domain: sdv sde

text:
Caroline Djian-Caporalino
email: caroline.caporalino@inra.fr

Thierry Mateille

Marc Bailly-Bechet

Nathalie Marteu

Ariane Fazari

Pierre Bautheac

Alizée Raptopoulo

Luan Van Duong

Johannes Tavoillot

Bernard Martiny

Claire Goillon

Philippe Castagnone-Sereno

Evaluating sorghums as green manure against root-knot nematodes

Keywords: Meloidogyne sp., Sorghum vulgare var. sudanense, sudangrass hybrids, cover crop, trap crop, biofumigation

Current restrictions on the use of chemical nematicides have led to an increase in rootknot nematode (RKN) damages in horticultural crops. The effects of two sorghums as summer cover crops, Sorghum sudanense sudangrass cv. 'Piper' or sudangrass hybrid [S. bicolor x S. sudanense] '270911', respectively with low and high dhurrin contents, were compared in their ability to suppress RKN in a vegetable production system. The use of both sorghums 'Piper' and '270911' as a green manure was found to be an effective strategy for decreasing RKN infestation in the soil, thereby protecting the subsequent planting of RKN susceptible crops (chard, lettuce or melon). Analytical experiments were further conducted in growth chamber and greenhouse pot experiments to investigate and compare the susceptibility of the sorghums and the factors affecting their efficacy for RKN management, in order to better explain the results obtained in the field trial. The two sorghums were poor hosts of RKN, acted as trap crops and as a biofumigant releasing hydrogen cyanide. Time of planting, time of biofumigation, and type of soil affected their efficacy for RKN management. For best RKN suppression, the sorghum cover crops need to be cultivated during one month or less and biofumigated for one month prior to crop planting. The trapping effect of both sorghums in clayey soil was less efficient than in sandy or sandy-loamy soils. Combining less than 30-days of sorghum culture and 10-days soil incorporation with solarization mulch was particularly efficient in suppressing nematodes. No effect relative to the sorghum type was detectable as long as they were used appropriately.

Introduction

Intensive monoculture of specialized horticultural crops is becoming vulnerable to damage from plant-parasitic nematodes (PPN), especially root-knot nematodes (RKN, Meloidogyne spp.) worldwide [START_REF] Jones | Top 10 plant-parasitic nematodes in molecular plant pathology[END_REF]. In Mediterranean regions, RKN are most destructive to vegetable farms [START_REF] Abd-Elgawad | Yield losses by phytonematodes: challenges and opportunities with special reference to Egypt[END_REF][START_REF] Djian-Caporalino | Root-knot nematodes (Meloidogyne spp.), a growing problem in French vegetable crops[END_REF][START_REF] Talavera | Perception of the impact of root-knot nematode-induced diseases in horticultural protected crops of south-eastern Spain[END_REF]. Accentuated by the current restrictions on the use of chemical nematicides [START_REF] Mbtoc | Report of the Methyl Bromide Technical Options Committee. Non-chemical Alternatives Adopted as Replacements to Methyl Bromide on a Large Scale[END_REF]EC Directive, 2009), there is an urgent need to develop innovative, low-input, ecologically sound and efficient solutions for managing these pests.

Use of cover crops in rotation to vegetable crop production has been recommended to farmers as a mean to improve fertility, physical and chemical properties of soil [START_REF] Fortuna | Optimizing nutrient availability and potential carbon sequestration[END_REF][START_REF] Séguy | Principes et fonctionnement des écosystèmes cultivés en semis direct sur couverture végétale[END_REF]. Cover crops improve the productivity of subsequent crops by reducing pressure of pests and pathogens [START_REF] Ratnadass | Plant species diversity for sustainable management of crop pests and diseases in agroecosystems: a review[END_REF], serving as non or poor hosts for RKN [START_REF] Djian-Caporalino | Nematicidal and nematode-resistant plants[END_REF], and thus contribute to nematode management [START_REF] Mcsorley | Multiple cropping systems for nematode management: a review[END_REF]. Cover crops documented to be capable of managing RKN include Phacelia spp., Avena sativa [START_REF] Carneiro | Seleçâo preliminar de plantas para rotaçâo de culturas em âreas infestadas por Meloidogyne incognita nos anos de 1979 e 1980[END_REF], brassicas, such as Sinapis alba, Eruca sativa, and Raphanus sativus [START_REF] Curto | The potential of Crotalaria juncea L. as a summer green manure crop in comparison to Brassicaceae catch crops for management of Meloidogyne incognita in the Mediterranean area[END_REF][START_REF] Kruger | Cover crops with biofumigation properties for the suppression of plant-parasitic nematodes: a review[END_REF], and grasses, such as Pennisetum glaucum, Sorghum bicolor 'forage sorghum' or Sorghum sudanense 'sudangrass', formerly classified as S. vulgare var. sudanense, and S. bicolor x S. sudanense 'sudangrass hybrids' [START_REF] Mcsorley | Host status of selected tropical rotation crops to four populations of root-knot nematodes[END_REF][START_REF] Mojtahedi | Suppression of Meloidogyne chitwoodi with Sudangrass cultivars as green manure[END_REF][START_REF] Sipes | Root-knot nematode management in dryland taro with tropical cover crops[END_REF][START_REF] Timper | Root-knot nematode resistance in pearl millet From West and East Africa[END_REF]. They have been tested as winter or summer cover crops for limiting RKN populations. These cover crops, which are used as green manure, can be used as trap crops if the nematodes penetrate the roots but cannot complete their cycle. They can also be used as biofumigant crops that release volatile compounds when soil incorporated [START_REF] Djian-Caporalino | Nematicidal and nematode-resistant plants[END_REF]. Interest in biofumigation for soilborne disease management has recently increased due to its compatibility with environmental friendly management [START_REF] Kruger | Cover crops with biofumigation properties for the suppression of plant-parasitic nematodes: a review[END_REF][START_REF] Prasad | Biofumigation: success and prospects in soilborne plant disease management[END_REF]. One group of cover crops with effective biofumigation effect against RKN is forage sorghum or sudangrass. Sorghum has been well known to release hydrogen cyanide (HCN) following the hydrolysis of dhurrin, a cyanogenic glycoside typically present in sorghum [START_REF] Chitwood | Phytochemical based strategies for nematode control[END_REF][START_REF] Curto | Evaluation of the activity of dhurrin and sorghum towards Meloidogyne incognita[END_REF][START_REF] De Nicola | A simple analytical method for dhurrin content evaluation in cyanogenic plants for their utilization in fodder and biofumigation[END_REF].

