Abhishek Sharma
email: abhishek.sharma@unt.edu

Bharat Gwalani

Sriswaroop Dasari

Deep Choudhuri

Yao-Jen Chang

Stéphane Gorsse

An-Chou Yeh

Rajarshi Banerjee
email: raj.banerjee@unt.edu

Stephane Gorsse

Insights into defect-mediated nucleation of equilibrium B2 phase in face-centered cubic high-entropy alloys

Keywords: Plastic deformation, defect structures, dislocations, B2, High Entropy Alloys, Complex concentrated Alloys, Heterogeneous Microstructures, Metastable, Intermetallic

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction:

Plastic deformation can result in a high density of crystalline defects in the form of boundaries, dislocations, and vacancies. These defects serve as high energy sites for initiating and accelerating phase transformations. Such defect mediated heterogenous nucleation is an essential microstructural design tool in the context of High Entropy Alloys (HEAs) or Complex Concentrated Alloys (CCAs) for tuning their mechanical properties [1][2][3]. The significance of heterogeneous nucleation is specifically escalated in the case of multicomponent HEAs/CCAs, as most of these alloys are associated with complex phase fields comprising of multiple equilibrium phases [4,5], The nucleation and growth of these phases, is influenced by competing processes such as recovery and recrystallization, typically associated with the aforementioned defect structures. Intentional introduction of defects can serve to increase such nucleation sites but can also impede the motion of mobile interphase boundaries (and hence, the growth of new phase domains). In such a scenario, the thermal activation results in a competition between defect annihilation by precipitation or by recrystallization which may also result in formation of nonequilibrium or metastable phases. Defects such as twins, sub grain boundaries, deformation bands, and dislocation tangles that are introduced during cold deformation/rolling act as potent sites for heterogenous nucleation due to the inherently high energy associated with them. Therefore, annealing after cold deformation can lead to heterogenous microstructures due to inhomogeneous rates of recrystallization and precipitation of equilibrium/non-equilibrium phases. Multiple reports exist highlighting the microstructural modification and mechanical properties of such heterogeneous microstructures created by following this approach [6][7][8].

HEA/CCAs offer a variety of phase transformation pathways leading to novel microstructures containing intermetallic phases such as L12, B2, , laves and  phases [9][10][11][12]. For example, in case of an FCC based Al0.2Ti0.3Co1.5CrFeNi1.5 alloy, it has been observed that precipitation annealing directly after cold rolling resulted in a discontinuously precipitated nano-lamellar/nanorod type microstructure consisting of L12 and FCC phases [8]. This is in contrast to the classical +' type microstructure comprising of spherical L12 precipitates which results from solutionizing prior to annealing after cold rolling and is a result of homogeneous precipitation of L12 phase within the FCC matrix. The microstructure resulting from such defect mediated discontinuous precipitation leads to improved mechanical properties as well. Similarly, in the case of Al0.3CoFeNi, solutionizing after cold rolling followed by annealing at 600°C, leads to eutectoid decomposition into (FCC+L12) and (BCC+B2) lamellae whereas direct annealing at 700°C followed by 600°C after cold rolling, results in ultrafine grained microstructure of FCC grains containing L12 precipitates, and BCC grains with B2 precipitates [13]. This also results in improved yield strength along with reasonable ductility.

For the case of an FCC based HEA, when the low temperature phase field comprises of FCC+BCC/B2, the classic nucleation of BCC/B2 based phase is difficult, owing to a high barrier to its nucleation in an FCC matrix. The presence of heterogeneous nucleation sites in this scenario, such as defect structures, can provide interesting possibilities for microstructural designing. The present paper demonstrates this by presenting results from three such FCC based HEAs where homogeneous nucleation of a BCC/B2 phase is energetically unfavorable but possible when mediated by high density of defect structures. The rejection of solute from a supersaturated FCC matrix via such heterogenous nucleation is additionally in competition with nucleation of metastable phases with a lower energy barrier. Thus, interesting possibilities in terms of microstructural design exist, based on the degree and extent of prior processing, choice of annealing temperature and the corresponding equilibrium phase field as well as the thermodynamic and kinetic barrier to nucleation of the related metastable phases. Fairly impressive mechanical properties often ensue with such a heterogenous microstructure resulting from defect mediated nucleation. We propose this route as a design template to microstructural modification extendable to a larger class of precipitation strengthened multicomponent HEAs/CCAs with possible gains in mechanical properties.

Materials and Methods:

Alloys with the nominal chemical compositions Al0.3CoFeNi (9.1Al-30.3Co-30.3Fe-30.3Ni at.%), Al0.5Co1.5CrFeNi1.5 (9.1Al-27.3Co-18.1Cr-18.2Fe-27.3Ni at.%) and Al0.3CoCrFeNi (7.0Al-23.3Co-23.3Cr-23.2Fe-23.2Ni at.%) were prepared using a conventional vacuum arc melter. The alloys were flipped at least three times to ensure homogenization of the melt. Microstructural characterization was carried out using Scanning Electron Microscopy (FEI Nova-NanoSEM 230 TM / Hitachi SU8010 FE-SEM), fitted with energy dispersive spectroscopy (EDS) and electron backscattered diffraction (EBSD) detectors. TSL OIM TM software was used to analyze the EBSD data. Transmission Electron Microscopy (FEI Tecnai G2 TF20™ operating at 200 kV/ JEOL JEM-3000F HRTEM operating at 300 kV) was used to obtain crystallographic information from the phases present in the material. For analysis of compositional partitioning, Atom Probe Tomography (APT) was performed using Cameca LEAP 5000XS 3D Atom Probe Microscope operating at a temperature of 30K with pulse energy of 50nJ, pulse rate of 100 kHz, and a detection rate maintained at 0.005-0.01 atoms/pulse in laser mode. The specimens for TEM and APT were prepared using a FEI Nova 200 dual beam focused ion beam (FIB). The heat-treatments of the respective alloys have been described in the individual sections. All the annealing treatments were performed in encapsulated quartz tubes backfilled with argon. The samples for microstructural analysis were machined using Kent wire saw submerged EDM. Thermodynamic predictions were performed using ThermoCalc and the TCHEA4 database.

Results and Discussions:

To study the defect mediated nucleation of B2 phase, we have selected compositions based on the alloy CoFeNi, which is nearly a single phase random solid solution [14,15]. Previous reports have observed that addition of aluminum to this system promotes ordering tendency and hence the formation of intermetallic precipitates [16][17][18][19][20][21][22][23]. Therefore, such aluminum bearing CoFeNi alloys are ideally suited to study the defect mediated nucleation of precipitating equilibrium phases. The pseudo-binary isopleths generated using CALPHAD, for three such systems, namely AlxCoFeNi, AlxCo1.5CrFeNi1.5 and AlxCoCrFeNi, are reproduced in Figure 1(a) which is an overlapping plot of the relevant sections from the three systems. From the plot, it is clear that the FCC+B2 phase field expands with these alloying additions which clearly established the effect of aluminum in promoting the formation of intermetallic phases. Based on such optimization of aluminum addition in these systems, three previously reported alloy compositions having high temperature FCC single phase-field coupled with an intermediate temperature (FCC+B2) phase-field, were selected for this study: Al0.3CoFeNi [6], Al0.5Co1.5CrFeNi1.5 [24] and Al0.3CoCrFeNi [25]. The corresponding phase fractions as a function of temperature are provided in Figures 1(b), (c) and(d).

To understand the heterogenous nucleation of B2 phase in contrast with its homogenous nucleation, two different processing routes were employed. The first route comprised of homogenizing and cold rolling the cast ingots followed by solutionizing within the respective single-phase FCC phase-fields. These solutionized ingots were then aged within the two-phase FCC+B2 phase fields to promote homogenous nucleation of B2 phase. The second route consisted of cold rolling the homogenized ingot to introduce high density and variety of defects. These defects provide favorable sites for heterogenous nucleation of B2 phase. The cold-rolled samples were then aged within the respective FCC+B2 phase fields. Additionally, depending on the composition, in some cases, metastable phases nucleate homogeneously during either of the processing routes. The resultant microstructures from the two routes are finally compared.

1) Promoting heterogenous nucleation of equilibrium B2 phase in Al0.3CoFeNi

Figure 1(a) shows a pseudo-binary isopleth for AlxCoFeNi, with the composition of Al0.3CoFeNi marked by a vertical line. Accordingly, the phase equilibrium at 1250°C is FCC, at 700°C is When this solutionized alloy is further annealed at 700°C (CRSA-700 condition), the microstructure consists of coarse FCC grains with fine B2 grains precipitating along the FCC grain boundaries. This is shown in Figure 2(a) which is a BSE contrast SEM image. Figure 2(b) is a phase map from a triple-junction, generated from EBSD, clearly showing presence of B2 phase along grain boundaries of FCC grains. The L12 phase is absent which is also supported by the phase stability predicted from CALPHAD (Figures 1(a) and (b)). Although CALPHAD calculations predict a finite onset-driving force for homogeneous nucleation of B2 phase in FCC matrix at 700°C, which is shown in Supplementary Figure S1(c), owing to a higher energy barrier to homogenous nucleation within the FCC matrix, B2 phase, despite being the equilibrium phase at this temperature, is unable to nucleate homogeneously within the grains [6]. Instead, it nucleates along the grain boundaries which serve as heterogenous nucleation sites with a much lower energy barrier to nucleation. The nucleation of B2 phase in this scenario is limited by the volume of grain boundary area available as nucleation sites which in turn depends on the grain size. Therefore, at a larger grain size, the actual volume fraction of B2 phase would be less than the expected equilibrium volume fraction. This suggests towards the possibility of achieving an increase in the B2 phase fraction by reducing the grain size. The second processing route precisely accomplishes this with the added advantage of the grain boundaries in this case being a more potent heterogenous nucleation site, owing to the highly strained microstructure. [6] Hence, when the homogenized and cold-rolled alloy is directly aged at 700°C (CR-700) avoiding the solutionizing step, the result is a fine-grained dual-phase microstructure consisting of FCC and B2 grains. This is depicted in the BSE contrast SEM image in Figure 2(c) along with the phase map from a similar region, shown in Figure 2(d). The crystal structures of the two phases are further confirmed via TEM. Figure 2(e) is a bright-field TEM image showing two adjacent FCC and B2 grains along with the SADP from the FCC grain in the inset. Figure 2(f) is a dark-field TEM image acquired using the B2 superlattice spot along with the corresponding SADP shown in inset, from the B2 grain. Because of a significantly finer FCC grain size and highly strained grain boundaries resulting from the cold-rolling step, a large number of nucleation sites in the form of these grain-boundaries are available for heterogenous nucleation of B2 grains. It appears that the stress concentrations along the grain boundaries such as dislocation pileups and slip steps are such potent heterogenous nucleation sites. The new strain free grains nucleating at such high energy sites eventually grow by consuming the nearby highly deformed grains. Additionally, in parallel to this, new FCC strain free grains may also nucleate and grow at such high energy sites, in process consuming the strained FCC grains, which is the usual mechanism observed during recrystallization. The microstructure is essentially getting recrystallized by both these processes. This has been observed in the case of Al0.3CoCrFeNi as well [7], which will be further discussed in a later section. This results in a fine distribution of both FCC and B2 grains and ultimately precipitation of a higher volume fraction of B2 phase (~21%) than that observed in the case of homogeneous nucleation (~6%).

