the development of mlignncy nd/or deth from cncer or sepsis. Mesures of llo-immunity nd NK cell function my e used to tilor immunosuppressive drugs to reduce cncer incidence, s reduction of immunosuppression hs een 1378 Kidney Interntionl (2) 88,

6 CM Hope et l.: Cncer nd immune function in trnsplnttion clinicl investigtion P=.722 P=.34 P= Spots per 3 1 PBMCs P=.1 Spots per 3 1 PBMCs drugs (n =1) drugs (n =19) + 3 drugs (n =26) drugs (n = 22) + MMF (n =12) MMF (n =17) + MMF (n =36) MMF (n = 12) c P=.83 Spots per 3 1 PBMCs P=.14 + CNI (n =13) CNI (n =16) + CNI (n =32) CNI (n = 16) Figure 4 Immunosuppressive drug regimen nd cncer ssocitions to llo-stimulted interferon-γ (IFN-γ) relese in kidney trnsplnt recipients (KTRs). A pnel of ox plots depicting medin (rnge) of llo-stimulted IFN-γ relese from 77 KTRs with vrious drug regimens nd with either current cncer (Cncer) or pst/no cncer (No Cncer). () IFN-γ relese from KTRs on triple therpy (three drugs) does not differ from their one to two drug counterprts. However, KTRs with cncer hd less IFN-γ relese thn the KTRs with no cncer for those on three drugs (P =.1). () KTRs on mycophenolte (MMF)-sed regimens do not differ from their non-mmf-sed counterprts. However, KTRs with cncer hve lower IFN-γ relese thn KTRs with no cncer when on MMF-sed regimens (P =.14). (c) KTRs on clcineurin inhiitor (CNI)- sed regimens do not differ from their non-cni counterprts. However, KTRs with cncer hve less IFN-γ relese thn the KTR with no cncer for those on CNI-sed regimens (P =.14). All sttisticl nlysis performed using Mnn Whitney tests. shown to reduce cncer previously Dntl et l. 11 showed dose reduction ws effective in rndomized controlled tril setting, ut the rejection rte ws reltively high. Importntly, the study ws performed in the 199s, efore the routine use of solid-phse ntiody detection nd the immunologicl ssys descried here, nd therefore it ws not known whether these KTRs with rejection hd preexisting llo-ntiodies, high PRT ELISPOT, or high NK function vlues. An lterntive therpy to immunosuppressive drug reduction is conversion to mmmlin trget of rpmycin inhiitor, which hs een shown to e effective in primry nd secondry prevention of cncer; 27,28 however, this clss of drug is frequently not well tolerted nd my e ssocited with the development of DSA in steroid-free regimens. 29 Therefore, it is importnt to develop pproprite tools to select ptients in whom it would e sfe to undertke dose reduction for cncer prevention. Within this study, KTRs with cncer were older nd hd incresed durtion of immunosuppression. These oservtions re not unexpected, s oth ge nd durtion of immunosuppression re known risk fctors for mlignncy. KTRs with cncer were less likely to e on MMF- nd CNIsed regimens. However, we were not le to demonstrte tht ny of these vriles hd n effect on the ssys over nd ove the presence or the sence of cncer in the ptients. The PRT ELISPOT nd NK function ssys did not relte to the stge or grde of the cncers nd, most importntly, were not ffected y drug regimen (Figures 3 nd 4) or durtion of immunosuppression. However, low PRT ELISPOT (o28 spots) vlues nd low NK function predicted metsttic cncer, cncer deth, nd septic deth (Figures 1 nd 2), nd it is this ssocition with outcome tht is surrogte mesure of disese severity. Although the levels of sttisticl significnce were P =. nd P =.63, oth PRT ELISPOT nd NK function ssy predicted douling of the risk of significnt immunosuppression-relted dverse events with prospective follow-up. If replicted in susequent studies, these ssys could ecome helpful clinicl risk ssessment nd mngement tool. The chnges in immune function documented in this study re not relted to immunosuppressive therpy ut rise the possiility tht mlignncy itself is contriuting to Kidney Interntionl (2) 88,