Sudangrass hybrids produce larger amounts of biomass than non-hybrid sudangrass. Both sudangrass and its hybrids have a long root system penetrating deep into the soil, and they are used as a green manure to improve soil porosity, and attract soil nutrients present at depth up towards the top soil layer [START_REF] Abawi | Impact of soil management practices on soil-borne pathogens, nematodes and root diseases of vegetable crops[END_REF][START_REF] Kratochvil | Evaluation of crop rotation and other cultural practices for management of root-knot and lesion nematodes[END_REF]. Sudangrass require little management, beside seed sowing and sprinkler irrigation after soil tillage. The cover crop grows for 1.5 to 2 months, and its biomass is then mowed and soil incorporated [START_REF] Delamarre | Désinfection des sols sous abri froid[END_REF]. A number of studies, summarized in the review by [START_REF] Quaranta | Effet des plantes de service sur les bio-agresseurs des cultures.-Etude[END_REF], have reported activity against several pests and diseases. In the case of PPN, forage sorghum or sudangrass have been reported to be effective at reducing the numbers of Helicotylenchus dihystera [START_REF] Wang | Winter cover crops and nematode densities[END_REF], Rotylenchus reniformis [START_REF] Asmus | Cover crops for reniform nematode suppression in cotton: greenhouse and field evaluations[END_REF] and Pratylenchus penetrans [START_REF] Lamondia | Integrated management of strawberry pests by rotation and intercropping[END_REF], but were found to facilitate the multiplication of Belonolaimus longicaudatus [START_REF] Rhoades | Effects of cover crops and fallowing on populations of Belonolaimus longicaudatus and Meloidogyne incognita and subsequent crop yields[END_REF][START_REF] Weingartner | Management strategies in potato for nematodes and soil-borne diseases in subtropical Florida[END_REF], Mesocriconema sp. [START_REF] Crow | Effect of sorghum Sudangrass and velvetbean cover crops on plant-parasitic nematodes associated with potato production in Florida[END_REF], Paratrichodorus minor [START_REF] Mcsorley | Nematode population changes and forage yields of six corn and sorghum cultivars[END_REF][START_REF] Mcsorley | Host status of selected tropical rotation crops to four populations of root-knot nematodes[END_REF][START_REF] Mcsorley | Effect of tropical rotation crops on Meloidogyne incognita and other plant-parasitic nematodes[END_REF] and Tylenchorhynchus sp. [START_REF] Crow | Effect of sorghum Sudangrass and velvetbean cover crops on plant-parasitic nematodes associated with potato production in Florida[END_REF]. [START_REF] Mcguidwin | Response of nematode communities to sudangrass and sorghum-sudangrass hybrids grown as green manure crops[END_REF] reported the maintenance or an increase in populations of Pratylenchus, Longidorus, Xiphinema and Paratrichodorus after the incorporation of some varieties of sudangrass and sudangrass hybrids. Conversely, these plants were considered to be non-hosts or poor hosts for RKN [START_REF] Colbran | Problems in tree replacement. III. The effects of cultural practices and soil fumigation on root lesion nematodes and on the growth of apple replants[END_REF][START_REF] Fay | An assessment of allelopathic potential in Avena germplasm[END_REF][START_REF] Carneiro | Seleçâo preliminar de plantas para rotaçâo de culturas em âreas infestadas por Meloidogyne incognita nos anos de 1979 e 1980[END_REF][START_REF] Hagan | Nematode suppressive crops[END_REF][START_REF] Ritzinger | Review: effect of fresh and dry organic amendments on Meloidogyne arenaria in greenhouse experiments[END_REF][START_REF] Védie | Lutte contre les nématodes a galles : test de différents engrais verts nématicides: compte-rendu d'essai 2005[END_REF], such as Meloidogyne incognita and M. hapla in particular [START_REF] Chitwood | Phytochemical based strategies for nematode control[END_REF][START_REF] Ferraz | Use of antagonistic plants and natural products[END_REF][START_REF] Mcsorley | Effect of tropical rotation crops on Meloidogyne incognita and other plant-parasitic nematodes[END_REF][START_REF] Viaene | Management of Meloidogyne hapla on lettuce in organic soil with Sudangrass as a cover crop[END_REF][START_REF] Wang | Winter cover crops and nematode densities[END_REF]. [START_REF] Mojtahedi | Suppression of Meloidogyne chitwoodi with Sudangrass cultivars as green manure[END_REF] and [START_REF] Widmer | Mechanism of suppression of Meloidogyne hapla and its damage by a green manure of Sudangrass[END_REF] also reported that they released HCN, with biofumigation effects against M. hapla or M. chitwoodi. [START_REF] Wang | Winter cover crops and nematode densities[END_REF] and [START_REF] Guerena | Nematodes: Alternative controls. National sustainable agriculture information service[END_REF] reported that the prior use of sudangrass hybrids as a cover crop decreases the density of populations of M. incognita on Secale cereale 'rye', Lupinus angustifolius 'lupin' or Glycine max 'soybean'. However, poor results were obtained when the crops were combined and sudangrass or sudangrass hybrids residues were incorporated into the soil while still green (Orfonedes, 1995;Widner and Abawi, 2002). The best results for RKN suppression in Florida were obtained with the use of forage sorghum or sudangrass hybrids as a rotation crop [START_REF] Dover | Nematode Management Using Sorghum and Its Relatives[END_REF][START_REF] Gill | Cover Crops for Managing Root-Knot Nematodes[END_REF], but the benefits of crop residues for nematode suppression were not assessed in this study. In France, sorghum residues have been found to be only partly effective in the field, often yielding variable results [START_REF] Abawi | Impact of soil management practices on soil-borne pathogens, nematodes and root diseases of vegetable crops[END_REF][START_REF] Collange | Root-knot nematode (Meloidogyne) management in vegetable crop production: The challenge of an agronomic system analysis[END_REF][START_REF] Mcguidwin | Response of nematode communities to sudangrass and sorghum-sudangrass hybrids grown as green manure crops[END_REF][START_REF] Thoden | Organic amendments and their influences on plant-parasitic and free-living nematodes: a promising method for nematode management?[END_REF]. However, interest in the use of biofumigation in vegetable crop rotations has recently increased [START_REF] Curto | Biofumigant effect of new defatted seed meals against the southern root-knot nematode, Meloidogyne incognita[END_REF][START_REF] Goillon | Utiliser le sorgho pour lutter contre les nématodes à galles[END_REF][START_REF] Prasad | Biofumigation: success and prospects in soilborne plant disease management[END_REF], and deserves futher work.

Sorghum is the most widely used green manure in vegetable cropping systems in the South of France. In this context, both analytical experiments under controlled conditions and a field experiment evaluating sorghums as green manure against RKN were performed. An innovative co-design process using a participatory approach involving scientists (geneticists, plant pathologists, agronomists), technical advisers and farmers, was used to take into account technical and socio-economic constraints [START_REF] Djian-Caporalino | Varietal and technical innovations for the sustainable and integrated managements of root knot nematodes[END_REF]). This 'system approach', recommended by the 'EIP-AGRI Focus Group IPM practices for soil-borne diseases ' (2015), allows the farmer to change some crops of the rotation and treatments according to market constraints and decision rules established during the co-design process. But the field experiment has to last at least two seasons. 'Piper' is the most commonly used sudangrass variety in France as green manure but it has low dhurrin content [START_REF] Mojtahedi | Suppression of Meloidogyne chitwoodi with Sudangrass cultivars as green manure[END_REF], containing only 2.7 mg/g of dry matter after three weeks of growth [START_REF] Gard | Le sorgho fourrager comme engrais vert à effet assainissant: évaluation agronomique et potentiel pour la biofumigation[END_REF]. Low dhurrin content in sudangrass is selected for low animal toxicity when used as feed [START_REF] Chambliss | Producing millets and sorghum[END_REF]. On the other hand, high level of dhurrin has been found in sudangrass hybrid '270911' which was developed for biofumigation, containing 12.7 mg/g dhurrin in dry matter after three weeks of growth [START_REF] Gard | Le sorgho fourrager comme engrais vert à effet assainissant: évaluation agronomique et potentiel pour la biofumigation[END_REF].

The objectives of this study were 1) to compare the effects as summer cover crops of the two sorghum varieties 'Piper' and '270911', respectively with low and high dhurrin content, in suppressing RKN in vegetable production systems; 2) to investigate in greenhouse conditions some factors affecting their efficiency in terms of nematode suppression; and 3) to screen both varieties and additional sorghum genotypes, either sudangrass or hybrids, for susceptibility to M. incognita.

Materials and methods

Nematode suppressive effects of sorghums in agroecosystem (field trial)

A four-year field trial was performed on a commercial organic farm near Lambesc (43.65N, 5.21E) located at Provence in southern France, with Mediterranean climate, from 2012 to 2016. The trial was carried out under a 80 m x 8 m x 3.5 m plastic cover plot. The soil was sandy-loamy (37.5% sand, 22.3% loam, 10.7% clay, 3.5% soil organic matter (OM) and with pH of 8.4) heavily infested with both M. arenaria and M. incognita as determined by their isoesterase phenotype [START_REF] Dalmasso | Molecular polymorphism and phylogenetic relationships in some Meloidogyne spp. : application to the taxonomy of Meloidogyne[END_REF]. During the experimental period, soil temperature at a depth of 15 cm varied from 5 to 15°C from November to February, and from 15 to 30°C from April to October.

The plot was divided into two subplots, one for planting sudangrass 'Piper', the standard cover crop commonly used by commercial farmers in France with low dhurrin content, the other for planting sudangrass hybrid '270911' with high dhurrin content known for biofumigation purposes. '270911' is a three-way hybrid generated from [Sorghum bicolor spp. bicolor x sudangrass] cross developed by UPL France SAS TM . The 4-year-field experiment lasted two seasons, the sorghum cover crop being grown in the first and third years in order to duplicate the experiment. The sorghum seeds were sown in July or August on rototilled soil with seedbed preparation before sowing, at a density of 50 kg seeds/ha for 'Piper' to reach 25 to 30 tons/ha of aboveground fresh weight. '270911' was sown at a density of 85 kg/ha in 2012 due to poor germination but at 30 kg seeds/ha in 2014 as recommended by UPL France to reach the same amendment rate (Table 1). One month after sowing, the leaves and shoots were mowed and soil incorporated with rotavator. The soil was rolled and left uncovered for one month to allow biofumigation. To prevent phytotoxicity, the next cash crops were planted three weeks after termination of biofumigation.

RKN-susceptible plant species were grown subsequent to these cover crops to assess the suppression of RKN. Two types of RKN-susceptible plants were used: 1) a very susceptible crop, i.e., Cucumis melo (melon) in spring, and 2) susceptible crops in winter when the RKN cycle is slower, such as Lactuca spp. (lettuce) or Beta vulgaris subsp. vulgaris (Swiss chard). Weak forms of R-crops were cropped during the summer in the second and fourth years, to determine whether the use of a green manure could decrease the RKN soil infestation potential enough to secure crop rotation with them. RKN-resistant tomatoes (R-tomato) whose Mi-1.2 resistance gene is inactivated at high temperature were used. Sweet pepper varieties grafted onto resistant pepper rootstock (R-pepper) that carries the major R-gene Me3 weakened in the highly susceptible genetic background Doux-Long-des-Landes, were also used and mixed with the R-tomatoes.