2) Competing nucleation tendencies of metastable L12 and the equilibrium B2 phases in

Al0.5Co1.5CrFeNi1.5

In the previous section, it was demonstrated that homogenous nucleation of equilibrium B2 phase within the FCC matrix, is energetically difficult as compared to its heterogenous nucleation along high energy sites such as strained grain boundaries. Additionally, in some cases, metastable phases may also interfere with this process whenever the barrier to nucleation for such phases is considerably lower than that for the equilibrium phase. Such competition has been captured in the present alloy. [24] with permission from Elsevier.

The Al0.5Co1.5CrFeNi1.5 alloy after homogenizing, and cold rolling to ~60% reduction in thickness, followed by solutionizing at 1250°C for 5 mins, was annealed at 750°C for 50 hrs (CRSA-750 condition). Figure 3(a) is a BSE contrast SEM image showing the microstructure in CRSA-750 condition which consists of coarse recrystallized FCC grains and B2 precipitates along the grain boundaries as well as within the grains near the grain boundaries, as shown in Figure 3(b) which is a higher magnification BSE contrast SEM image from near a grain boundary. Figure 3(c) is a SADP from one such precipitate acquired along the [001] zone axis of the B2 precipitate. This microstructure is similar to that observed for the previous alloy in CRSA-700 condition. However, in the present condition, additionally within the FCC grains, fine scale L12 precipitates are also present which appear as fine pockets dispersed in FCC matrix between the coarse B2 plates. This is also shown in Figure 3(b). Figure 3(d) is an SADP along [001] zone, captured from within the FCC grains showing superlattice spots confirming presence of L12 precipitates. Such L12 precipitates have been captured in APT reconstruction from the FCC grain, as shown in Figure 3(e) along with the corresponding proxigram showing the composition of the L12 precipitate and the matrix. Based on the composition estimated from APT, it appears that the precipitates may have the stoichiometry corresponding to (Ni,Fe,Co)3(Al,Cr), assuming an Ni3Al type stoichiometry [26,27]. For the case of current alloy composition, although L12 is not an equilibrium phase at this temperature as per CALPHAD calculations [24], it stills forms as a metastable transient phase having a low barrier to its homogenous nucleation within the super-saturated FCC matrix. The B2 phase has a higher onset-driving force for homogenous nucleation within the FCC matrix which is depicted in Figure 4. But despite being an equilibrium phase (based on CALPHAD), B2 is unable to nucleate due to a high barrier to homogenous nucleation in an FCC matrix. Such high barrier to nucleation of B2 is due to the high interfacial energy associated with the FCC/B2 interface as compared to that for L12/FCC [7,24]. Hence, although L12 is a meta-stable phase at this temperature, it is able to nucleate homogeneously whereas B2, despite being equilibrium phase, is only able to nucleate heterogeneously along grain boundaries.

To understand which way this competition between a metastable phase and an equilibrium phase is biased by promoting heterogenous nucleation, the alloy was annealed directly at 750°C after cold-rolling step, entirely avoiding the solutionizing step, i.e. CR-750 condition. Figure 5(a) is a BSE contrast SEM image showing the microstructure in CR-750 condition which consists of fine recrystallized pockets distributed within un-recrystallized FCC regions. The recrystallized pockets consist of fine FCC recrystallized grains devoid of L12 along with fine-scale B2 precipitates both within the grains as well as along grain boundaries resembling the microstructure observed in CR-700 condition in previous section. Examples of these two cases are depicted in Figures 5(c) and (d), both of which are dark-field TEM images. Just like in previous section for the case of Al0.3CoFeNi, the strain build-up along grain boundaries due to cold rolling, aid in nucleation of B2 grains along with concurrent recrystallization of the microstructure. Detailed report of this condition observed that some of these intra-granular B2 precipitates follow Nishiyama-Wasserman (N-W) orientation relationship whereas some follow the Kurdjumov-Sachs (K-S) orientation relationship with the parent FCC matrix [24]. Such an occurrence of B2 phase in multiple orientation relationships with the parent FCC lattice has been reported previously in case of Al0.3CoCrFeNi [28]. For the same alloy, it was observed that K-S oriented B2 plates nucleate along broad faces of twins whereas N-W oriented B2 pockets nucleate along regions where two different variants of twins intersect within a single grain. Nucleation along such intersections is aided owing to a high degree of locally altered matrix orientation. Pitsch oriented B2 pockets were also observed to nucleate along some of such intersections.

The mechanism of B2 nucleation in FCC HEAs along twin interfaces has been discussed in detail previously [29] where it was discovered that the FCC to B2 transformation is a mixed-mode [24] with permission from Elsevier. [25] with permission from Elsevier.

(c) Dark-field TEM image acquired using superlattice spot from [011] zone axis of B2 phase in recrystallized region. (d) Dark-field TEM image acquired using superlattice spot from [011] zone axis of B2 plate along a twin in unrecrystallized region. (e) APT reconstruction showing an FCC/B2 interface, along with the corresponding proxigram depicting FCC/B2 compositions in (f). Figures reprinted from

(b) HRTEM image of a deformation twin along with corresponding FFT shown in inset. (c) Dark-field TEM image showing nucleation of B2 plates along twin boundaries after 30 minutes of annealing at 700°C. (d) Low magnification dark-field TEM image acquired using superlattice spot from B2 phase showing B2 plates decorating twin boundaries after annealing for 30 mins. Figures reprinted from

In the present sample, the unrecrystallized region consists of coarse FCC grains without any L12, but fine-scale B2 precipitates aligned along deformation bands/twins which formed during coldrolling. The composition of these B2 precipitates was examined via APT. Figure 5(e) is an APT reconstruction along with the corresponding proxigram shown in Figure 5(f). From the composition data, it appears that the B2 precipitates follow the stoichiometry:

(Ni,Co)(Al,Co,Cr,Fe) assuming NiAl type stoichiometry [26,27]. Although B2 phase has a higher barrier to homogenous nucleation within an FCC matrix, the high density of defects structures such as grain boundaries, deformation bands, twins, and dislocation tangles, serve as potent sites for heterogenous nucleation of B2 phase, resulting in a microstructure with much higher volumefraction of B2 than that in previous case (i.e. CRSA750). This also indicates that the nucleation and growth of B2 phase results in depletion of the solute saturation from within the FCC matrix because the metastable L12 phase no longer precipitates out along with the B2, as was observed in the case of CRSA-750 condition. Hence, employing defect mediated nucleation of equilibrium phase may inhibit the formation of metastable phases which might be otherwise unavoidable via conventional processing route.

3) Competing nucleation tendencies between metastable L12 and multiple equilibrium phases in Al0.3CoCrFeNi

In the foregoing section, it was established that the competition between nucleation of metastable and equilibrium phases can be influenced by introducing a large density of heterogenous nucleation sites in the microstructure via cold rolling. The present section explores the microstructural evolution when nucleation of multiple equilibrium phases compete with that of a metastable phase.

In this case, the homogenized Al0.3CoCrFeNi alloy was cold rolled to ~50% reduction in thickness which was then directly annealed at 700°C for 96 hrs [25]. The CALPHAD calculations shown in Figure 1(a) predict the equilibrium phase field to consist of (FCC+B2+σ). As explained above, the annealing temperature was selected to ensure a ternary phase-field in the present case. Samples were analyzed from intermediate stages of annealing as well, to understand the microstructural evolution with annealing time [25]. The microstructure in the cold rolled sample consists of high density of deformation twins. This is shown in the dark-field TEM image shown in Figure 6(a) and the HRTEM image in Figure 6(b). With as short as 30 minutes of aging, B2 plates start [25] with permission from Elsevier.

nucleating along these twins as shown in Figures 6(c Figure 8 shows that the homogeneous nucleation of B2 directly within the FCC matrix at 700°C is associated with a higher onset-driving force, but again due to a high nucleation barrier, it is instead nucleating heterogeneously along various high energy defects such as triple-junctions, grain boundaries, twin-interfaces, and dislocation tangles. Also, Type-II and Type-III B2 precipitates both maintain K-S orientation relationship with the parent FCC matrix.

As discussed previously, although L12 phase is not an equilibrium phase at this temperature as predicted by CALPHAD (see Figures 1(a) and (d)), it forms in the current system owing to a finite driving force (see Figure 8 [25] with permission from Elsevier.

Nucleation and subsequent growth of Ni and Al rich Type-I B2 grains at prior FCC grainboundaries leads to Cr enrichment adjacent to these B2 precipitates which is conducive for heterogenous nucleation of σ phase here which is otherwise difficult. Just like in the case of B2 phase, the difficulty in homogenously nucleating sigma arises out of a prohibitively high energy barrier to nucleation within an FCC matrix despite having a finite onset-driving force, which can be seen from Figure 8. Formation of Ni and Al rich Type-II B2 precipitates also result in pockets of Cr enrichment along twin boundaries with BCC structure which may be precursors to σ phase.

Being embedded within the FCC matrix perhaps prevents their transformation owing to a high barrier for nucleation of σ phase within an FCC matrix which is not the case with those pockets which are situated along grain boundaries. Therefore, multiple occurrences of sigma grains along prior grain boundaries have been observed in the present condition whereas no instances of sigma phase within an FCC grain was observed.

Conclusion

In the present study, defects induced during processing of HEAs/CCAs are used to promote heterogenous nucleation of equilibrium B2 phase along high energy defects such as grain boundaries, deformation twins, annealing twins, deformation bands, dislocation tangles, etc.