7 clinicl investigtion CM Hope et l.: Cncer nd immune function in trnsplnttion 2 High NK function 2 P =.26 1 Low NK function (n =13) 3 4 (n =1) 4 6 (n =9) egfr (ml/min/1.73 m 2 ) >6 (n =3) Positive proteinuri (n =13) Negtive proteinuri (n =44) c d P =.473 Spots per 3 1 PBMCs Spots per 3 1 PBMC (n = ) 3 4 (n = 14) 4 6 (n = 13) >6 (n = 3) Positive proteinuri (n =18) Negtive proteinuri (n =3) egfr (ml/min/1.73 m 2 ) Figure Associtions of kidney grft function nd proteinuri (4 mg/dy) to nturl killer (NK) cell function nd llo-stimulted interferon-γ (IFN-γ) relese in kidney trnsplnt recipients (KTRs). ( nd ) Br grphs depicting proportions of KTRs with high (lck) nd low (gry) NK cell function dichotomized on medin NK cell function. () Br grph of 62 KTRs. Estimted glomerulr filtrtion rte (egfr) does not ssocite to dichotomized NK cell function (P =.276), using χ 2 -nlysis. () Br grph depicting 7 KTRs. There is lrger proportion of KTRs with high NK function (8%), with proteinuri (4 mg/dy) compred with KTR with lower or no detectle proteinuri (48%, P =.26) using Fisher s exct test. (c nd d) Box plots depicting the medin (rnge) of llo-stimulted IFN-γ (IFN-γ) relese (Spots) from 3 1 peripherl lood mononucler cells (PBMCs) mesured y enzyme-linked immunospot (ELISPOT) in KTRs. (c) For 77 KTRs tested, there re no differences etween the different groups of egfr nd mount of IFN-γ relese (P =.86), using Kruskl Wllis test. (d) Out of 71 KTRs, there is no difference etween those KTRs with proteinuri (n = 18) nd those without (n = 3, P =.473), using Mnn Whitney test. immunosuppression. A vriety of cncers elorte immunologicl suppressive fctors (i.e., regultory T cells or cytokines such s trnsforming growth fctor-β nd interleukin-1) to evde immune detection This my e nlogous to chnges in immune phenotype (regultory T cells nd NK cells) previously documented in KTRs with cncer resection. 19 It is possile tht high PRT ELISPOT vlue is mrker of immune ctivtion nd this my e protective for mlignncy. There is lso evidence tht high PRT ELISPOT vlues determine risk of cell-medited rejection nd renl function. 14, However, our dt show tht KTRs with current nd pst cncer hd similr mesures of grft function to KTRs with no cncer ut with lower PRT ELISPOT vlues. These current nd pst cncer ptients with PRT o28 spots were t significnt risk of developing complictions of immunosuppression. Therefore, we propose tht the IFN-γ ELISPOT llo-response mesures oth risk of cncer nd nti-rejection immunity. Innte NK cell function is required for effective nticncer immunity ut hs lso een implicted in cute nd chronic rejection Although the design of this study initilly excluded ptients with unstle grft function nd could hve ised the results, there were still ptients who developed trnsplnt glomerulopthy or grft filure with prospective follow-up nd ll hd PRT 428 spots nd 8% hd high NK function, nd ll were found to hve DSA on repet testing. Dose reduction for mlignncy in ptients with these immune profiles my therefore precipitte rejection. In conclusion, this is the first study to show tht there re immune function prmeters tht could e used to risk strtify ptients for significnt dverse events relted to immunosuppression. Further prospective studies should e performed to determine the utility of these ssys in the mngement of immunosuppression in KTRs. MATERIALS AND METHODS Internl review ord pprovl ws otined from the Royl Adelide Hospitl Reserch Ethics Committee (protocol numer 9) nd the study ws performed in ccordnce with 138 Kidney Interntionl (2) 88,