Sorghum biomass was estimated at the end of the cover crop by weighing the aerial part of plants sampled from a 1 m 2 microplot before and after drying at 60°C for 48 h (Table 1). Rhizosphere soil from eight 250 ml samples per subplot were sampled at a depth of 15 cm, before the experiment and after each susceptible crop was terminated. Throughout the experiment, samples were systematically taken from the same core site, to minimize the effects of heterogeneity in the distribution of nematodes over the plot. RKN were extracted by the [START_REF] Seinhorst | Extraction Methods for Nematodes Inhabiting Soil[END_REF] elutriation procedure and counted under a stereomicroscope (x60 magnification) [START_REF] Merny | Les techniques d'échantillonnage des peuplements de nématodes dans le sol[END_REF]. Nematode population densities were determined and expressed per dm 3 of fresh soil (mean of 8 replicates). To analyse the effect of the four-year-cropping systems on RKN soil populations, a multiplication rate Pf/Pi (final population Pf = nematode density in the soil at the end of the crop; initial population Pi = nematode density in the soil at planting or sowing) was compared between the plots. To determine the impact of the system on susceptible and resistant crops, RKN damage was estimated by determining the gall index (GI) on a scale of 0-10 [START_REF] Zeck | A rating scheme for field evaluation of root-knot nematode infestations[END_REF]) for the root system of a representative subsample (36 plants per subplot) at the end of each crop. The number of RKN-infected plants was also recorded. a After planting, the crop was irrigated every three days initially and less frequently thereafter; b after incorporation with rotavator, the soil was rolled and left uncovered for one month to allow biofumigation

Effects of soil types, time of planting and time of biofumigation on nematocidal effects of sorghums (greenhouse pot experiment)

A greenhouse pot experiment was conducted to compare, in terms of nematode suppression, two times of planting and of biofumigation for both sorghum varieties, 'Piper' and the hybrid '270911' in three soil types: 1) sandy (82% sand, 9% loam, 4% clay, 1.6% OM), 2) clayey (21% sand, 24% loam, 33% clay, 3.9% OM), and 3) sandy-loamy (53% sand, 22% loam, 12% clay, 3.3% OM). M. incognita, obtained from the collection maintained at INRA Sophia Antipolis, France, was inoculated at 1000

J2s.kg -1 soil in 12-liter pots. Experiments were conducted in a 2×3×2 (sorghum variety × soils × termination time) factorial designed experiment with 4 replications and repeated twice. Nine seeds of '270911' and 15 seeds of 'Piper' were sown per pot, in accordance with UPL France SAS TM recommendations of 30 kg seeds ha -1 to reach the same amendment rate (1% after two months, 0.3% after one month in g of fresh sorghum material per 100 g of soil). Sorghums were grown in sandy, clayey and sandy-loamy soils for one or two months. The plants were chopped into pieces then soil incorporated. The pots were irrigated to establish anaerobic soil conditions and tarped with a virtually impermeable film (VIF) usually used as solarization mulch. The effect of biofumigation on RKN was examined at 10 days after sorghum residues were incorporated into the soil. During this time, soil temperature varied from 20 to 40°C. To evaluate the effect of time of biofumigation, sorghums were grown in sandy-clayey soil for one month, incorporated into the soil, irrigated and tarped with VIF and examined at 10 or 30 days. The experiment was conducted twice with 4 replicates each time. Soil infestation potential (SIP) was evaluated by subsampling 1 kg of soil from each pot at a depth of 15 cm before sowing the sorghum seeds, after the growth period (one or two months), and after biofumigation (10 or 30 days). Two-month-old tomato plants (RKN-susceptible cv Saint-Pierre, provided by Vilmorin TM France) were transplanted into each pot filled with these soil samples and maintained in the greenhouse. After six weeks, SIP was evaluated in each pot by immersing tomato roots in cold aqueous eosin yellow solution as described by [START_REF] Roberts | Resistance in Lycopersicon peruvianum to isolates of Mi gene-compatible Meloidogyne populations[END_REF], observing under a magnifying glass and determining the number of egg masses (EMs) present on them. The multiplication rates Pf/Pi were compared between each modality. (1983).

Variety screening for susceptibility (growth chamber conditions)

Statistical analysis

For the field trial, two-way ANOVA was performed to compare nematode concentration in soil in function of time and sorghum type. Time points were not compared altogether but three comparisons were done: T0 vs T3, T24 vs T27 (to measure the short-term impact of sorghum culture) and T0 vs T43 (to measure the long-term effect of using sorghum culture). Interactions were included. Another set of two-way ANOVA were performed to compare galls index on similar cultures at different time points for both sorghum types. Cultures tested were melon, winter cultures (chard and lettuce together, or lettuce only), and resistant cultures together (pepper and tomato). Interactions were included.

For the greenhouse experiment, multifactorial ANOVA with all interactions was done on the mean SIP data (over replicates) of differences between EM at the time considered and start of the experiment. Two analyses were done, separately before and after biofumigation (with factors time x soil x sorghum type, i.e. 2x3x2).

Data from the host suitability assay in controlled conditions were assessed in a global Kruskal-Wallis test followed by post-hoc pairwise Wilcoxon tests with Benjamini-Hochberg correction for multiple testing, with a threshold of P=0.05 [START_REF] Benjamini | Controlling the false discovery rate: a practical and powerful approach to multiple testing[END_REF].

Results

Impact of the sorghum cropping system on RKN in the field experiment

Mean RKN abundance was high over the entire plot in the trial at Lambesc (initial rate = 20,766 ±5,620 individuals/dm 3 ) after the first melon crop (Fig. 1A). We observed a strong and significant short term diminution of RKN abundance in soil by using both sorghums. At the beginning of the study, the relative RKN concentration loss was of 94% for '270911' and of 95% for 'Piper' (ANOVA, p_time<0.02, p_sorghum>0.5, no interaction). RKN populations subsequently remained low in the soil, below the initial rate, after the cultivation of highly susceptible chard or lettuce winter crops just after sorghum.

Following chard or lettuce, the summer crops with low resistance to RKN (pepper in 2013, pepper and tomato in 2015) allowed an increase of RKN populations in the soil significantly higher for '270911' plot than for 'Piper' plot. At mid-study, the relative concentration loss was of 81% for '270911' and of 73% for 'Piper' (ANOVA, p_time<0.01, p_sorghum>0.5, no interaction). Throughout the four years of the experiment, the alternation of sorghum green manure and partially resistant crops maintained a decrease in the RKN population and the long-term trend was still beneficial, with a global diminution of 84% for '270911' and 79% for 'Piper' (ANOVA, p_time<0.01, p_sorghum>0.5, no interaction) (final rate = 2,850±1,365 individuals/dm 3 in 'Piper' plot and 4,725±600 individuals/dm 3 in '270911' plot).

After one month of cultivation in summer, no gall was present on the roots of 'Piper' or '270911' (Fig. 1B). GI was similar and moderate (less than 4) on melon culture in 2012 and 2014 after both sorghum types (p_time=0.46, p_sorghum=0.32, no interaction). For resistant cultures, very few galls were observed and 40 to 70% of plants exhibited no gall during all the experiment, tomato showing an average GI lower than pepper, with no difference in both sorghum plots (p_crop<10 -3 , p_sorghum=0.73, no interaction). The picture is more complex for winter cultures, with a significant difference between chard and lettuce, chard being more infected than lettuce (ANOVA, p<0.05) and 'Piper' sorghum plot being less infected than '270911' sorghum plot (ANOVA, p<0.05, no interaction with crop type). Moreover, results for lettuce only showed some variability, with early lettuce crops (harvested in October) being significantly less infected than late crops for both sorghum types (ANOVA, p_time<10 -16 , p_sorghum>0.05). This late analysis also showed a weak interaction between time points and sorghum type (ANOVA, p<0.02) essentially due to a diminution of GI on lettuce at the end of the experiment, only in plots cultivated with 'Piper', diminution which was not significant at other time points. GI reached almost 4 and only 3% of plants had no gall when no sorghum was used in the fourth year compared to GI < 2 and 30 to 45% of plants with no gall when sorghums were used before winter crops.

Effects of soil types, time of planting and time of biofumigation on nematocidal effects of sorghums in pot experiment

Before the sorghum was planted in the 2 x 3 x 2 factorial experiment, the number of nematodes in the soil, as evaluated by determining SIP, was moderate in clayey and sandy-loamy soils, to heavy in sandy soils (Fig. 2). After one month of cultivation, the number of nematodes greatly decreased in the three types of soils, for both sorghum varieties: Pf/Pi was less than 0.4. The experiment being repeated twice with the same mean values, results of both experiments (thus for 8 pots) were combined and shown in Fig. 2. A multifactorial ANOVA on data before biofumigation (time x soil x sorghum type, i.e. 2x3x2, Tables 2 and3) showed a strong time effect (p<10 -11 ) with an increase in EM numbers when comparing two months cultivation relative to one month (0.4 < Pf/Pi after two months < 2.1 ; 0.1 < Pf/Pi < 0.4 after one month), and another strong soil effect (p<10 -15 ) showing a strong increase in clayey soils (Pf/Pi > 2). No effect relative to the sorghum type was detectable.

Moreover, an advanced analysis including interaction terms showed that the increase in EM numbers due to two months-cultivation in clayey soils still exists, but in a much weaker form (p<10 -3 ). Repeating the same analysis after 10 days of biofumigation (Tables 2 and3) replicated the same results, with slightly different p-values: p<10 -4 for the increase in EMs number with two months cultivation (0.3 < Pf/Pi after two months < 1.4; 0.1 < Pf/Pi < 0.6 after one month), p<10 -14 for the soil effect with the same effect as previously, and still no sorghum type effect. This showed that 10 days of biofumigation did not remediate to the increase in EM numbers due to the two months-cultivation. In sandy-loamy soil after one month-cultivation, 30 days of biofumigation were slightly more efficient (Pf/Pi <0.003) than 10 days of biofumigation (Pf/Pi < 0.04), resulting in the almost complete abolition of SIP in some pots. The temperature in the soil under the VIF varied from 22 to 47°C over this 30-day period. and SsSs, and about 500 ± 50 µm in diameter on the other types of sorghum, including Sg 'Piper' and SbSg '270911'. Moreover, EMs were often found inside sorghum roots, as seen with acid fuchsin staining, whereas those on tomato roots were always outside the gall (Fig. 4). Small numbers of second, third and fourth stage juveniles, females and EMs were observed in the roots of the poor hosts. Very few J2s penetrated the roots of the non-host SbSg1 and they did not develop into J3s after six weeks. Necrotic cells (hypersensitive-like reaction) were visible as a darkening of the orange staining in the epidermis and cortex of the roots at a higher frequency in the roots of Sg 'Piper' than in SbSg '270911', where more juveniles of all stages were observed. No hypersensitive-like reaction was observed in the roots of SbSg1.