Conventional processing route of solutionizing after cold rolling followed by annealing, may not always result in the equilibrium phases in their predicted volume fractions, for these alloys. While this is true for all the three alloys reported in this study, sometimes a metastable L12 phase may nucleate homogeneously in competition with the equilibrium phase (the case of Al0.5Co1.5CrFeNi1.5 alloy). This stems from a high nucleation barrier for the equilibrium phases in FCC matrix while a much lower barrier to nucleation of the metastable L12 phase exists. A large density of defect induced heterogenous nucleation sites bias this competition in the favor of the equilibrium B2 phase (the case of Al0.5Co1.5CrFeNi1.5). The scenario is further complicated when more than one equilibrium phase competes for nucleation with the metastable L12 phase with a lower nucleation barrier which was the case for Al0.3CoCrFeNi alloy. Whereas annealing this alloy in cold rolled condition within a ternary phase field results in nucleation of multiple generations of B2 precipitates along twin boundaries, within the FCC grains, and along prior FCC grain boundaries with σ grains, system favors the nucleation of L12 precipitates over B2 in the FCC matrix, again owing to a lower nucleation barrier of L12 as compared to B2. In all three alloys, such heterogeneously templated microstructure results from a prior step of introducing high defect density via processing, before annealing conventionally. Previously published literature has highlighted achievement of an attractive balance in mechanical properties from such a microstructure, when compared to the conventionally processed counterparts [6,24,25]. This has been possible due to the existence of multiphase equilibrium domains within the complex phase diagrams associated with HEAs/CCAs. In summary, such an approach can be exploited to tailor the microstructure in case of other HEAs as well to gain a handle over their mechanical properties.

Figure 1 :

 1 Figure 1: (a) Overlapping pseudo-binary isopleths generated using ThermoCalc, for Al x CoFeNi, Al x Co 1.5 CrFeNi 1.5 and Al x CoCrFeNi systems. Corresponding phase-fractions as a function of temperature for (b) Al 0.3 CoFeNi. (c) Al 0.5 Co 1.5 FeNi 1.5 , and (d) Al 0.3 CoCrFeNi.

Figure 2 :

 2 Figure 2: (a) BSE contrast SEM image, and (b) Phase-map from EBSD acquired from a triple junction, both showing microstructure of Al 0.3 CoFeNi in CRSA-700 condition. (c) BSE contrast SEM image, and (d) Phase-map from EBSD, both showing microstructure of Al 0.3 CoFeNi in CR-700 condition. (e) Bright-field TEM image and (f) Dark-field TEM image to confirm the presence of FCC and B2 grains, in CR-700 condition. Figures reprinted from[6]

Figure 3 :

 3 Figure 3: (a) Low magnification, and (b) High magnification BSE contrast SEM image from Al 0.5 Co 1.5 FeNi 1.5 in CRSA-750 condition (c) SADP acquired along [001] zone axis of B2 precipitate. (d) SADP acquired along [001] zone axis of FCC matrix. (e) APT reconstruction from FCC matrix along with a L1 2 precipitate. (f) Corresponding proxigram depicting FCC matrix and L1 2 precipitate compositions. Figures reprinted from[24] with permission from Elsevier.

Figure 4 :

 4 Figure 4: Normalized onset-driving forces for homogeneous nucleation within an FCC matrix for various phases in alloy Al 0.5 Co 1.5 CrFeNi 1.5 determined using ThermoCalc, as a function of temperature. Figure reprinted from[24] with permission from Elsevier.

Figure 5 :

 5 Figure 5: (a) BSE contrast SEM image from Al 0.5 Co 1.5 FeNi 1.5 in CR-750 condition (b) Higher magnification phase-map acquired using EBSD, from the same condition. (c) Dark-field TEM image acquired using superlattice spot from [011] zone axis of B2 phase in recrystallized region.(d) Dark-field TEM image acquired using superlattice spot from [011] zone axis of B2 plate along a twin in unrecrystallized region. (e) APT reconstruction showing an FCC/B2 interface, along with the corresponding proxigram depicting FCC/B2 compositions in (f). Figures reprinted from[24] with permission from Elsevier.

  transformation including a displacive structural component along with solute diffusion. Plastic deformation results in formation of metastable intermediate structures in vicinity of twin interfaces, comprising of structural motifs which closely resemble BCC crystallographic symmetry necessary for K-S oriented B2 nucleation. Such structures are compositionally indistinguishable from the surrounding FCC matrix at the time of formation. Eventually, solute partitioning entails with annealing which drives the transformation of these pockets to B2 phase.

Figure 6 :

 6 Figure 6: (a) Dark-field TEM image from cold-rolled sample showing high density of deformation twins oriented along (111) planes. (b) HRTEM image of a deformation twin along with corresponding FFT shown in inset. (c) Dark-field TEM image showing nucleation of B2 plates along twin boundaries after 30 minutes of annealing at 700°C. (d) Low magnification dark-field TEM image acquired using superlattice spot from B2 phase showing B2 plates decorating twin boundaries after annealing for 30 mins. Figures reprinted from[25] with permission from Elsevier.

Figure 7 :

 7 Figure 7: (a) Low magnification BSE contrast SEM image, and (b) High magnification BSE contrast SEM image, both from Al 0.3 CoCrFeNi in CR-96 condition. (c) Bright-field TEM image showing presence of Type-I B2 along prior FCC grain boundary (d) Brigth-field image showing Type-III B2 precipitate along a twin boundary and a Type-III B2 precipitate within the FCC matrix. (e) Darkfield TEM image acquired using superlattice reflection showing presence of L1 2 precipitates. (f) Bright-field TEM image showing nucleation of grain-boundary sigma phase adjacent to a Type-I B2 grain. Figures reprinted from[25] with permission from Elsevier.

  ) and (d), which are both dark-field TEM images. The density of such B2 plates increase eventually with aging time. With about 4 hours of annealing (CR-4 condition), nucleation of B2 along grain boundary starts. With increasing annealing time, EBSD indicates an increase in twin-boundary density owing to increase in number of annealing twins which then serve as additional nucleation sites for B2 phase. As a result, larger heterogenous nucleation sites are available for B2 precipitates to form leading to an increase in B2 density with aging time. After 96 hours of annealing (CR-96 condition), multiple generations and types of precipitates have nucleated along various defects in the microstructure as well as within the FCC matrix. a. Type I: These are fine-scale recrystallized B2 grains nucleated along prior FCC grain-boundaries and triple-junctions. Figure7(a) is a BSE contrast SEM image showing their presence along FCC grain boundaries. Further, a bright-field TEM image from such a precipitate is also shown in Figure 7(c). b. Type II: These are elongated, pancake shaped B2 precipitates forming along twin boundaries within the FCC grains. They are marked in Figure 7(a) along with a representative bright-field TEM image showing their presence along twin boundaries in Figure 7(d). c. Type III: These are faceted B2 precipitates nucleating within FCC grains in regions away from twin boundaries, probably either along dislocation tangles or nucleating homogeneously. These are also marked in Figure 7(a) along with an example shown in Figure 7(d). d. Type IV: These are fine-scale metastable L12 precipitates in regions within adjacent twin bands decorated with Type-II B2 precipitates. These are marked in Figure 7(b) which is a BSE-contrast SEM image. Their presence was also confirmed by darkfield TEM imaging acquired using the superlattice reflection, as shown in Figure 7(e). e. σ grains nucleate adjacent to Type-I B2 grains along prior-FCC grain-boundaries as shown by the bright-field TEM image in Figure 7(f). f. Cr-rich BCC pockets adjacent to Type-II B2 precipitates along twin interfaces. An example is shown in Supplementary Figure S2(a) which is an STEM-EDS map of Cr showing Cr rich pocket along a twin boundary, along with micro-diffraction pattern shown in inset of Supplementary Figure S2(b) which is a DF-TEM image.

  )[7] coupled with a low nucleation barrier to homogenous nucleation within the FCC matrix. Such L12 precipitates only exist in regions farther away from twin bands decorated with B2 phase because the nucleation and growth of B2 phase, renders the regions surrounding twin bands, poorer in Ni and Al hence, making nucleation of L12 compositionally challenging.

Figure 8 :

 8 Figure 8: Normalized onset-driving forces for homogeneous nucleation within an FCC matrix for various phases in alloy Al 0.3 CoCrFeNi, determined using ThermoCalc, as a function of temperature. Figure reprinted from[25] with permission from Elsevier.

Acknowledgments:

The work was supported by the US Air Force Office of Scientific Research under grant FA9550-17-1-0395. The authors acknowledge the Materials Research Facility (MRF) at the University of North Texas for use of microscopy and characterization facilities. BG would like to acknowledge the support by Pacific Northwest National Laboratory's (PNNL) open call laboratory directed research and development (LDRD) program. PNNL is a multi-program national laboratory operated by Battelle for the U.S. DOE under Contract DE-AC05-76RL01830.

Declaration of Competing Interest:

The authors declare that they have no conflicting interests which may influence the results reported in this paper.


MD Arnaud Scherpereel
email: arnaud.scherpereel@chru-lille.fr

Julien Mazières

Laurent Greillier

Sylvie Lantuéjoul

Pascal Do

Olivier Bylicki

Isabelle Monnet

Romain Corre

Clarisse Audigier-Valette

MD Myriam Locatelli-Sanchez

Julien Mazieres

MD Sylvie Lantuejoul

Pascal Dô

Myriam Locatelli- Sanchez

Olivier Molinier

Florian Guisier

Thierry Urban

Catherine Ligeza-Poisson

MD David Planchard

Elodie Amour

Franck Morin

Denis Moro-Sibilot

Gérard Zalcman

Prof A Scherpereel

Prof D Moro-Sibilot

Prof G Zalcman

Nivolumab or nivolumab plus ipilimumab in patients with relapsed malignant pleural mesothelioma (IFCT-1501 MAPS2): a multicentre, open-label, randomised, non-comparative, phase 2 trial

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Background There is no recommended therapy for malignant pleural mesothelioma (MPM) progressing after 1 st -line pemetrexed-platinum treatment. Disease control rate (DCR) was below 30% with all 2 nd -line drugs tested. Preliminary results suggested anti-PD-1 monoclonal antibody (mAb) could be active in this setting. We thus aimed to prospectively assess anti-PD-1 mAb alone or in combination with anti-CTLA-4 in MPM.

Methods

In this multicentre randomised non-comparative, open-label phase 2 trial, patients >18 years old with PS 0-1, histologically-proven MPM progressing after 1 st -/2 nd -line treatments involving pemetrexed/platinum, and measurable disease, were randomly allocated

(1:1) to nivolumab (3 mg/kg bodyweight intravenously) every 2 weeks), or nivolumab plus ipilimumab (3 mg/kg every 2 weeks plus 1 mg/kg, every 6 weeks, intravenously), given until progression or unacceptable toxicity. Central randomization stratified by histology (epithelioïd vs non-epithelioïd), treatment line (second vs third line), and chemosensitivity to previous pemetrexed-platinum doublet, used a minimization method with a 0.8 random factor.