8 CM Hope et l.: Cncer nd immune function in trnsplnttion clinicl investigtion Strengthening the Reporting of Oservtionl Studies nd Epidemiology guidelines. 36 Ptients nd study design All KTRs were pproched t mulnt outptient clinic visits t the Centrl nd Northern Adelide Renl nd Trnsplnt Centre etween July 29 nd August 214. The exclusion criteri were s follows: unstle grft function, current infection, o24 months posttrnsplnt, or could not give informed consent. There were two recruitment periods nd selection of ptients. The first recruitment period ws etween July 29 nd August 213 with smples eing frozen. The ssys were performed on these smples t rndom, no selection ws pplied. The second recruitment period ws etween Ferury 214 nd August 214. All KTRs with new dignosis of solid orgn cncer (n = 16) were pproched nd ll consented to enrol in the study t this time. Concurrent KTRs ttending routine weekly outptient clinic were pproched nd ll consented (n = 26). The cohort consisted of 17 with SCC, 9 with 42 SCC, 6 with metsttic SCC, 3 with loclly dvnced SCC lung, 2 with erlystge lung denocrcinom, 1 with metsttic renl crcinom, 2 with metsttic lung denocrcinom, 1 with metsttic lung cncer, 1 with melnom, 1 with metsttic cholngiocrcinom, 2 with erly-stge owel cncer, 4 with erly-stge genitourinry cncer, 1 erly-stge renl crcinom, 1 Dukes B colonic cncer, 4 with posttrnsplnt lymphoprolifertive disese nd 1 cererl lymphom. Ptient clinicl prmeters Grft function ws mesured y serum cretinine levels, estimted glomerulr filtrtion rte using the Modifiction of Diet in Renl Disese formul, proteinuri, nd previous rejection episodes proven y histologiclly ssessed iopsies. Other ptient chrcteristics including ge, gender, immunosuppression tretments, DSA, nd ptient history were collected from locl dtses. Blood collection nd PBMC seprtion Approximtely 3 ml of KTR lood ws collected y venesection in Lithium-Heprin vcuum tues nd processed within 2 h. Plsm ws collected from centrifuged lood (2 g,1min,no rke)nd stored t 8 C. The KTR PBMCs were seprted y Lymphoprep (Axis- Shield, Oslo, Norwy) centrifugtion techniques nd re-suspended in either RPMI 164 medium (Invitrogen, Grnd Islnd, NY) supplemented with 7% (v/v) fetl clf serum (Bovogen, Kielor Est, VIC, Austrli), 1 mmol/l L-glutmine (Sigm, São Pulo, Brzil), 1 mmol/l Penicillin (Commonwelth Serum Lortories, Prkville, VIC, Austrli)/1 mmol/l Gentmycin (Gico, Grnd Islnd, NY), nd 1% (v/v) dimethyl sulfoxide (VWR Interntionl, Fonteny-sous- Bois, Frnce) for cryopreservtion medi or resuspended in RPMI 164 supplemented with 2% (v/v) fetl clf serum, 1 mmol/l L-glutmine, 1 mmol/l Penicillin/Gentmycin, nd plced in % CO 2, 37 C incutor overnight. Vile cell concentrtions were determined vi Trypn-lue (BDH Chemicls, Poole, Englnd) exclusion ssy. DSA ssy The Lifecodes Tepnel ID, LSA1, nd LSA2 screen kits (Immucor, Norcross, GA) were used to determine nti-hla molecules. A men fluorescence intensity cutoff vlue of 4 units ws used. 17 DSA rectivity ws determined y investigting the PRA-positive KTR (ID kit) with single ntigen eds (LSA1 nd LSA2) kits. B-cell expnsion Helthy, HLA-typed B cells were isolted with EsySep B-cell enrichment kit s per the mnufcturer s protocol (StemCell Technologies, Vncouver, BC, Cnd) nd expnded s previously pulished. 37 Concisely, B cells were co-incuted with irrdited (43 Grys) NIH-3T3-CD4 firolsts (generously donted y Professor P.S. Heeger, Mount Sini, New York, NY), in the descried medi. Before cryopreservtion, the B cells were nlysed for mjor histocomptiility complex-i (HLA-ABC, clone: B9.12.1, BD Phrmingen, Sn Jose, CA) nd mjor histocomptiility complex-ii (HLA-DR, clone: L243, BD Bioscience, Sn Jose, CA) expression y flow cytometry nd superntnts exmined for Epstein-Brr virus RNA y PCR nlysis. PBMC/NK cell lctte dehydrogense relese ssy The KTR PBMCs were thwed or collected from incutor, wshed nd resuspended in AIM-V AluMAX serum-free medi (Invitrogen, Crlsd, CA) supplemented with 1 mmol/l L-glutmine. The coculture consisted of /ml (thwed) or /ml (fresh) PBMCs with (thwed) or (fresh) (2:1) K62 cells/ml or 1 μl medi, in triplicte, for 6 h. After the incution, lctte dehydrogense relese ws mesured using CytoTox 96 Non-Rdioctive Cytotoxicity Assy (Promeg, Fitcherg, WI) s per the mnufcturer s procedures. Pltes were red on Bio-rd Model 68 (Bio-Rd, Hercules, CA) microplte reder t 49 nm wvelength with Microplte Mnger v.1 softwre (Bio-Rd). PRTIFN-γ ELISPOT ssy Ptient PBMCs were thwed nd resuspend t /ml in AIM-V AluMAX serum-free medi supplemented with 1 mmol/l L-glutmine. The KTR responders were run in triplicte in medi, test wells, nd Phytohemgglutinin (Sigm-Aldrich, St Louis, MO) s positive control. The mixed lymphocyte rection consisted of 3 1 thwed KTR PBMCs plted on n nti IFN-γ ntiody (2G1, Thermo-Scientific, Wourn, MA)-coted ELISPOT plte (Millipore, Belleric, MA) co-incuted with four or five B-cell lines t 1 4 / per B-cell line, two B-cell lines per well (totl of 1 1 B cells/well), or with the fifth B-cell line t 1 1 B cells/well, for 24 h. These B-cell lines hd either 17 or 21 disprte ntigens, with the most similr cell lines pired. Next, the pltes were hndled s per previously pulished protocol, 23 with hlf the pltes wshed using n ELx Microplte strip wsher (BioTek, Winooski, VT). The imges were cptured with n ImmunoSpot S6 Micro Anlyzer (Cellulr Technology Ltd (CTL), Shker Heights, OH) with ImmunoCpture 6.4 softwre (CTL). The spots counted using CTL ImmunoSpot. (CTL) nd qulity controlled y inspecting every well nd djusting sensitivity. The IFN-γ ELISPOT pltes were nlysed t the Clinicl Trils of Orgn Trnsplnttion center in Mount Sini School of Medicine, New York. Sttisticl nlyses To compre non-normlly distriuted continuous vriles (e.g., trnsplnt durtion, mesures of grft function, drug doses nd levels, nd NK cell numer) from KTRs with current, pst, nd no cncer, multiple comprison Kruskl Wllis tests were employed. When compring ge, multi-comprison nlysis of vrince ws used. When compring IFN-γ Spots/3 1 PBMCs etween two KTR groups, Mnn Whitney U-tests were used. In ddition, when compring ctegoricl vriles (gender, rejection episodes, multiple trnsplnts, DSA, drug usge, nd high/low NK cell function), Kidney Interntionl (2) 88,

9 clinicl investigtion CM Hope et l.: Cncer nd immune function in trnsplnttion Person s χ 2 - or Fishers exct test were used s pproprite. Prospective llo-response ssy sensitivity nd specificity ws determined using receiver opertor chrcteristic curve. Trde-off ws determined y identifying single vlue tht hd oth the highest likelihood rtio nd Youden s index. This trde-off or medin NK cell function ws used to determine hzrd rtios, 9% confidence intervls, nd log-rnk P-vlues. All sttisticl nlyses were run in Grph-Pd Prism v6.2 (GrphPd Softwre, L Joll, CA). DISCLOSURE P.S.H. receives grnt funding from Alexion Phrmceuticls; the other uthors of this mnuscript hve no conflicts of interest to disclose. ACKNOWLEDGMENTS We thnk the Jcquot Reserch Estlishment Awrd from the Austrlin nd New Zelnd Society of Nephrology, the Mrk Cocks Awrd from Temlife Austrli, the Deprtment of Medicine of the University of Adelide, nd the Wlter & Dorothy Duncn Trust for providing funding. 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Inhiition of nturl killer cells results in cceptnce of crdic llogrfts in CD28-/- mice. Nt Med 21; 7: McNerney ME, Lee KM, Zhou P et l. Role of nturl killer cell susets in crdic llogrft rejection. Am J Trnsplnt 26; 6: von Elm E, Altmn DG, Egger M et l. The Strengthening the Reporting of Oservtionl Studies in Epidemiology (STROBE) sttement: guidelines for reporting oservtionl studies. Lncet 27; 37: Znd MS, Bose A, Vo T et l. A renewle source of donor cells for repetitive monitoring of T- nd B-cell llorectivity. Am J Trnsplnt 2; : Kidney Interntionl (2) 88,