Discussion

In this study, the effects of two sorghums as summer cover crops, the hybrid '270911' with high dhurrin content and the commercial 'Piper' with low dhurrin content, were compared in a vegetable production system by determining their impact on two main components of crop protection against RKN: the ability to decrease parasite levels in the soil and the potential to protect subsequent crops (susceptible or partially resistant) in the rotation. Analytical experiments in controlled conditions (growth chamber and greenhouse) were designed to provide explanatory elements to what was observed in the field.

Both sorghums with low and high dhurrin contents used as summer cover crop had the same potential to control RKN:

The results of the four-year trial and greenhouse pot experiment clearly demonstrated that both sorghums, cultivated for one month or less to avoid RKN multiplication and then buried for one month, were able to control RKN in vegetable production systems with the same efficiency: they significantly reduced RKN populations in the soil by up to 70% compared to the rate before planting, thereby protecting crop rotations including forthcoming susceptible hosts. The farm had sandy-loamy soil and the greenhouse experiment confirmed the high efficiency of both sorghums in this type of soil as long as they are used appropriately.

In the same way, both types of sorghum appeared to minimize damage to subsequent susceptible winter crops in the rotation: Swiss chard and lettuce exhibited a very low mean GI after both types of sorghum, whereas GI was higher if sorghum was not grown. Nevertheless, GI for these plants also varied according to the date of plantation (1.5 < GI < 4 for October plantation and 0 < GI < 0.2 for November and January plantations). Date of plantation is strongly related tothe soil temperature; indeed, M. incognita and M. arenaria are not able to infect plants, develop and reproduce at temperatures below 15°C (Evans and [START_REF] Perry | Survival mechanisms[END_REF][START_REF] Thomason | Rate of reproduction of Meloidogyne spp. as influenced by soil temperature[END_REF][START_REF] Vrain | Influence of low temperature on rate of development of Meloidogyne incognita and M. hapla larvae[END_REF]. In October, the mean soil temperature in Lambesc plot was around 15-20°C and in November, around 7-15°C. Thus, we cannot conclude that the GI reduction observed on these winter crops was only due to the cultivation of both sorghums.

Both types of sorghum helped protect weak R-summer crops (tomato and pepper): very few galls were observed on the roots of these summer crops (GI < 1), and 40 to 70% of plants had no gall, even during the second crop rotation. Decreasing the number of parasites in the soil may increase the durability of R-genes, because the appearance and early increase in the frequency of virulence alleles in the pathogen population depend on the balance between mutation rates and population size [START_REF] Consortium | Heterogeneity of selection and the evolution of resistance[END_REF]. However, longer experiments are required for firm conclusions about efficacy to be drawn, because these weak R-summer crops did not keep inoculum levels low in the soil. Nevertheless, the alternation of sorghum as summer cover crop and partially R-Solanaceae over a four-year period resulted in the sustainable control of RKN populations, with 0.05 < Pf/Pi < 0.36.

Time of cultivation, time and quality of biofumigation, and type of soil affected sorghums efficacy in terms of nematode suppression:

The sorghums were cultivated only one month in summer before incorporation of residues into the soil of the field trial because they were suspected of favoring the long term multiplication of a few starting RKN. Experiments in controlled conditions confirmed this hypothesis, both sorghums being only poor hosts. Thus, duration of the growing period before burial of fresh material in the soil was of crucial importance: RKN significantly increased in the soil after 75 days cultivation (Pf/Pi up to 2), while they always significantly decreased with only 30 days cultivation (0.1 < Pf/Pi < 0.4), before completion of the RKN life cycle. This temporary trapping, reducing RKN infestation in the soil, masks the high risk of RKN multiplication if the sudangrass crop is destroyed too late.

Moreover, young sorghums have a higher dhurrin content than old ones [START_REF] Adewusi | Turnover of dhurrin in green sorghum seedlings[END_REF]. It was confirmed by [START_REF] Gard | Le sorgho fourrager comme engrais vert à effet assainissant: évaluation agronomique et potentiel pour la biofumigation[END_REF]: 12.7 and 2.68 mg/g of dry matter for 3-week-old '270911' and 'Piper', respectively, versus 8.7 and 2.3 mg/g of dry matter for 4-week-old '270911' and 'Piper', respectively, decreasing to 4.5 and 0.7 mg/g of dry matter for 6-week-old old '270911' and 'Piper', respectively.

Older tissues are also more bulky and may break down more slowly, thereby releasing smaller amounts of HCN into the soil [START_REF] Viaene | Management of Meloidogyne hapla on lettuce in organic soil with Sudangrass as a cover crop[END_REF].

The soil and temperature conditions may also affect the degradation of the fresh material buried in the soil and, thus, the release of HCN (Viane and [START_REF] Viaene | Management of Meloidogyne hapla on lettuce in organic soil with Sudangrass as a cover crop[END_REF]. It may explain why increasing the biofumigation time under VIF (that holds in the gaseous breakdown products; [START_REF] Gamliel | Characterization of antifungal volatile compounds evolved from solarized soil amended with cabbage residues[END_REF] to 30 days in the greenhouse experiment, although a less important factor than the growing time, may nevertheless allow the almost complete elimination of RKN from soils when combined with the trapping effect. [START_REF] Wang | Effects of cover cropping, solarization, and soil fumigation on nematode communities[END_REF] also found cover crop solarization to be an effective combined treatment for decreasing Meloidogyne spp. infestation, with an efficacy similar to that of methyl bromide fumigation at crop harvest, the most effective treatment used against soil-borne diseases and pests in conventional agriculture, before its prohibition.

The duration of burial, measured under controlled conditions, must be adjusted according to the growing conditions. Under cover in the South of France, this period may be estimated to be four weeks in spring, and three weeks in summer. In open-field conditions in northern France, this period could be extended to five weeks in spring and four weeks in summer. For farmers who consider green manure as essential and can wait for two months before incorporating sorghum residues into the soil, an innovative double-sowing technique (two lots of sorghum grown one after another for three weeks each and then buried) could be proposed as it is currently tested efficiently in some farms following results obtained from the Lambesc farm experiment.

All these required conditions may explain why some authors found sorghums efficient to control RKN populations in vegetable cropping systems [START_REF] Everts | Root-knot and root-lesion nematode suppression by cover crops, poultry litter, and poultry litter compost[END_REF][START_REF] Kratochvil | Evaluation of crop rotation and other cultural practices for management of root-knot and lesion nematodes[END_REF][START_REF] Mojtahedi | Suppression of Meloidogyne chitwoodi with Sudangrass cultivars as green manure[END_REF][START_REF] Viaene | Management of Meloidogyne hapla on lettuce in organic soil with Sudangrass as a cover crop[END_REF][START_REF] Widmer | Mechanism of suppression of Meloidogyne hapla and its damage by a green manure of Sudangrass[END_REF], whereas other obtained disappointing results [START_REF] Collange | Root-knot nematode (Meloidogyne) management in vegetable crop production: The challenge of an agronomic system analysis[END_REF][START_REF] Crow | Effect of sorghum Sudangrass and velvetbean cover crops on plant-parasitic nematodes associated with potato production in Florida[END_REF][START_REF] Kokalis-Burelle | Evaluation of cover crops with potential for use in anaerobic soil disinfestation (ASD) for susceptibility to three species of Meloidogyne[END_REF][START_REF] Védie | Protection des nématodes à galles: intérêt de méthodes alternatives sur l'interculture d'été[END_REF]. Now, the sustainable management of RKN populations should not only be considered in terms of managing these nematodes but also in terms of managing the pathogenicity and the biodiversity of the whole plant-parasitic and non-parasitic nematode communities (i.e., ecological sustainability) [START_REF] Mateille | Control and management of plant-parasitic nematode communities in a soil conservation approach[END_REF], because competitive interactions between nematodes may increase the sustainabilty of the management strategy (Mateille et al., submitted).

The mode of action of sorghums to control RKN may depend on their genotypes:

Only one variety, SbSg1, was a non-host for M. incognita (no EM) and was as dhurrin-rich as '270911' (7.5 ± 0.2 mg/g of dry matter after one month according to [START_REF] Gard | Le sorgho fourrager comme engrais vert à effet assainissant: évaluation agronomique et potentiel pour la biofumigation[END_REF]: it could therefore be grown for more than four weeks before burial without the risk of RKN multiplication. The SbSg1 hybrid was obtained from a [Sorghum bicolor x sudangrass] cross. It is not yet developed, registered or commercialized. Its dhurrin content reached 7.5 ± 0.2 mg/g of dry matter after one month [START_REF] Gard | Le sorgho fourrager comme engrais vert à effet assainissant: évaluation agronomique et potentiel pour la biofumigation[END_REF], a level slightly lower than that of SbSg '270911' after one month (8.7 ± 0.2 mg/g).

All the other varieties, SsSs hybrid, Sb 'Nutrigrain' and SbSb hybrids were more susceptible than Sg 'Piper' and SbSg hybrids, including SbSg '270911'. [START_REF] Birchfield | Wheat and grain sorghum varietal reaction to Meloidogyne incognita and Rotylenchulus reniformis[END_REF], [START_REF] Davis | Identification of widely varying levels of resistance to Meloidogyne incognita in sweet sorghum[END_REF], [START_REF] Fortnum | Host suitability of grain sorghum cultivars to Meloidogyne spp[END_REF] and [START_REF] Mojtahedi | Suppression of Meloidogyne chitwoodi with Sudangrass cultivars as green manure[END_REF] also reported a varietal effect on susceptibility of grain and sweet sorghum Sb (S. bicolor), Sg (sudangrass), SbSg (Sorghum-sudangrass hybrids)

genotypes tested against various RKN species. This differential level of resistance cannot be accounted for by dhurrin content, because Sg 'Piper' (low level of dhurrin) and SbSg '270911' (high level of dhurrin) were both very poor hosts, with no significant difference in EM numbers on their roots.