The primary endpoint was 12-week DCR ≥40%, assessed by blinded central review, on the first 108 required eligible patients. Last patient was accrued in August 2016; we report here the final analysis. ClinicalTrials.gov: NCT02716272.

Findings

During a 5-months period, 125 patients were recruited. The with nivolumab or the combination respectively. The most frequent G3 adverse event was asthenia, with nivolumab (n=1, 1.6%) or with the combination (n=3, 4.9%), AST or ALT asymptomatic increase (n=4, 6.6%, all in the combination arm), lipase asymptomatic increase, with nivolumab (n=2, 3.6%) or with the combination (n=1,1.6%), with one additional G4 lipase increase (1.6%) in both arms. There were 0 and 3/62 (4.8%) toxic deaths, in the nivolumab and the combination arm respectively.

Interpretation:

Single-drug anti-PD-1 nivolumab or anti-PD-1 plus anti-CTLA-4 ipilimumab combination both showed efficacy in pre-treated MPM patients, without unexpected toxicity, deserving confirmation by larger clinical trials.

Funding: The French Cooperative Thoracic Intergroup (IFCT), France

Research in context

Evidence before this study On November 15 th 2015, we searched PubMed for studies evaluating immunotherapeutic antibody use, PD-1-blocking or CTLA4-blocking antibodies, in mesothelioma patients using the following search terms: "mesothelioma" and "nivolumab OR pembrolizumab OR

Implications of all the available evidence

These findings, involving 125 randomized patients, clearly show that immune-checkpoint inhibitors (ICIs) do achieve significant clinical activity in relapsed MPM, either as single or combination therapy, at standard doses. The data revealed ICIs are capable of inducing antitumour objective responses according to RECIST-meso criteria, as well as significant median progression-free and overall survival rates, with a tolerable safety profile, in this orphan-disease population. While these results require further confirmation in larger-scale trials, they could at present justify ICI use in relapsed MPM patients who have no other efficient therapeutic options available.

Introduction

Malignant pleural mesothelioma (MPM) is a rare but aggressive malignancy of the pleural surface, commonly associated with occupational asbestos exposure, and its incidence is increasing worldwide. [START_REF] Zalcman | Bevacizumab for newly diagnosed pleural mesothelioma in the Mesothelioma Avastin Cisplatin Pemetrexed Study (MAPS): a randomised, controlled, open-label, phase 3 trial[END_REF] Nonetheless, an optimal second-line MPM treatment has not yet been defined by most recent guidelines. [START_REF] Scherpereel | Guidelines of the European Respiratory Society and the European Society of Thoracic Surgeons for the management of malignant pleural mesothelioma[END_REF][4][START_REF] Woolhouse | British Thoracic Society Guideline for the investigation and management of malignant pleural mesothelioma[END_REF][START_REF] Kindler | Treatment of Malignant Pleural Mesothelioma: American Society of Clinical Oncology Clinical Practice Guideline[END_REF][7] Our understanding of MPM pathogenesis has significantly improved over recent years, leading to innovative drugs and strategies, [START_REF] Bueno | Comprehensive genomic analysis of malignant pleural mesothelioma identifies recurrent mutations, gene fusions and splicing alterations[END_REF]9 with targeted therapies and immunotherapies sparking new hope for MPM patients. [START_REF] Zalcman | Bevacizumab for newly diagnosed pleural mesothelioma in the Mesothelioma Avastin Cisplatin Pemetrexed Study (MAPS): a randomised, controlled, open-label, phase 3 trial[END_REF][9][START_REF] Yap | Novel insights into mesothelioma biology and implications for therapy[END_REF][START_REF] Grosso | Nintedanib Plus Pemetrexed/Cisplatin in Patients With Malignant Pleural Mesothelioma: Phase II Results From the Randomised, Placebo-Controlled LUME-Meso Trial[END_REF] By instigating chronic inflammation and localized tumour immunosuppression, the immune system has proven paramount in MPM pathogenesis, with improved outcomes correlating with higher intra-tumour infiltration by cytotoxic T CD8+ cells [START_REF] Yamada | CD8+ tumor-infiltrating lymphocytes predict favorable prognosis in malignant pleural mesothelioma after resection[END_REF] . Conversely, high tumour expression of programmed cell deathligand 1 (PD-L1), as it inhibits T-cell function via binding the programmed cell death-1 (PD-1), has been associated with poor prognosis in mesothelioma (mOS: 5•0 months in PD-L1positive patients vs. 14•5 months in PD-L1-negative patients). 9, [START_REF] Mansfield | B7-H1 expression in malignant pleural mesothelioma is associated with sarcomatoid histology and poor prognosis[END_REF][START_REF] Cedrès | Analysis of Expression of Programmed Cell Death 1 Ligand 1 (PD-L1) in Malignant Pleural Mesothelioma (MPM)[END_REF] Among the different immunotherapies evaluated so far to restore anti-tumour immune response in MPM, immune checkpoint inhibitors (ICI) have garnered the most attention based on their efficacy, particularly in melanoma and non-small cell lung cancer (NSCLC). 9,10

Cytotoxic T lymphocyte-associated protein (CTLA-4) is a checkpoint inhibitor that blocks interactions between antigen-presenting cells, such as dendritic and naïve T cells, occurring early in the anti-tumour cycle. Following encouraging Phase II trial results, tremelimumab, an anti-CTLA-4 antibody, was tested alone vs. placebo in 2 nd -or 3 rd -line treatment in MPM in a large randomised Phase IIb trial ('DETERMINE'), yet no survival gain was obtained compared to placebo. [START_REF] Maio | Tremelimumab as second-line or third-line treatment in relapsed malignant mesothelioma (DETERMINE): a multicentre, international, randomised, double-blind, placebo-controlled phase 2b trial[END_REF] In contrast, several studies assessing ICIs targeting the PD-1/PD-L1 pathway generated promising results. 9, [START_REF] Alley | Clinical safety and activity of pembrolizumab in patients with malignant pleural mesothelioma (KEYNOTE-028): preliminary results from a non-randomised, open-label, phase 1b trial[END_REF] In a Phase Ib trial, 25 patients with PD-L1-expressing MPM (≥1% positive tumour cells) treated with the anti-PD-1 antibody pembrolizumab, mostly as second-line, exhibited a 20% overall response rate, with 52% achieving stable disease (SD). The median duration of response (DOR) was 12 months, with no safety concerns. [START_REF] Alley | Clinical safety and activity of pembrolizumab in patients with malignant pleural mesothelioma (KEYNOTE-028): preliminary results from a non-randomised, open-label, phase 1b trial[END_REF] Other trials assessing anti-PD-1/PD-L1 antibodies in MPM have demonstrated similar response rates. Prior to inclusion, all patients provided written informed consent to participate in the trial.

Randomisation and masking

We used an interactive web-response system to generate random, non-masked treatment allocation. We randomly assigned patients enrolled by investigators (1:1) to the two treatment groups. Randomisation was centrally performed by computer. We used a minimisation method (random factor of 0.8) and stratified patients by histology (epithelioid vs sarcomatoid or mixed-histological subtypes), treatment line (second vs third line), and chemosensitivity to pemetrexed-platinum doublet (progression >3 months after completing pemetrexed-platinum doublet or <3 months).

Procedures

Patients received intravenous anti-PD-1 nivolumab (Nivo) at 3mg/kg of their body weight (60 Treatment delay of Ipi interruption for >12 weeks also required treatment discontinuation, except for delays due to drug-related adverse events (DRAEs) needing slow steroid tapering off until <10mg daily steroid dose for full recovery from immunotherapy-related DRAE.

min
Criteria for permanent treatment discontinuation included the following treatment-related adverse events: grade 3 non-skin events lasting 7 days or more, grade 3 laboratory abnormalities of thrombocytopenia or liver function test, and all grade 4 events, as well as laboratory abnormalities, except for asymptomatic amylase or lipase elevations.

Other potential treatment termination reasons included tumour progression, death, intercurrent illness, protocol violation, non-compliance, and patient consent withdrawn.

Following progression or unacceptable toxicity, further treatment lines could be initiated at the discretion of the investigators, although crossover and further Ipi or Ipi plus Nivo were not permitted in the Nivo arm.

Baseline laboratory tests required to assess eligibility included white blood cell counts, neutrophils, platelets, haemoglobin, serum creatinine, alanine aminotransferase, aspartate amino transferase, alkaline phosphatase, total bilirubin, albumin, lipase, and was performed at baseline and at every treatment infusion. TSH was measured at baseline and monitored prior every 12 weeks.

Adverse event monitoring was performed prior to each treatment infusion thus every two weeks until off protocol therapy and until three months post-discontinuation.

At baseline, disease assessment was performed using chest CT-scan including abdominal exploration and brain magnetic resonance imaging (MRI) or CT. Tumour node metastasis (TNM) classification was centrally assessed post-hoc by expert thoracic radiologists, blinded to the allocation arm, on thoracic CT-scans at diagnosis, and by reviewing the pathological and surgical reports in cases involving initial diagnostic thoracoscopy, according to the 8th TNM classification for MPM. [START_REF] Rusch | IASLC Staging and Prognostic Factors Committee, advisory boards, and participating institutions. The IASLC Mesothelioma Staging Project: Proposals for the M Descriptors and for Revision of the TNM Stage Groupings in the Forthcoming (Eighth) Edition of the TNM Classification for Mesothelioma[END_REF] We performed CT scans every 12 weeks from randomisation, at the same time points in both arms, with response assessed at 12 weeks by modified Response Evaluation Criteria in Solid Tumours criteria for mesothelioma. [START_REF] Byrne | Modified RECIST criteria for assessment of response in malignant pleural mesothelioma[END_REF] CTs were centrally reviewed by three blinded independent MPM-experienced radiologists. Patients were contacted every 12 weeks in order to assess survival upon follow-up. Adverse events (AEs) and laboratory abnormalities were graded according to the National Cancer Institute

Common Terminology Criteria for Adverse Events, Version 4.0.