In 2015, Harris-Shultz et al. mapped a major quantitative trait locus (QTL) to sorghum chromosome 3, accounting for the resistance of one of Sb varieties to M. incognita race 3. Other inherited resistance factors may be present in the sudangrass genome, accounting for the lack of RKN reproduction on Sg 'Piper' and the hybrids. Sg 'Piper' roots contained only a few RKN and showed many hypersensitivelike reaction (HR) sites, indicating a response to infection similar to that in Mi-1.2 resistant tomato plants [START_REF] Paulson | Ultrastructure of the hypersensitive reaction in roots of tomato, Lycopersicon esculentum L. to infection by the root-knot nematode, Meloidogyne incognita[END_REF]; SbSg '270911' plants contained larger numbers of juveniles of all stages, concealed within the roots, with little sign of a HR. Both sorghums acted as trap plants for RKN because only few EMs were produced. In the roots of the non-host sorghum SbSg1, only a few scattered J2s, with no further development, and a total absence of HR were observed. Thus, SbSg1 roots may repel juveniles, due to the toxic root exudates, as reported for several other non-host plants (summarized in [START_REF] Djian-Caporalino | Nematicidal and nematode-resistant plants[END_REF], including two SbSg (Sorghum bicolor x S. sudanense 'SX-15' and 'SX-17') [START_REF] Czarnota | Evaluation of seven sorghum (Sorghum sp.) accessions[END_REF]. Sorgoleone, the phenolic compound identified as a predominant constituent in exudates, could be potentially responsible for the suppressive effect of SbSg1. This hypothesis should be tested in future studies. This sorghum could be a more usable candidate for farmers as it may be cultivated more than one month before burying for biofumigation because it is non-host for RKN.

Conclusion

The use of sorghums, sudangrass or hybrids, as a green manure was found to be an effective strategy for decreasing the RKN infestation potential of soils, thereby protecting crop rotations including both susceptible and partially resistant hosts. But the efficacy of sorghums clearly depends on the management strategy to be set up in the field. In particular, incorporating sorghums into the soil before the end of the RKN cycle plays a key role in the efficient and sustainable control of these parasites. No effect relative to the sorghum type (with low or high dhurrin content) was detectable as long as they were used correctly, i.e., cultivated during one month or less and left for one month for biofumigation. This study thus provides information potentially useful to breeders and farmers for the sustainable management of RKN in protected vegetable systems.

"Declarations of interest: None"

Color should be used for any figure. Article online only.

30-days of sorghum culture as nematocidal cover crop

Melon roots infested by Meloidogyne arenaria and M. incognita Melon roots after sorghum

Fig. 1 .

 1 Fig. 1. (A) Kinetics of RKN populations in the soil over the 4-year field experiments in Lambesc: means (n=8) ± standard deviation followed by stars indicate significant differences (p < 0.05) between both sorghum plots at the sampling date; and (B) Gall index (GI) on plants: mean (n=36) ± standard deviation followed by different letters indicate significant differences. P = 'Piper', S = '270911'; chard = Swiss chard; lettuce = Batavia salad or oak leaf lettuce; R-pepper = several varieties of sweet peppers grafted onto a R-pepper rootstock carrying Me3 R-gene in the susceptible genetic background Doux-Long-des-Landes; R-tomato = Mi-1 R-tomato.

292 12Fig. 2 .

 2922 Fig. 2. Changes over time in RKN soil infestation potential (SIP) with 'Piper' and '270911' in greenhouse experiments, as expressed by the number of egg masses (EMs) on susceptible tomato plants maintained for six weeks in pots filled with 1 kg rhizosphere soil sampled from each pot: means (n=8) ± standard deviation. A multifactorial ANOVA was done on the mean variation in EM numbers along the experiment before and after biofumigation (time x soil x sorghum type, i.e. 2x3x2). In sandy soil (A) and clayey soil (B), both sorghums were grown for either one or two months, buried, irrigated, and tarped with VIF (a solarization mulch) during 10 days. In sandy-loamy soil (C), both sorghums were grown for one month, buried, irrigated, and tarped with VIF during 10 or 30 days. Soil effect was compared for 'Piper' (D) and '270911' (E) grown for one month, buried, irrigated, and tarped with VIF during 10 days. Dotted lines indicate biofumigant effects. Note that panels D and E are based on the same experiments and data as panels A, B and C, and are presented under this format for easier comparison between sorghum types.

Fig. 3 .Fig. 4 .

 34 Fig. 3. Mean number of EMs per plant counted on several sorghum varieties and on tomato (as a control) maintained in a controlled-climate growth chamber after inoculation with 400 juveniles of M. incognita: means (n=16) ± standard deviation followed by different letters indicate significant differences (p < 0.05). Sg 'Piper' = Sudan grass (Sorghum bicolor ssp. sudanense) variety 'Piper'; SbSg '270911' = [Sorghum bicolor spp. bicolor x Sudan grass] 3-way hybrid; Sb 'Nutrigrain'= Sorghum bicolor; SbSg1,2,3,4 = [Sorghum bicolor x Sudan grass] hybrids; SbSb1,2,3 = [Sorghum bicolor x Sorghum bicolor] hybrids; SsSs = [S. bicolor ssp. saccharatum x S. bicolor ssp. saccharatum] hybrid.

Table 1 .

 1 Cropping schedule for cover crop in the commercial farm located in Lambesc 43.65N, 5.21E (80 m x 8

	m x 3.5 m plastic cover plot)
	Year	2012	2014
	Sorghum '270911' plot
	Sowing date a	23/07	16/08
	Sowing density (kg/ha) (% germination)	85 (50%)	30 (100%)
	Aboveground fresh weight (tonnes/ha)	29.2	29
	Burial date b	23/08	12/09
	Winter crop planting date a	03/10 (chard)	03/10 (lettuce)
	Control 'Piper' plot
	Sowing date a	23/07	16/08
	Sowing density (kg/ha) (% germination)	50 (100%)	50 (100%)
	Aboveground fresh weight (tonnes/ha)	24.6	29
	Burial date b	23/08	12/09
	Winter crop planting date a	03/10 (chard)	03/10 (lettuce)

  'Piper' (Sg 'Piper') and'270911' (SbSg '270911') were compared for susceptibility to M. incognita. Nine other sorghum varieties including Sorghum bicolor (Sb 'Nutrigrain'), four [Sorghum bicolor x sudangrass] hybrids (SbSg1,2,3,4), three [Sorghum bicolor x Sorghum bicolor] hybrids (SbSb1,2,3) and one [S. bicolor ssp. saccharatum x S. bicolor ssp. saccharatum] hybrid (SsSs) provided by UPL France SAS TM were also analyzed for susceptibility to M. incognita. The susceptible tomato cv Saint-Pierre was used as a control. Seeds of each variety were grown in 400 mL pots containing steam-sterilized sandy soil covered by a 1 cm layer of loam and maintained in growth chambers (16 h light/8 h dark cycle, mean temperature of 24±2°C, relative humidity of 60-70%). Each variety was replicated in 8 pots and the experiment was conducted twice. At 4 weeks after seeding, 400 M. incognita J2s, obtained from the collection maintained at INRA Sophia Antipolis, France, were inoculated per pot. At 6 weeks after nematode inoculation, the number of EMs per root system for each plant was estimated as described above. Plants were considered to be susceptible if EMs > 100,

non-host if EMs =0, and poor host if 1 < EMs ≤ 100. After observation, the same roots were stained to visualize RKN infection into the plant tissues by acid fuchsin method as described by

Byrd et al.

Table 2 .

 2 ANOVA of soil infestation potential data before or after biofumigation and at the beginning of

		Before biofumigation	After biofumigation
	Variable	df	Sum sq.	p-val	df	Sum sq.	p-val
	Time	1	27572673	< 10 -11	1	2220990	< 10 -4
	Soil	2	9309191	< 10 -15	2	11691741	< 10 -14
	Sorghum type	1	40747	0.59	1	295017	0.09
	Time*Soil	1	1867127	<10 -3	1	1968328	<10 -4
	Time*Sorghum type	1	9350	0.79	1	67002	0.42
	Soil*Sorghum type	2	1262920	0.014	2	4202318	<10 -6
	Time*Soil*Sorghum type 1	138751	0.32	1	14354	0.70

the experiment. df: degrees of freedom, Sum sq: sum of squares, p-val: p-value associated to the variable considered.

Table 3 .