We assessed quality of life (QoL) using the Lung Cancer Symptom Score (LCSS)-Meso at baseline, treatment initiation and every 12 weeks. For each scale or item in LCSS-Meso, a linear transformation was applied to standardise the raw score to a 0-100 range (100=best possible function or QoL for functional scales and highest symptom burden for symptom scales and symptom items). A 10-point change in an item or domain was considered clinically meaningful [START_REF] Zalcman | Bevacizumab for newly diagnosed pleural mesothelioma in the Mesothelioma Avastin Cisplatin Pemetrexed Study (MAPS): a randomised, controlled, open-label, phase 3 trial[END_REF] . QoL was defined as improved when a ≥10-point increase was recorded for functioning scales and ≥10-point reduction for symptom domains or items between baseline and 12-week assessments. We deemed QoL stable when variations of <10 points were recorded for functioning scales and symptom domains and items, and as worsened with ≥10point decreases for functioning scales and ≥10-point increases for symptom domains or items.

To compute treatment exposure, we calculated the ratio of the dose intensity measured as a proportion of the theoretical dose intensity. Lantuejoul). [START_REF] Lantuejoul | PD-L1 Testing for Immune Checkpoint Inhibitors in Mesothelioma: For Want of Anything Better[END_REF] The pathologist in charge of analysing the specimens was blinded to treatment and patient response. Expression was categorized according to tumour proportion scores (i.e., percentage of tumour cells with membranous PD-L1 staining, regardless of intensity). An exploratory cut-off threshold of 25% tumour cells presenting membranous PD-L1 staining was chosen based on data resulting from a post-hoc analysis to ensure there were sufficient numbers of patients in each subset.

Outcomes

The primary outcome was the rate of patients with controlled disease (disease control rate, DCR), defined as the number of patients with complete response, partial response or stable disease at 12 weeks post-randomisation, assessed by the independent central review involving three radiologists blinded to treatment arm, using modified RECIST criteria for mesothelioma [START_REF] Byrne | Modified RECIST criteria for assessment of response in malignant pleural mesothelioma[END_REF] , divided by the number of patients in each group.

Secondary outcomes were OS (defined as the time from randomisation to death from any cause), PFS (time from randomisation to documented disease progression or death, whichever occurred first), QoL as evaluated by Lung Cancer Symptom Scale (LCSS) questionnaires at each infusion, safety and correlation between PD-L1 tumour expression and ORR, DCR, PFS and OS.

Patients were considered as assessable for response or survival provided they had the 12weeks tumour evaluation (for response) or if they progressed before the 12 weeks evaluation (either clinically or by CT-scan evaluation), and provided they were eligible.

Statistical analysis

The primary study endpoint included the first 108 eligible patients, while the intention-to-treat (ITT) population included all randomised patients for efficacy analyses; the 'safety population' for safety analyses included all patients who received at least one cycle of study treatment.

We assumed a ≤20% DCR at 12 weeks (null hypothesis), thus of no therapeutic interest (i.e., DCR below which the treatment would be deemed inactive), validated by a blinded independent central review by one of three radiologists, and a target ≥40% DCR (alternative hypothesis), thus indicating clinical activity, along with a one-sided α error of 0•05. We thus calculated that a total of 54 eligible patients in each arm (108 in total) would enable us to detect an effect on the primary outcome with 95% power. Assuming 5% were ineligible, we had to recruit 57 patients to each arm. Based on these assumptions, ≥17 failure-free patients had to be independently observed at 12 weeks in either arm, using a one-step Fleming procedure, to enable us to conclude on the activity of the corresponding regimen. No interim analysis was planned.

For patients exhibiting no events, the cut-off point was last contact. We plotted PFS and OS by means of Kaplan-Meier curves, with follow-up censored on December 28 receiving Nivo+Ipi. Since accrual ran faster than anticipated, 11 patients providing consent on the last day of accrual were enrolled in addition to the 114 initially planned (Figure 1).

Five patients were deemed ineligible in the nivolumab arm, since one had no evidence of progression at inclusion, three had received more than three treatment lines at inclusion, and one had only a cytological evidence to sustain the MPM diagnosis. Two additional patients were deemed ineligible in the combination arm, one having no evidence of progression at inclusion, another one having received more than three previous treatment arms. In between both arms (Table 1).

At the cut-off date, all but one patient had received at least one dose of Nivo or Nivo+Ipi, and one Nivo+Ipi patient died before receiving the allocated treatment (Figure 1 toxicity (detailed in Figure 1), with one patient having died without toxicity or progression, another exhibiting a second unrelated cancer, one patient presenting an intercurrent disease, one patient having decided to stop treatment by his own choice, two patients for both toxicity and progression, only four remaining patients continuing treatment (Figure 1). In the Nivo+Ipi arm, 58 patients withdrew from the study, 38 of whom (65•5%) had disease progression and 13 (22•4%) toxicity (detailed in Figure 1), five patients having died without toxicity or progression, one patient having an intercurrent disease and one patient by decision of the investigator, with only three patients still on treatment at the data cut-off point (Figure 1).

To conform to CONSORT guidelines, we report the primary efficacy analysis as estimated by DCR at 12 weeks post-randomisation in the first 108 eligible patients (54 patients in each arm), the population anticipated by the statistical analysis plan. This primary endpoint which was met in both arms, with a 44 3). There were 3/63 (4.8%) and 17/61 (27.8%) all-grade serious drug-related AEs in the nivolumab and the combination arms respectively (Table 3). Three DRAEs of 63 patients (4.8%) and 13/61 (21.3%) led to treatment discontinuation in the nivolumab and the combination arms respectively (Table 3). There were three treatment-related deaths reported by investigators in the combination arm (Table 3): one fulminant hepatitis, one encephalitis (normal cerebrospinal fluid cellular and biochemical composition, normal brain MRI and no blood cerebrospinal fluid neuronal self-antibodies found) and one acute kidney failure in a patient with (end of life) disease progression exhibiting recurrent pleural and peritoneal effusions needing daily punctures. These three lethal events (4•9%) occurred within the first 4 study months, with no other toxic deaths reported later on in the trial. No treatment-related death was reported in the nivolumab arm. The incidence of Grade 4 adverse events proved low in both arms (1•6%, n=1, for Nivo; 3•3%, n=2, for Nivo+Ipi). 3 (webappendix p.5).

Patient-reported outcomes (PROs) were collected using the LCSS questionnaire, expressed as a percentage of patients who reported deteriorating quality of life between the 12-week and baseline questionnaires. A graphical representation of the rate of decline for 10 items is depicted in the supplemental Figure 1 (webappendix p.2). There were no major differences detected between arms in the numbers of patients reporting score decline at 12 weeks in each item, taking into account the exploratory nature of such unpowered analyses precluding any formal statistical test. Longitudinal QoL studies, using time until definitive deterioration (TUDD), and thus long-term QoL data, will be presented in a separate paper.

An exploratory analysis of PD-L1 tumour expression, using both 28.8 and SP-263 anti-PD-L1 assays, was performed, with centralized immunohistochemistry and pathological review, on samples containing a minimum of 100 viable tumour cells. Such exploratory study was possible in only 104 patients for SP263 and 99 for 28.8 antibodies, since there were no longer any tissue remaining for the other patients. 28.8 Dako pharmDx™ (supplementary Table 4, webappendix p.6) PD-L1 expression in ≥1% of cells was found to be significantly associated with objective response to immunotherapy (39•0% vs. 12•1% ORR, p=0•002), yet not with 12-week DCR. Typically, the same two cut-offs are used in the literature to define patients with high PD-L1 tumour expression: 25% or more tumour cells expressing PD-L1 regardless of the intensity [START_REF] Adam | Multicenter harmonization study for PD-L1 IHC testing in non-small-cell lung cancer[END_REF][START_REF] Lantuejoul | PD-L1 Testing for Immune Checkpoint Inhibitors in Mesothelioma: For Want of Anything Better[END_REF] or 50% or more [START_REF] Hellmann | Nivolumab plus ipilimumab as first-line treatment for advanced non-small-cell lung cancer (CheckMate 012 : results of an open-lable, phase 1, multicohort stud[END_REF] . A post-hoc analysis was performed with the 25% cutoff since there were not enough patients with 50% cells or more expressing PD-L1 in this series (zero and three in the nivolumab and combination groups respectively).When (p=0•047 or 0.003) with 28.8 and SP-263 assays, respectively. Thus, while the concordance kappa index was low (κ = 0•56), reflecting differences in sensitivity for low PD-L1 expressing tumours, analyses of response rates or DCR were similar whether using the 28.8 or Dako PD-L1 (SP263) assay. This observation further supports how consistently valuable PD-L1 expression is in predicting response to ICI and survival.

comparing
Lastly, we conducted exploratory subgroup post-hoc analyses for known prognostic factors in MPM, using an adjusted Cox model for the stratification variables, represented as a forest plot, in both arms, separately (Figure 5).

Discussion

Our findings show that nivolumab monotherapy and nivolumab plus ipilimumab provide clinically meaningful activity for patients with pre-treated MPM who progressed after one or 22 two lines of treatment including pemetrexed-platinum doublet, despite an excess of adverse events and three toxic deaths in the combination arm. Indeed, following the publication of a few small-sized trials suggesting that anti-PD-1/anti-PD-L1 antibodies exert activity in relapsed or refractory MPM, the MAPS-2 academic phase 2 randomised trial reached its disease control rate primary endpoint in both groups of patients treated also leading to remarkable overall survivals reaching or exceeding one year.

The nivolumab plus ipilimumab combination has likewise been evaluated in several different doses and schedules as first-line therapy in advanced NSCLC patients, such as in Checkmate CA209012 Phase I trial [START_REF] Hellmann | Nivolumab plus ipilimumab as first-line treatment for advanced non-small-cell lung cancer (CheckMate 012 : results of an open-lable, phase 1, multicohort stud[END_REF] . Early cohorts evaluated two different dosing schedules, of which nivolumab 3mg/kg plus ipilimumab 1 mg/kg, every 3 weeks resulted in significant toxicity, with 37% of patient discontinuing treatment due to treatment-related adverse events. Thus, four other combination cohorts were studied, including nivolumab 3 mg/kg every 2 weeks + ipilimumab 1mg/kg every 6 weeks, which was selected for the recently published NSCLC phase 3 first-line trial CheckMate 227 [START_REF] Hellmann | Nivolumab plus ipilimumab in lung cancer with a high tumor mutational burden[END_REF] and then was selected for our MPM trial.

The stratified, randomised nature of this trial ensured that both groups were well balanced.