 3 Linear model table of the soil infestation potential values before or after biofumigation. First column indicates variable and the corresponding reference level, second column the modality under study, third, forth and fifth columns the estimate for the model, the std. deviation of the estimate and the associated p-value. Interactions are reported only for those showing a p-val<0.1. Data are identical to those of the ANOVA table 2. EMs were about 1 ± 0.05 mm in diameter on the roots of susceptible tomato plants, but only about 650 ± 60 µm in diameter on sorghums Sb, SbSb3

			Before biofumigation		After biofumigation
	Variable:ref	Modality	Effect	Std. Dev p-val	Effect	Std. Dev p-val
	Soil:clayley	Sandy	-844.32 132.45	<10 -4	-844.32 132.45	<10 -4
		Sandy-loamy -384.18 166.04	0.023 -384.18 166.04	0.023
	Time:Short	Long	1219.88 187.32	<10 -8	1219.88 187.32	<10 -8
	Sorghum type:'Piper' S'270911'	156.63 187.32	0.40	156.63 187.32	0.40
	Soil*time	Sandy*Long	-874.53 264.90	<10 -2	-874.53 264.90	<10 -2
	Soil*Sorghum type	Sandy-loamy*	-420.07 232.13	0.073 -420.07 232.13	0.073
		S'270911'
	3.3. Tests of host suitability in controlled conditions and observations of RKN developmental stages in
	the roots
	The experiment in growth chamber being repeated twice with the same mean values, results of
	both experiments (thus for 16 plants) were combined and shown in Fig. 3. Based on EM production,
	both the commonly used variety Sg 'Piper' and the recommended variety with high dhurrin SbSg
	'270911' were equally resistant to M. incognita. SbSg2 exhibited the same EM numbers. Only SbSg1
	was non-host. SbSg3, SbSg4, and SbSb1 could be compared to SbSg '270911' and designated as

poor hosts. SbSb2, SbSb3, Sb 'Nutrigrain' and SsSs were more susceptible to RKN compared to Sg 'Piper' and SbSg '270911'. The galls induced by M. incognita on the roots of all sorghum plants were much smaller than those observed on tomato roots.

Acknowledgments

The authors would like to thank O. Arnaud for supplying the field site and performing all the cropping practices. This work was part of the GEDUNEM project (Varietal and technical innovations for the sustainable and integrated management of root-knot nematodes in protected vegetable


Guillaume Manson

Jean-Baptiste Mear

Charles Herbaux

Jean-Marc Schiano

Olivier Casasnovas

Aspasia Stamatoullas

Bénédicte Deau

Anna Schmitt

Georges Garnier

Caroline Régny

Caroline Regny

Krimo Bouabdallah

Marie-Pierre Moles-Moreau

Hervé Ghesquieres

Adrian Tempescul

Remy Dulery

Emmanuelle Nicolas-Virelizier

Alain Delmer

Cecile Borel

Adrien Chauchet

Diane Damotte

Laurent Dercle

Pauline Brice

Roch Houot
email: roch.houot@chu-rennes.fr

Prof Roch Houot

Long-term efficacy of anti-PD1 therapy in Hodgkin lymphoma with and without allogenic stem cell transplantation

Keywords: Immunotherapy, Hodgkin Lymphoma, Checkpoint inhibitors, anti-PD1, nivolumab, Allogenic hematopoietic stem cell transplantation

Introduction

Classical Hodgkin Lymphoma (HL) is highly curable with standard treatments. However, relapse can occur in up to 30% of patients with advanced disease [START_REF] Gallamini | Early chemotherapy intensification with escalated beacopp in patients with advanced-stage hodgkin lymphoma with a positive interim positron emission tomography/computed tomography scan after two abvd cycles: Long-term results of the GITIL/FIL HD 0607 tria[END_REF][START_REF] Press | US intergroup trial of response-adapted therapy for stage III to IV Hodgkin lymphoma using early interim fluorodeoxyglucose-positron emission tomography imaging: Southwest Oncology Group S0816[END_REF]. In relapsed patients, salvage therapy followed by autologous hematopoietic stem cell transplantation (autoHSCT) can induce prolonged remissions [START_REF] Rancea | High-dose chemotherapy followed by autologous stem cell transplantation for patients with relapsed/refractory Hodgkin lymphoma[END_REF]. Patients who relapse after autoHSCT have a poor outcome with a median overall survival (OS) ranging from 10.5 to 27.6 months in the prebrentuximab-vedotin era [START_REF] Arai | Defining a Hodgkin lymphoma population for novel therapeutics after relapse from autologous hematopoietic cell transplant[END_REF][START_REF] Crump | Management of Hodgkin lymphoma in relapse after autologous stem cell transplant[END_REF]. Although brentuximab-vedotin has shown encouraging overall response rates in relapsed or refractory (R/R) HL, only ~20% of patients might be cured with this single-agent therapy [START_REF] Chen | Five-year survival and durability results of brentuximab vedotin in patients with relapsed or refractory Hodgkin lymphoma[END_REF].

In recent years, checkpoint blockade with anti-PD1 antibodies demonstrated remarkable efficacy in patients with R/R HL [START_REF] Ansell | PD-1 Blockade with Nivolumab in Relapsed or Refractory Hodgkin's Lymphoma[END_REF][START_REF] Armand | Programmed Death-1 Blockade With Pembrolizumab in Patients With Classical Hodgkin Lymphoma After Brentuximab Vedotin Failure[END_REF][START_REF] Engert | Nivolumab for relapsed/refractory classical Hodgkin lymphoma after autologous transplant: full results after extended follow-up of the multicohort muticenter phase 2 CHECKMATE 205 trial[END_REF][START_REF] Armand | Nivolumab for Relapsed/Refractory Classic Hodgkin Lymphoma After Failure of Autologous Hematopoietic Cell Transplantation: Extended Follow-Up of the Multicohort Single-Arm Phase II CheckMate 205 Trial[END_REF]. Studies evaluating anti-PD1 therapy in these patients, which account for more than 450 patients in total, showed overall response rates (ORR) of around 70% and complete remissions (CR) of up to 20%. These results led to the approval of nivolumab and pembrolizumab for R/R HL by the Food and Drug Administration in 2016 and 2017, respectively. In 2015, an Early Access Program (EAP) was started in France allowing treatment of R/R HL patients with nivolumab.

Despite remarkable immediate efficacy, many questions remain unanswered regarding the long-term efficacy and optimal management of HL patients treated with anti-PD1, including the duration of treatment and the need for a consolidation with allogenic stem cell transplantation (alloHSCT). We have previously addressed the first issue and demonstrated that prolonged remissions can be achieved after nivolumab discontinuation in patients with CR [START_REF] Manson | Prolonged remissions after anti-PD1 discontinuation in patients with Hodgkin lymphoma[END_REF]. Here, we evaluated the role of alloHSCT consolidation in HL patients treated with anti-PD1.

There is limited data regarding the long term efficacy of anti-PD1 therapy since the median follow-up of previously published studies was rather short (median = 7-18 months [START_REF] Armand | Nivolumab for Relapsed/Refractory Classic Hodgkin Lymphoma After Failure of Autologous Hematopoietic Cell Transplantation: Extended Follow-Up of the Multicohort Single-Arm Phase II CheckMate 205 Trial[END_REF][START_REF] Chen | Phase II Study of the Efficacy and Safety of Pembrolizumab for Relapsed/Refractory Classic Hodgkin Lymphoma[END_REF][START_REF] Beköz | Nivolumab for relapsed or refractory Hodgkin lymphoma: real-life experience[END_REF]).

Furthermore, the role of alloHSCT after anti-PD1 therapy remains poorly defined. A retrospective study by Merryman et al (N=31 HL patients) suggested that patients undergoing alloHSCT after nivolumab might experience a lower relapse rate compared to historical controls [START_REF] Merryman | Safety and efficacy of allogeneic hematopoietic stem cell transplant after PD-1 blockade in relapsed/refractory lymphoma[END_REF]. However, this strategy was not compared to patients receiving anti-PD1 treatment without subsequent alloHSCT.

Here, we report the results of R/R HL patients consolidated with alloHSCT after nivolumab treatment in comparison to patients treated with nivolumab monotherapy alone in the French EAP.

Methods

We conducted a retrospective, nationwide study of patients aged ≥ 18 years with R/R HL who were treated with nivolumab in the French EAP. EAP included patients with HL relapsing or refractory after three lines of chemotherapy (including Brentuximab-Vedotin) and autoHSCT, or four lines of chemotherapy if the patient was not eligible for HSCT due to age, insufficient stem-cell collection or chemorefractory disease. Patients with active auto-immune disease and/or steroid or immunosuppressive treatment-requiring disease were not eligible.

All patients who had received at least one dose of nivolumab as part of the French EAP were eligible for the study. Nivolumab was administered at 3 mg/kg IV over 60 min every 2 weeks in an outpatient setting until progression, death of any cause, unacceptable toxicity, consent withdrawal, or treating physician's decision. Patients were allowed to undergo subsequent alloHSCT according to primary physician's decision.

Primary endpoint was best response (ORR, CR, partial response (PR), stable disease (SD) and progressive disease (PD) as defined by the primary physician using the Cheson 1999 [START_REF] Cheson | Report of an international workshop to standardize response criteria for non-Hodgkin's lymphomas[END_REF] or Cheson 2007[16] criteria depending on the current practice in each center at the time of evaluation) at any time during nivolumab treatment.

Secondary endpoints included other efficacy parameters (including duration of response, progression-free survival, and overall survival), safety analysis and the impact of alloHSCT.

OS and progression-free survival (PFS) were defined as the duration from the first dose of nivolumab to death of any cause and disease progression or death of any cause, whichever occurred first, respectively. Both OS and PFS were censored at the date of last information and were estimated using the Kaplan-Meier method. Exact 95% confidence intervals (CI) were used when appropriate. All data analysis was carried out using SAS v.9.3 software. Safety and tolerability were evaluated and reported by investigators according to the National Cancer Institute Common Terminology Criteria for Adverse Events version 4.03.

The protocol was approved by the Comité Consultatif sur le Traitement de l'Information en matière de Recherche dans le domaine de la Santé -CCTIRS (Approval n° 16.861). All patients have been informed and consented before registration.