Another strength of this study was its very fast accrual for such a disease that, given its rare incidence, could have been limited to an overly restrictive selection of patients. However, we acknowledge that our patients population could still be selected, good-prognosis group of patients, taking into account the favourable PS selection (PS 0-1), usual for a clinical trial but not necessarily representative of a wider population of 2 nd or 3 rd -line MPM patients.

We made a pragmatic choice to select the 12-weeks disease control rate (DCR) as the primary endpoint rather than overall response rate since patients displaying long-term control without any formal objective response criteria could also drive an essential part of the survival effect.

This methodological choice should be considered in the time context in which the trial was Group (iMig) meeting, similarly assessing the value of nivolumab (240mg every 2 weeks in contrast to 3mg/kg every 2 weeks in MAPS-2) plus ipilimumab (1mg/kg every 6 weeks) as second-/third-line treatment in MPM (85% of patients) or peritoneal mesothelioma [START_REF] Baas | Phase II trial of Nivolumab and Ipilimumab in patients with malignant mesothelioma[END_REF] . Their tolerance and efficacy results were likewise similar to those of the MAPS-2 trial. However, these exciting data now require confirmation with a comparative randomised Phase 3 trial that could determine whether the nivolumab+ipilimumab combination is superior or not to nivolumab alone or another single chemotherapy drug, such as vinorelbine or gemcitabine, which are commonly used in this setting, despite the lack of any data from prospective randomised trials. In fact, larger randomised trials are also needed in order to assess the reproducibility of the survival results, as well as evaluating their external validity, to confirm that the MAPS-2 patients are wholly representative of standard pre-treated MPM patients.

This would also be valuable in excluding the possibility that our patients who exhibited good general status after one or two treatment lines might exhibit more indolent tumour biology than the majority of the MPM population.

Some large Phase 3 randomized trials are, in fact, already ongoing in mesothelioma patients testing ICIs alone or in combination with chemotherapy or targeted therapies, as frontline or second/third-line treatment. 9 Taking into account for the increased toxicity observed in the combination arm, with 22.4% of withdrawals due to toxicity, three toxic deaths, we recognize that this regimen could be debatable in such 2 nd and 3 rd -line setting, although the 15.9 months long-term survival is also appealing. Only a Phase 3 trial would be able to provide definitive conclusions on that issue. At least, a large randomised Phase 3 trial ("CheckMate BMS CA209-743"; NCT 02899299; n=600) is currently assessing the benefit of nivolumab+ipilimumab vs. standard frontline chemotherapy in MPM patients, with PFS and OS as co-primary endpoints. Another ongoing Phase 3 trial is evaluating pembrolizumab+cisplatin-pemetrexed vs. cisplatin-pemetrexed in the same setting (Canadian Cancer Trials Group; NCT 02784171). Finally, two single-arm Phase 2 trials are assessing durvalumab+cisplatin-pemetrexed in the USA (NCT 02899195; n=55) and Australia ("DREAM" trial; n=54). The response results for the first 31 patients of this last trial were presented at the 2018 ASCO meeting, reporting an excellent dose-intensity of both chemotherapy (95%) and durvalumab (94%), a median PFS of 7.3 months, 95% CI [5.8-11],

leading to a 65% 6-month PFS and a remarkable 84% DCR [START_REF] Nowak | DREAM: A phase II study of durvalumab with first line chemotherapy in mesothelioma -First results[END_REF] .

While MPM patients have been shown to display low tumour mutational burden (TMB) [START_REF] Bueno | Comprehensive genomic analysis of malignant pleural mesothelioma identifies recurrent mutations, gene fusions and splicing alterations[END_REF] , a potential predictive biomarker for ICI in other tumour types, the pathogenesis of MPM seems to be mostly driven by inflammation. Accordingly, the MAPS-2 results suggest that nivolumab or nivolumab+ipilimumab efficacy may be strongest in PD-L1+ MPM patients, and particularly in high PD-L1 expressers (>25% positive tumour cells: PD-L1 hi>25% ). These subjects exhibit a 71•4% ORR and 62•5% ORR, respectively (28.8 assay is less sensitive than SP263 assay, respectively). However, there is a weakness with this finding related to the posthoc choice of a 25% cut-off point (data driven), given that, as previously reported in the literature [START_REF] Cedrès | Analysis of Expression of Programmed Cell Death 1 Ligand 1 (PD-L1) in Malignant Pleural Mesothelioma (MPM)[END_REF][START_REF] Antonia | Durvalumab after chemordiotherapy in stage III non-small-cell lung cancer[END_REF] , we found relatively low numbers of tumours with very high PD-L1 expression (i.e., over 50% of positive tumour cells) in our patients, compared to NSCLC patients as previously reported [START_REF] Mansfield | B7-H1 expression in malignant pleural mesothelioma is associated with sarcomatoid histology and poor prognosis[END_REF][START_REF] Cedrès | Analysis of Expression of Programmed Cell Death 1 Ligand 1 (PD-L1) in Malignant Pleural Mesothelioma (MPM)[END_REF][START_REF] Kindler | Biomarkers of pembrolizumab (P) activity in mesothelioma (MM): Results from a phase II trial[END_REF] . Another weakness comes from the fact this biomarker study was only possible in roughly 80% of patients with enough tumour tissue remaining. The 25% cutoff point was therefore a pragmatic choice taken from among all the different thresholds used in the literature so as to ensure a sufficient patient number in each subset to allow for statistical comparison. Similarly, as the non-comparative nature of our trial did not enable analysis of PD-L1 prognostic value arm by arm, we simply observed that the two groups of patients were well balanced in terms of PD-L1 high expression between (Table 1).

In an Australian cohort, PD-L1 high>50% subjects exhibited 50% ORR vs. 22% in PDL1 low <5% patients [START_REF] Rivalland | Outcomes of anti-PD-1 therapy in mesothelioma and correlation with PD-L1 expression[END_REF] , with a similar trend observed in a US cohort. [START_REF] Kindler | Biomarkers of pembrolizumab (P) activity in mesothelioma (MM): Results from a phase II trial[END_REF] A subgroup of mesothelioma patients thus seemingly benefit from ICIs, as already described in melanoma and NSCLC.

Nevertheless, it is still unclear how to accurately select the patients best suited for such immunotherapy. [START_REF] Kindler | Biomarkers of pembrolizumab (P) activity in mesothelioma (MM): Results from a phase II trial[END_REF] The complex interplay of tumour-infiltrating lymphocytes and immune checkpoints probably impacts on response to ICIs in MPM. More in-depth studies of immunohistochemistry markers and tumour infiltration by immune cells in MAPS-2 patients are thus ongoing. Therefore, it is of paramount importance that patient clinical characteristics (histologic subtype, previous chemosensitivity, PS) and biomarkers (such as tumour PD-L1

IHC status, CD8 or myelomonocytic infiltration extent, TMB, genomic signatures, [START_REF] Bueno | Comprehensive genomic analysis of malignant pleural mesothelioma identifies recurrent mutations, gene fusions and splicing alterations[END_REF] expression of multiple checkpoint inhibitors, specific mutations, etc. [START_REF] Reck | Pembrolizumab versus Chemotherapy for PD-L1-Positive Non-Small-Cell Lung Cancer[END_REF][START_REF] Thapa | The Immune Microenvironment, Genome-wide Copy Number Aberrations, and Survival in Mesothelioma[END_REF][START_REF] Suzuki | Chronic inflammation in tumor stroma is an independent predictor of prolonged survival in epithelioid malignant pleural mesothelioma patients[END_REF][START_REF] Ujiie | The tumoral and stromal immune microenvironment in malignant pleural mesothelioma: A comprehensive analysis reveals prognostic immune markers[END_REF][33][START_REF] Ayers | IFN-γ-related mRNA profile predicts clinical response to PD-1 blockade[END_REF] ) be prospectively investigated in all future immunotherapy trials for MPM patients. [START_REF] Lantuejoul | PD-L1 Testing for Immune Checkpoint Inhibitors in Mesothelioma: For Want of Anything Better[END_REF] We reported relatively high rates of drug-related adverse events with close to 90% rates of allgrade AEs in both arms and more Grade 3 AEs in the combination arm vs. the nivolumab arm (approximately 23% vs. 13%, respectively). However, It should be noted that all the three treatment-related deaths (all in the combination arm) occurred in the first months of the trial, with no other toxic deaths subsequently occurring over 14 months. This observation may suggest that our investigators experienced a learning curve in identifying immune-related adverse events and optimising treatment for these patients, as well as simultaneously optimising care for the NSCLC patients they started to treat routinely with anti-PD-1 at that time. It should also be underlined that the safety profiles of nivolumab alone or combined with ipilimumab compared favourably with what has already been posited in the literature for platinum-based chemotherapy, and that AEs observed in our trial were similar in type to those reported for immunotherapy drugs used in other settings and in numerous previous trials.

This AE incidence could certainly raise questions regarding the chosen dosing schedule in the combination arm. As mentioned above, we chose what was considered to be the most tolerable of six schemes previously tested in the Checkmate 012 Phase 1 trial [START_REF] Hellmann | Nivolumab plus ipilimumab as first-line treatment for advanced non-small-cell lung cancer (CheckMate 012 : results of an open-lable, phase 1, multicohort stud[END_REF] , and this scheme being subsequently selected for the NSCLC Phase 3 Checkmate 227 trial [START_REF] Hellmann | Nivolumab plus ipilimumab in lung cancer with a high tumor mutational burden[END_REF] and the ongoing first-line Phase 3 Checkmate 743 trial in MPM patients (NCT02899299). It is also paramount to note that most AEs were classed as Grade 1-2, including a substantial number that were purely biological or asymptomatic, rapidly resolving either spontaneously or once treatment was interrupted. Moreover, our preliminary QOL analysis failed to detect any obvious consequences on patient-reported outcome items at Week 12. Taking into account potential immunotherapy-induced harm, it was crucial to early report some QOL data, even if these data were unpowered and incomplete. We are aware that the scientific considerations regarding estimating a minimally clinically important difference (MCID) are challenging and that our choice of a 10-point change in the scores as a cut-off point has not been prospectively validated for MPM patients in the literature. To resolve this, additional studies of healthrelated quality of life data from the MAPS-2 trial are underway, modeling longitudinal QOL based on the time until definitive deterioration (TUDD), the results of which will be reported in a separate paper dedicated to this outcome. Additionally, it should be underlined that the study's open-label design could have influenced the QoL analysis.