Results

Patients' characteristics

Seventy-eight patients from 35 French centers were included (Supplementary Figure 1). The characteristics of the patients are summarized in Table 1. Median age at nivolumab initiation was 37 (range, 18 -77) years. Performance status was ≥2 in 27% of the patients,27% of them had B symptoms and 77.5% had stage III/IV disease. The median number of prior lines of treatment was 6 (range, 2 -13). All patients had been previously treated with brentuximab-vedotin, 62% and 28% of them had undergone prior autologous or allogenic HSCT, respectively. The median exposure to nivolumab was 5.2 (range, 0 -38.3) months, and the median number of cycles was 9.5 (range, 1 -84). The median follow-up was 34.3 months (range, 0.1 -39.7). At the time of analysis, 85.9% of patients had discontinued nivolumab, mostly because of progression (50.7%), consolidation with alloHSCT (19.4%) or toxicity (9.0%).

Outcome of the entire cohort Among evaluable patients (n=76), the best ORR was 65.8% (90% CI [55.82; 74.80]), including 38.2% CR and 27.6% PR. The median time to response was 2.6 months (95% CI [2.0; 4.0]) and the median time to best response was 2.7 months (6 patients reached CR at the second evaluation and 2 others later). The median duration of response was 24.3 months (95% CI [9.9; not evaluable]). Median PFS was 12.1 months (95% CI [7.3; 26.2]) and median OS was 38.7 months (95% CI [38.7; not evaluable]). At three years of follow-up, the PFS and OS rates were 32% and 65%, respectively (Figure 1 A &B). Patients achieving a CR upon nivolumab had a significantly longer PFS than those reaching a PR (median PFS = not reached vs 9.3 months, p<0.001). There was no difference in OS between those two groups (Figure 1 C &D). Among 50 responders (CR or PR), 23 (46%) patients had relapsed/progressed at the time of analysis: 7 out of 29 (24%) CR patients, 16 out of 21 (76%) PR patients, and up to 94% of the PR patients who did not undergo subsequent alloHSCT. Overall, 45 (57.7%) patients relapsed or progressed after nivolumab treatment.

Thirty-six (80%) of them received a salvage therapy. Among 23 patients evaluated, 13 (56.6%) experienced an objective response and 8 (34.8%) achieved CR.

A total of 107 adverse events (AE) were reported in 40 (51.3%) patients. Among these, there were 62 AE of grade ≥3 in 29 (37%) patients and 28 serious AE (SAE) in 16 (20.5%) patients. Most frequent clinically relevant, immune-related and serious AE are summarized in Supplementary Table 1.

At the time of analysis, 54 (69.2%) patients were alive. The main cause of death was lymphoma progression in 14 (58.3%) patients.

Outcome of patients according to subsequent alloHSCT consolidation

In our cohort, 17 patients proceeded to alloHSCT after nivolumab therapy (Figure 2 & 3, Supplementary Table 2). One patient reached a PR upon nivolumab then progressed before starting the conditioning regimen and thus received radiotherapy before transplantation.

Three patients had a PD upon nivolumab therapy of whom 2 received a salvage therapy before transplantation. Characteristics of allo-transplanted patients are summarized in Supplementary Table 2.

At the time of analysis, 14 out of the 17 transplanted patients were alive and 13 remained disease-free after a median follow-up of 34.0 months from nivolumab initiation and 29.2 months from transplantation (Figure 3). The PFS and OS at one year from alloHSCT were 76% and 82%, respectively.

In order to assess the benefit of alloHSCT consolidation, we compared the outcome of patients who achieved an objective response (CR or PR) upon nivolumab monotherapy and underwent (N=13) or not (N=37) immediate subsequent alloHSCT. Characteristics of these patients are summarized in Table 2. At nivolumab initiation, patients with subsequent alloHSCT were younger (median age = 30.5 vs 38.0 years, p=0.029). There was no significant difference between the two groups regarding disease stage or number of prior lines of systemic therapy. The duration of treatment with nivolumab was shorter in patients receiving subsequent alloHSCT (median duration = 2.89 vs 6.1 months, p=0.012) probably due to the initiation of the allograft procedure. The median time to response from nivolumab initiation was not significantly different between the two groups (2.0 vs 1.8 months, p=0.420) but the time to best response was shorter in the transplanted group (2.4 vs 1.8 months, p=0.039). Indeed, all patients who underwent alloHSCT, except one, achieved a CR at first evaluation. At the time of analysis, 23 out of 37 (62.2%) patients without subsequent alloHSCT had relapsed (7 out of 20 CR patients and 16 out of 17 PR patients) whereas all patients who underwent alloHSCT remained disease-free after a median follow-up of 33.3 months from nivolumab initiation (p<0.001). Median PFS was 22.4 (95% CI [7.3; 32.0]) months for the non-transplanted group and was not reached for the transplanted group (p=0.003; Figure 2A). In terms of PFS, alloHSCT consolidation tended to benefit both CR (p=NS) and PR (p=0.01) patients (Supplementary Figure 2 A &C). In the non-transplanted group, 12 out of 20 (60%) patients in CR and 0 out of 17 patients in PR were alive and progression-free at the time of analysis. In the transplanted group, 2 patients died of toxicity at day 155 and 192 after alloHSCT, respectively (causes of death are described below).

There was no difference in OS between the transplanted and non-transplanted groups (Figure 2B). In the non-transplanted group, 19 out of 23 relapsed patients received a salvage therapy. Among 12 patients evaluated, 9 (75%) experienced an objective response including 5 CR (41.7%).

All the 17 transplanted patients experienced acute and/or chronic-GVHD (Supplementary Table 2). Fourteen patients experienced acute GVHD (4 grade IV), 5 of which were steroidrefractory and required second line treatment with anti-IL2R antibody (inolimomab and basiliximab), anti-thymocyte globulin, or extra-corporal photopheresis. Seven patients experienced chronic GHVD, 5 of which were steroid-refractory and had not resolved at the time of analysis (4 cutaneous and 1 hepatic GVHD). One patient experienced veno-occlusive disease which resolved after defibrotide treatment and 2 patients presented a non-infectious febrile syndrome responding to corticosteroids. Treatment-Related Mortality (TRM) was 5.9%, 11.8% and 17.6% at day 100, 6 months and 12 months, respectively. Three patients died: one patient had undergone prior alloHSCT and received a second (haploidentical) graft after reaching a PR upon nivolumab. He presented with a grade IV cutaneous and hepatic GHVD, and an unexplained encephalitis leading to death 5 months after transplantation. The second death occurred in a patient who underwent geno-identical alloHSCT after reaching a CR upon nivolumab. He first presented a non-infectious febrile syndrome that was efficiently treated with corticosteroids and then developed a steroid-refractory cutaneous and gastrointestinal GVHD. He died of massive and unexplained hemoptysis 6 months after transplantation. The third death occurred in a patient with infectious acute pneumonia.

Discussion

Our study has a particularly long follow-up compared to previously published studies (median = 34.3 months versus 7-18 months [START_REF] Armand | Nivolumab for Relapsed/Refractory Classic Hodgkin Lymphoma After Failure of Autologous Hematopoietic Cell Transplantation: Extended Follow-Up of the Multicohort Single-Arm Phase II CheckMate 205 Trial[END_REF][START_REF] Chen | Phase II Study of the Efficacy and Safety of Pembrolizumab for Relapsed/Refractory Classic Hodgkin Lymphoma[END_REF][START_REF] Beköz | Nivolumab for relapsed or refractory Hodgkin lymphoma: real-life experience[END_REF]). This gave us the opportunity to evaluate long term efficacy of anti-PD1 therapy in 78 R/R HL patients. Although initial response to anti-PD1 was high (ORR=65.8%), our results show that most patients eventually relapse/progress, notably those who are unable to achieve a CR (76% of all PR patients and 94% of PR patients without subsequent alloHSCT). In responding (CR or PR) patients, the relapse rate was significantly lower in patients consolidated with alloHSCT (0% vs 62.2%, p<0.001). Furthermore, five out of 6 patients who were not in CR at the time of transplantation (4 PR and 1 PD) converted into a CR after alloHSCT.

Our results are consistent with the studies previously published by Armand et al [START_REF] Armand | Nivolumab for Relapsed/Refractory Classic Hodgkin Lymphoma After Failure of Autologous Hematopoietic Cell Transplantation: Extended Follow-Up of the Multicohort Single-Arm Phase II CheckMate 205 Trial[END_REF], Chen et al [START_REF] Chen | Phase II Study of the Efficacy and Safety of Pembrolizumab for Relapsed/Refractory Classic Hodgkin Lymphoma[END_REF] and Beköz et al [START_REF] Beköz | Nivolumab for relapsed or refractory Hodgkin lymphoma: real-life experience[END_REF] showing an ORR of 69%, 69%, and 64%, respectively (Supplementary Table 3). Similarly, the median PFS in our study was comparable to the one in the study by Armand et al (12.1 months versus 14.7 months, respectively).

We were also able to analyze the outcome of patients who underwent consolidation with alloHSCT and compare them to patients who were not consolidated with alloHSCT. To reduce the bias, we limited the final comparison to patients who experienced an objective response after nivolumab monotherapy.