We provide exploratory subset analysis, although carefully avoiding to directly compare the two groups of patients, according to methodological rules for a randomized Phase 2 trial, which was not powered for such comparisons. Dual PD-1/CTLA-4 inhibition in "more indolent" tumours (i.e,. patients relapsing more than 3 months after pemetrexed-based chemotherapy was stopped) provided a 0.35 [0.19-0.67] adjusted HR for death vs. those with more "aggressive" cancers. However, such exploratory analyses should be considered as purely hypothesis-generating and must thus not be over-interpreted, taking into account the wide-ranging 95% confidence intervals observed, reflecting the low patient numbers in each subset, and considering, although these analyses were adjusted for stratification factors, that some biases could have been induced by the prognosis influence of other variables. Hence, the optimal strategy in view of a better efficacy/safety (and cost) ratio remains to be defined for all patients. Of note, we suspected in our trial that a few patients would exhibit hyperprogression under ICIs, as previously described in some NSCLC cases. [START_REF] Champiat | Hyperprogressive Disease is a new pattern of progression in cancer patients treated by Anti-PD-1/PD-L1[END_REF] Additional studies for these patients are on-going, in particular to assess whether their tumour growth rate, defined as a >50% increase in tumour volume over time, could actually correspond to the still-debated criteria for hyper-progressive disease, thus requiring to analyze two CT scans performed before the ICI treatment was initiated.

In conclusion, as previously observed in melanoma and NSCLC patients, immunotherapy appears to offer real hope for patients with MPM, a cancer which, until now, had very few therapeutic options. Thus, ICIs are likely to change our standard of care in MPM, as already emphasized for nivolumab alone or combined with ipilimumab in the NCCN guidelines 7 . Yet many questions remain unanswered, and more data is required from randomised Phase 2 or 3 trials to select the best candidates suited for ICIs (pretreated vs. frontline patients, biomarkers, tolerance, etc.) and to define the long-term survival benefit, as well as the optimal treatment regimen (anti-PD- To enter data, just enter in each line the upper limit, the lower limit of the confidence interval and the estimate point in this sequence. If you want each line to have a label in the graph, type it in the column label.

Unfortunately, the Excel does not allow us to rotate the graph, so you have to use another trick: Copy the graph to a Power Point blank slide. Select the graph. In the Draw menu click in "Ungroup", accept the prompt and then in "Group". In the same menu, choose "Rotate or Flip" and then "Rotate Left". Be careful to not click in other places while in the group/ ungroup process. You will have to select all parts of the ungrouped graph if this happens.

In Excel, its possible to change any of the parameters that you want, by changing the proprieties of the graph. This sheet can be changed and distributed free of charge. We ask you to give us the appropriate credit using any kind of citation we included this sugestion in the sheet

  atezolizumab OR avelumab OR durvalumab OR ipilimumab OR tremelimumab OR PD-1 OR PD-L1, OR CTLA-4". Additionally, we examined abstracts from the 2015, 2016 and 2017 editions of the American Society of Clinical Oncology (ASCO) annual meeting. Though several studies confirmed that mesothelioma tumour cells do express immune checkpoint proteins, including PD-L1, and that mesothelioma specimens at times demonstrate high stromal infiltration by immune cells like lymphocytes or mono-macrophage and dendritic cells, we found no published clinical studies investigating the safety or efficacy of the anti-PD-1 nivolumab and anti-CTLA-4 ipilimumab combination or anti-PD-1 nivolumab monotherapy in human malignant pleural mesothelioma (MPM) patients. One large randomised Phase IIb trial evaluating tremelimumab, an anti-CTLA-4 antibody, tested this agent administered alone versus placebo as second-or third-line treatment in 564 MPM patients, yet no survival gain was demonstrated compared to placebo. Also, a Phase Ib trial was recently published reporting 20% overall response rate (ORR) (95% CI: 6•8-40•7) and 52% stable disease in 25 patients with PD-L1-expressing MPM (>1% positive tumour cells) treated with the anti-PD-1 antibody pembrolizumab, mostly as second-line treatment, along with a median duration of response (DOR) of 12 months (95% CI: 3•7-not reached), without any safety concerns. Lastly, a single-arm Phase II trial ("NIBIT-MESO-1"; NCT02588131) assessed the combination of 1mg/kg anti-CTLA-4 tremelimumab and 20mg/kg anti-PDL-1 durvalumab, intravenously injected every 4 weeks in four doses and followed by maintenance durvalumab, as first-or second-line treatment in unresectable malignant mesothelioma patients. The authors reported evidence of clinical activity, demonstrated by 27•5% (out of 40 patients) exhibiting immune-related (ir) partial response (median DOR: 16•1 months) and another 65%-patient subset exhibiting ir disease control, resulting in a median ir PFS of 8 months and mOS of 16•6 months.Added value of this studyOn Nov. 15 th 2015, no second-or third-line treatment had as yet demonstrated efficacy in MPM patients who had received first-line pemetrexed-platinum-based chemotherapy with or without the anti-angiogenic agent bevacizumab, namely the current reference first-line strategy in unresectable MPM patients. Furthermore, no PD-1/PD-L1-directed antibodies, non-PD-1-directed targeted immunotherapies or dual immunotherapies had been approved for MPM indications. There is thus a significant unmet need for new therapeutic strategies assessing immunotherapies in relapsed MPM patients. This study achieved its statistical endpoint and was, to our best knowledge, the first to assess the safety and efficacy of singletherapy anti-PD-1 nivolumab or anti-PD-1 nivolumab and anti-CTLA-4 ipilimumab combination therapy in MPM patients as second-or third-line following first-line pemetrexedplatinum-based chemotherapy. These findings provide supporting evidence that both single therapy with nivolumab and combination therapy with both nivolumab and ipilimumab are effective in treating relapsed MPM patients.

Formalin-fixed tumour

  samples were collected and PD-L1 expression was assessed by immunohistochemistry at a central laboratory (Lyon, Léon Bérard Cancer Center) by PD-L1 IHC, using both 28.8 Dako pharmDx™ and SP-263 monoclonal antibody clones, on a Dako AS Link48 platform, according to previously-described laboratory-developed tests for SP-263 clones using the Dako platform 20 with formalin-fixed tumour samples obtained by thoracoscopy or CT-guided core-needle biopsies by the National Reference Center (NRC) for MPM pathological diagnosis certification MESOPATH (F. Galateau-Sallé and S.

  ). Drug delivery was relatively good: approximately 70% received the first six infusions (Nivo or Nivo+Ipi) as initially planned, at 100% of the planned drug dose; 49•2% Nivo patients (n=31) and 38•7% Nivo+Ipi (n=24) received 10 planned infusions (both drugs injected by infusion on Days 1, 4, 7 and 10 in the Nivo+Ipi arm). After a median follow-up of 20•1 months (IQR: 18•4-20•8), 59 patients had discontinued treatment in the Nivo arm, 50 of whom (84•7%) exhibited disease progression, three (5•1%)

  Figure 2 presents waterfall plots of percentage changes in tumour size between baseline and 12 weeks in Nivo vs. Nivo+Ipi groups for the 114 patients evaluable at 12 weeks, revealing major tumour response irrespective of histological subtype. In contrast, as early as the 12 th week of treatment at the first tumour response evaluation, 6 out of 59 (10.1%) and 2 out of 55 (3.6%) patients treated with nivolumab or nivolumab+ipilimumab, respectively, exhibited over 80% increase in size of their target lesions suggesting hyper-progression, with no obvious correlation with the histological subtype found. Treatment duration, time to response and treatment response duration are presented on the swimmer plots in Figure 3 for patients with objective response. Median DORs were 7•4 months (4•1-11•9) and 8•3 months (3•0-14•0) in Nivo and Nivo+Ipi, respectively, with four (one still on Nivo) and seven (two still on the combination) patients still responding at 15 months in Nivo and Nivo+Ipi, respectively. After a median follow-up of 20•1 months (95% CI: 19•6-20•3), median PFS were 4•0 months (95% CI: 2•8-5•7) and 5•6 months (95% CI: 3•1-8•3) in Nivo and Nivo+Ipi, respectively with 58/63 and 53/62 events (i.e. progression or death) recorded at study cut-off (Figure 4A

Figure 1 :Figure 2 :Figure 3 :Figure 4 :Figure 5 :

 12345 Figure 1: Patient flow chart

  et Ipilimumab Instructions for the use of Forest Plot Excell sheet: This sheet does not allow you to calculate anything, or to perform a meta-analysis. It only plots results in a forest-plot like graph. It's previously set to have nine entries, but you can change this easily.

  [START_REF] Robinson | Malignant mesothelioma[END_REF] MPM patients display usually a dismal prognosis, with a median overall survival (mOS) of approximately 12 months, even if some selected MPM patients could exhibit surprisingly long tumour doubling time indicative of a more indolent tumour, specifically in second or third-line setting. Moreover, they often exhibit strong resistance to

chemotherapy, and only few patients are suitable candidates for multimodal treatment including "radical" surgery.

[START_REF] Scherpereel | Guidelines of the European Respiratory Society and the European Society of Thoracic Surgeons for the management of malignant pleural mesothelioma[END_REF]

In 2015, our group initiated a Phase 3 randomised open-label IFCT 0701 MAPS (Mesothelioma Avastin Cisplatin Pemetrexed Study) trial that revealed an overall survival (OS) benefit when adding bevacizumab to standard cisplatin plus pemetrexed chemotherapy (18•8 vs. 16•1 months; hazard ratio [HR]=0•77; p=0•017).

  Nivolumab, Ipilimumab and an unrestricted research grant were provided by Bristol-Myers-Squibb (BMS) (France). The Intergroupe Francophone de Cancérologie Thoracique (IFCT) designed the whole trial, collected and interpreted the data. IFCT investigators and staff participated in study design and data analysis. BMS had no role in designing the research, analyzing the data or writing the report. The corresponding author (AS) and co-principal investigator (GZ) had full access to all study raw data and took final responsibility in the decision to publish the trial data.

	Role of the funding source
	L1 tumour expression
	(28.8 PharmDX or SP263 Assays) using a non-adjusted Cox model. This IFCT-1501 trial was
	registered beforehand with the European Union Clinical Trials Register (number 2015-
	004475-75) and ClinicalTrials.gov (number NCT 02716272). The whole protocol study is

th , 2017. In subgroup analyses, HRs and 95%CIs were estimated using a Cox model adjusted for stratification factors. For statistical analyses, SAS software Version 9.4 was employed, with all p values and CIs two-sided. We tested the prognostic effect of PDavailable in the supplementary web appendix (pp 1-75).