In our cohort, 17 patients had undergone alloHSCT after nivolumab therapy (Figure 3). Among them, 13 patients were transplanted after reaching an objective response upon nivolumab monotherapy (9 CR and 4 PR). Of note, 4 additional patients were transplanted: one with a progressive disease and 3 after salvage therapy. Interestingly, 5 out of 6 patients who were not in CR at the time of transplantation (4 PR and 1 PD) converted into a CR after alloHSCT. Among the 17 transplanted patients, 14 are alive and 13 remain disease-free after a median follow-up of 29.2 months from transplantation. One-year PFS and OS from alloHSCT were 76% and 82%, respectively. All patients experienced graft-versus-host disease (GVHD), either acute (N=14, 82%) and/or chronic (N=7, 41%) GVHD, including 7 (41%) patients with grade III-IV GVHD. At the time of analysis, GVHD had resolved in 9 out of 13 disease-free patients. Two patients experienced non-infectious febrile syndrome which was no significant difference in OS between the transplanted and non-transplanted groups (Figure 2B). Whether the benefit observed in PFS may eventually translate into an increase in OS remains uncertain and should be re-evaluated after a longer follow-up. Emerging data suggest that anti-PD1 therapy may "re-sensitize" Hodgkin lymphoma tumors to standard chemotherapy [START_REF] Rossi | Efficacy of chemotherapy or chemo-anti-PD-1 combination after failed anti-PD-1 therapy for relapsed and refractory hodgkin lymphoma: A series from lysa centers[END_REF]. Thus, patients who relapse or progress after anti-PD1 therapy may respond to salvage chemotherapy even if they were previously refractory. In our cohort, despite a great number of prior lines of treatment, most patients who progressed/relapsed after anti-PD1 were able to receive a salvage therapy (80%), most of whom achieved an objective response (56.5% among evaluated patients). These results suggest that a subset of patients who fail anti-PD1 therapy may still be rescued.

The relapse rate after alloHSCT following anti-PD1 therapy seems much lower compared to patients who are transplanted after conventional chemotherapy. Indeed, the one-year relapse rate in our study was 5.8% versus 26 -41% in historical controls [START_REF] Anderlini | Gemcitabine, Fludarabine, and Melphalan for Reduced-Intensity Conditioning and Allogeneic Stem Cell Transplantation for Relapsed and Refractory Hodgkin Lymphoma[END_REF][START_REF] Devetten | Unrelated Donor Reduced-Intensity Allogeneic Hematopoietic Stem Cell Transplantation for Relapsed and Refractory Hodgkin Lymphoma[END_REF][START_REF] Marcais | Impact of disease status and stem cell source on the results of reduced intensity conditioning transplant for Hodgkin's lymphoma: A retrospective study from the French Society of Bone Marrow Transplantation and Cellular Therapy (SFGM-TC)[END_REF][START_REF] Sureda | Reduced-intensity conditioning compared with conventional allogeneic stem-cell transplantation in relapsed or refractory Hodgkin's lymphoma: An analysis from the lymphoma working party of the European Group for Blood and Marrow Transplantation[END_REF][START_REF] Armand | The addition of sirolimus to the graft-versus-host disease prophylaxis regimen in reduced intensity allogeneic stem cell transplantation for lymphoma: A multicentre randomized trial[END_REF][START_REF] Armand | Allogeneic Transplantation with Reduced-Intensity Conditioning for Hodgkin and non-Hodgkin Lymphoma: Importance of Histology for Outcome[END_REF][START_REF] Robinson | Reduced intensity conditioning allogeneic stem cell transplantation for Hodgkin's lymphoma: identification of prognostic factors predicting outcome[END_REF], suggesting that prior anti-PD1 therapy may enhance the graft-versus-lymphoma effect of alloHSCT.

Although our study suggests a benefit of alloHSCT consolidation, these results should be interpreted with caution given the retrospective nature of this analysis. Despite our attempt to limit the bias associated with the response to anti-PD1 therapy by limiting the comparison to responding patients, other biases -including age and proportion of complete respondersmay have influenced the differences observed between transplanted and non-transplanted groups. The decision to perform an alloHSCT was left to the decision of the primary physician and thus could not be controlled. Finally, the number of patients in the two subgroups is limited and may have reduced the power of the study.

Overall, our results show that, with a long follow-up (median = 34.3 months), most R/R HL patients eventually progress during anti-PD1 therapy, notably patients who are unable to achieve a CR. Conversely, consolidation with alloHSCT can convert incomplete responses into CR and is associated with a very low relapse-rate. Although patients undergoing alloHSCT after anti-PD1 may experience increased toxicities, these adverse events appeared manageable and reversible in most patients. Thus, consolidation with alloHSCT may be considered in R/R HL treated with anti-PD1, particularly in those not reaching a CR. However, the absence of benefit in OS and the possibility to induce a response with salvage chemotherapy after anti-PD1 "re-sensitization" should be considered when evaluating the risk/benefit ratio. Prospective studies are needed to further define which patients may benefit from consolidation with alloHSCT after anti-PD1 therapy.

***

Figure 1 :Figure 2 :Figure 3 :

 123 Figure 1: PFS (A & C) and OS (B & D) of the entire cohort (A & B) and according to best response upon nivolumab monotherapy (C & D)

•*Figure 1 .Figure 3 .

 13 Figure 1. PFS (A & C) and OS (B & D) of the entire cohort (A & B) and according to best response upon nivolumab monotherapy (C & D)

Table 1 . Patients' characteristics for the entire cohort Characteristics at nivolumab initiation

 1

	All
	N = 78

Nivolumab treatment and response Number of nivolumab injections, median [range]

	9.5 [1 -84]

Duration of anti-PD1 therapy, months, median [range] 5

  .2 [0 -38.3]

	Permanent treatment discontinuation	(85.9%)
	Reason for treatment discontinuation
	• Disease progression • Toxicity • Consolidation with allogenic-HSCT • Consolidation with autologous transplant • Decision of the clinician with no further treatment • Death • Other	(49.3%) (9.0%) (20.9%) (1.5%) (10.4%) (3.0%) (6.0%)
	Concomitant radiotherapy, No (%)	(9.6%)
	Concomitant chemotherapy, No (%)	(6.8%)
	Follow-up, months, median [range]	34.3 [0.1 -39.7]

Table 2 . Characteristics of patients in CR or PR after nivolumab monotherapy according to subsequent allogenic-HSCT Characteristics at nivolumab initiation No alloHSCT consolidation (N=37)

 2

	p
	AlloHSCT consolidation (N=13)

Acknowledgments

Elodie Gat, Laure Flament, Fanny Cherblanc, Florence Broussais, Nadine Morineau, Jean-Pierre Marolleau, Elena Loppinet, Sophie Lefort, Isabelle Roche-Lachaise, Sophie Ducastelle, Benoît Bareau, Mohamed Touati, Franck Morschhauser, Fabien Lebras, Marjan Ertault, Frédéric Peyrade, Caroline Regny, Thomas Gastine, Julien Lazarovici, Stéphanie Guidez, Luc-Mathieu Fornecker, Katell Le Dû, Georges Garnier, Barbara Burroni, Aiping Chen and Fatima-Zohra Mokrane.

Funding

This study was supported and funded by Bristol-Myers Squibb. The views expressed in this article are the authors' own and not an official position of Bristol-Myers Squibb or their respective institutions.

resolved with corticosteroids and one patient experienced a sinusoidal obstructive syndrome. The 6 or 12 months TRM was 11.8% and 17.6%.

Although the number of transplanted patients is limited, our results are consistent with other previously published studies (Supplementary Table 4). In their study, Merryman et al reported the outcome of 39 patients (31 HL and 8 NHL) who underwent alloHSCT after PD1 blockade [START_REF] Merryman | Safety and efficacy of allogeneic hematopoietic stem cell transplant after PD-1 blockade in relapsed/refractory lymphoma[END_REF]. Among HL patients, one-year PFS and OS were 74% and 90%, respectively.

Incidence of grade III-IV GVHD was 23% and 4 patients died of treatment-related toxicity.

Seven patients (18%) presented a non-infectious febrile syndrome requiring corticosteroids and 3 patients (8%) a sinusoidal obstructive syndrome. In Checkmate-205, 44 patients out of 243 patients proceeded to alloHSCT, with a median follow-up of 5.5 months after alloHSCT [START_REF] Armand | Nivolumab for Relapsed/Refractory Classic Hodgkin Lymphoma After Failure of Autologous Hematopoietic Cell Transplantation: Extended Follow-Up of the Multicohort Single-Arm Phase II CheckMate 205 Trial[END_REF]. The 6-months cumulative incidence of TRM and disease-progression were 13% and 7%, respectively. All grade incidence of acute and chronic GVHD were 48% and 15%, respectively.

To assess whether responding patients should be consolidated with alloHSCT or continue treatment with anti-PD1, we compared the outcome of patients in CR or PR upon nivolumab monotherapy with (n=13) and without (N= 37) subsequent alloHSCT. Although the patients in the transplanted group were younger (median age = 30.5 vs 38.0 years, p=0.029), and with a trend for more CR, the other characteristics at baseline were not significantly different between the 2 groups (Table 2). Twelve out of 20 patients (60%) who achieved a CR upon nivolumab monotherapy and did not undergo alloHSCT remained disease-free at the time of analysis, suggesting that some of them may be cured with anti-PD1 alone, even after nivolumab discontinuation [START_REF] Manson | Prolonged remissions after anti-PD1 discontinuation in patients with Hodgkin lymphoma[END_REF]. Conversely, 16 out of 17 (94.1%) patients who were unable to reach a CR upon nivolumab and did not undergo alloHSCT eventually progressed (Supplementary Figure 2). In transplanted patients, the relapse rate was markedly lower compared to non-transplanted patients (0% vs 62.2%, p<0.001, Figure 2A), both in CR and PR patients (Supplementary Figure 2). Interestingly, 5 out of 6 patients who were not in CR