Results

Between

March 24, 2016, and August 25, 2016, we randomly assigned 125 patients to Nivo (63 [50•4%]) or Nivo+Ipi (62 [49•6%]), with 63 (100%) receiving Nivo and 61 (>98%)

  MPM histology was epithelioid in 52 (83%) and biphasic or sarcomatoid in 11 (17%) of the Nivo patients; vs. 53 and nine (85% and 15%) in the Nivo+Ipi patients, respectively, as assessed by central review of the NRC MESOPATH (Table1). Most patients experienced

	progression beyond a period of 3 months after receiving first-line pemetrexed-platinum-based
	chemotherapy (59% Nivo, n=37; 66% Nivo+Ipi, n=41); the majority of patients had received
	only one therapy line prior to inclusion (70% and 68%, respectively). Blood counts were
	balanced (leukocytes, red cells, estimated by haemoglobin concentration, and platelets)
	total, 63
	patients were randomized in the nivolumab arm and all received at least one infusion of
	treatment, 62 were randomized in the combination arm, with only 61 treated since one patient
	died before receiving any treatment.
	Median age was 72•3 [32•5-87•2] in the Nivo arm and 71•2 [48•1-88•1] in Nivo+Ipi. The
	Nivo arm comprised 25% (n=16) women and Nivo+Ipi 15% (n=9); 89% of Nivo (n=56) and

82% of Nivo+Ipi (n=51) patients exhibited Stage III-IV disease, while 31% of Nivo (n=19) and 40% of Nivo+Ipi (n=25) patients had ECOG PS 0.

Table 1

 1

	•4% [31•2-57•7%] 12-week DCR (n=24/54) in the Nivo group
	and 50% [36•7-63•3%] 12-week DCR (n=27/54) in Nivo+Ipi, as centrally assessed by an
	independent blinded radiological expert panel. Objective response rates were 18•5% [8•2-
	28•9%] (n=10/54) and 27•8% [15•8-39•7%] (n=15/54) in Nivo and Nivo+Ipi, respectively. In
	the ITT population, comprising 125 patients, the 12-week DCR was 39•7% [27•6-51•8%]
	(n=25/63) and 51•6% [39•2-64•1%] (n=32/62) in Nivo and Nivo+Ipi, respectively
	(supplementary

, webappendix p.3).

Table 2

 2

	3•2% and 5/61, 8•2% for Nivo and Nivo+Ipi, respectively). All-grade diarrhoea events
	occurred frequently in Nivo+Ipi and Nivo patients (9/63, 14•3% and 18/61, 29•5 % for Nivo
	and Nivo+Ipi, respectively). All-grade pruritus were frequent in both arms (6/63, 9•5% and
	15/61, 24•6% for Nivo and Nivo+Ipi, respectively). No differences in drug-related
	haematological AE rates were noted (0% Grade 3-4 in both arms). Immune-related AEs
	(irAEs), listed in Table 3, ranged from 0 to 4/63, 6.34% and 0 to 7/61, 11•4% in Nivo and
	Nivo+Ipi, respectively, for any-grade toxicities. No Grade 4 irAEs were reported, except for
	1•6% of cases exhibiting increased lipase levels in both arms. Most all-grade immune-related
	toxicities were biological, causing neither relevant clinical consequences nor treatment

presents all drug-related non-haematological AEs of any grade, in 124 patients evaluable for safety, for all treatment injections, involving 10% of patients, with very low Grade 4 (0 and 1/61, 1•6% for Nivo and Nivo+Ipi, respectively) or Grade 3 incidences (2/63, interruption. There also were all-grade asymptomatic elevations of aminotransferase ALT levels in both arms (n=1/63, 1•6% in Nivo vs. n= 8/61, 13•1% in Nivo+Ipi). The incidence of Grade 3, 4, 5 and all-grade AEs is summarized in the dataset of Supplementary Table

  designed, namely in mid-2015. At that time, we only had scarce data on the efficacy of immunotherapy in MPM or the ICI tolerance of MPM patients, who are slightly older than patients with NSCLC or melanoma patients, typical cancer types evaluated in the vast majority trials assessing ICI. We also had no idea about the best regimen to choose, either single anti-PD-1 mAb or dual immunotherapy combination, and thus were undecided about the optimal statistical comparative hypothesis to propose with survival (PFS or OS) as primary endpoint, supposing a preconceived idea about the best ICI regimen. Such considerations led us to choose a classical non-comparative design for a randomised phase 2 trial, not powered for face to face comparisons, but instead allowing to study simultaneously two ICI regimens. The Phase 2 nature of this trial was a conservative choice aimed solely to detect early efficacy (and tolerance) signals of two different immunotherapy regimens at the same time, with no preconceptions, and to select at least one of these regimens for a future comparative Phase 3 trial.

	profiles, providing clear data for selecting adequate experimental arms in future prospective
	comparative trials. Of note is that the MAPS-2 results do already support a recent NCCN
	panel decision (NCCN guidelines Malignant Pleural Mesothelioma, Version 2.2018-February
	26, 2018) to recommend nivolumab or nivolumab+ipilimumab as options for second/third-
	line therapy in MPM. 7 Interestingly, another small and non-randomised trial ("INITIATE";

An impressive response was achieved with nivolumab and nivolumab+ipilimumab in secondor third-line MPM treatment. Additionally, ORR as assessed by central review were clinically meaningful, and mOS were also noteworthy in that setting (up to the significant values of 11.9 and 15.9 months, respectively), compared to what was previously reported with standard chemotherapies or investigational targeted therapies. Thus, the MAPS-2 study was able, by its randomised design to assess that both regimens demonstrated activity signs yet distinct safety n=34) by P. Baas et al. was recently presented at the 2018 International Mesothelioma Interest

  1 monotherapy vs. combination with ICIs, chemotherapy or targeted therapy, or even surgery or radiotherapy). It is essential that MPM expert centres from all over the world collaborate to speed-up the recruitment of patients with this rare cancer into large, randomised trials and translational studies, to bring new hope and real progress in the future of mesothelioma patient care.CAV reports personal fees from Roche, personal fees from Pfizer, personal fees from Boehringer Ingelheim, personal fees from Novartis, personal fees from AstraZeneca, personal fees from Lilly, personal fees from Amgen, personal fees from Bristol-Myers Squibb,

	personal fees from Sysmex, personal fees from MSD, personal fees from Clovis
	Oncology, from Abbvie, outside the submitted work;
	OM reports personal fees from Bristol-Myers Squibb, outside the submitted work;
	FG reports personal fees and non-financial support from 3MS, personal fees from
	MSD/MERCK US, personal fees from AstraZeneca, personal fees from Roche, grants and
	personal fees from Boehringer Ingelheim, non-financial support from Chugai, outside the
	submitted work;
	DP reports personal fees from Astrazeneca, personal fees from Boehringer, personal fees from
	BMS, personal fees from Celgene, personal fees from Novartis, personal fees from MSD,
	personal fees from Pfizer, personal fees from Roche, outside the submitted work;
	DMS reports personal fees and non-financial support from BMS, during the conduct of the
	study; personal fees from Roche, personal fees from MSD, personal fees from lilly, personal
	fees from Novartis, personal fees from Pfizer, personal fees from astra zeneca, personal fees
	from Boehringer Ingelheim, personal fees from Takeda, personal fees from leurquin
	mediolanum, outside the submitted work;
	GZ reports grants, personal fees and non-financial support from BMS, during the conduct of
	the study; non-financial support from ROCHE, personal fees and non-financial support from
	ASTRA-ZENECA, non-financial support from MSD, non-financial support from PFIZER,

personal fees from DA VOLTERA, outside the submitted work; JM, SL, PD, EA, FM, CLP, TU and MLS have nothing to disclose

Table 1 : Baseline patient characteristics per treatment arm txNumInclusion Response RECIST_MESO TRTGRP marquage_mb_numerique dec__typhisto histo marquage_mb_numeriquebis toto Position Partial response Disease progression Stable disease

 1

	/L	17 (27)	19 (31)
	PD-L1 status available (28•8 mAb, Dako PharmDx™)	50 (79%)	49 (79%)
	Negative	31 (62%)	27 (55%)
	≥1%	19 (38%)	22 (45%)
	≥25%	2 (4%)	5 (10%)
	≥50%	0	3 (6%)
	Nivo=nivolumab. Nivo+Ipi=nivolumab plus ipilimumab.SD=standard
	deviation. Pem= pemetrexed. TNM=Tumour Node Metastasis classification.
	PD-L1=programmed cell death-ligand 1. mAb=monoclonal antibody

  : Clark O; Djulbegovic B. Forest plots in excel software(Data sheet). 2001. Available at www.evidencias.com.

	Otavio Clark
	Benjamin Djulbegovic
	H Lee Moffitt Cancer Center
	Tampa, FL, USA
	clark@evidencias.com

Acknowledgments

Bristol-Myers-Squibb (BMS) (France) supplied nivolumab/ipilimumab and a research grant to Intergroupe Francophone de Cancérologie Thoracique (IFCT) for the MAPS-2 biomarkers studies. We thank the participating patients and their families as well as the study teams involved in the trial, the clinical research assistants, study coordinators and IFCT operations staff, as well as Dr. G. Cremer at Cremer Consulting (Strasbourg, France) for medical writing support. We thank also the French National Cancer Institute (INCa) and the French League against Cancer.

Contributors

AS and GZ were the principal investigators of the trial and were involved in conception and study design. All investigators, including FM and EA, were involved in the data collection. SL contributed to pathological review and diagnostic certification, immunohistochemistry analysis. All authors contributed to the data analysis, data interpretation, the writing and the approval of the final manuscript.

Declaration of interests

AS reports personal fees and non-financial support from BMS, personal fees and nonfinancial support from Roche, personal fees and non-financial support from MSD, personal fees from Boehringer-Ingelheim, outside the submitted work;

LG reports grants, personal fees and non-financial support from ROCHE, personal fees and non-financial support from LILLY, grants, personal fees and non-financial support from NOVARTIS, personal fees and non-financial support from PFIZER, personal fees and nonfinancial support from BRISTOL-MYERS SQUIBB, personal fees and non-financial support from BOEHRINGER INGELHEIM, personal fees and non-financial support from ASTRA ZENECA, personal fees and non-financial support from ABBVIE, personal fees and nonfinancial support from MSD, outside the submitted work; OB reports personal fees and non-financial support from MSD, non-financial support from Roche, outside the submitted work; IM reports other from BMS (medical congress), during the conduct of the study; RC reports participation to board with Roche and BMS.

Data sharing statement

The Data Sharing statement is available in the webappendix (p. 8) Lipase increase

Tables