Prof Martin J Van Den Bent

C Mircea

S Tesileanu

Wolfgang Wick

Prof Marc Sanson

Alba Ariela Brandes

Paul M Clement

Sarah Erridge

Michael A Vogelbaum

Prof Anna K Nowak

MD Walter Taal

MD Iris De Heer

French Phd

Prof Johan M Kros

Jean Français Baurain

Warren P Mason

Helen Wheeler

Olivier L Chinot

Sanjeev Gill

Matthew Griffin

Leland Rogers

Roberta Rudà

Prof Michael Weller

Catherine Mcbain

Jaap Reijneveld

MD Roelien H Enting

Francesca Caparrotti

Thierry Lesimple

Susan Clenton

Anja Gijtenbeek

Elizabeth Lim

Ulrich Herrlinger

Prof Peter Hau

Frederic Dhermain

Kenneth Aldape

Hendrikus Robert B Jenkins

Jan Dubbink

Pieter Wesseling

Sarah Nuyens

MD Vassilis Golfinopoulos

PhD Thierry Gorlia

Pim French

Brigitta G Baumert

Brain Tumor Center (

published or not. The documents may come

Adjuvant and concurrent temozolomide for 1p/19q non-co-deleted anaplastic glioma (CATNON; EORTC study 26053

Introduction

In 2005, a randomised controlled trial reported improved survival for patients with glioblastoma treated with concurrent and adjuvant temozolomide in addition to radio therapy. 1 However, at that time, no survival improvement was seen in patients with anaplastic oligodendroglioma treated with adjuvant procarbazine, lomustine, and vincristine chemotherapy after radio therapy. 2,3 Additionally, patients with anaplastic glioma without combined 1p/19q loss (1p/19q noncodeleted) had worse outcomes than those with 1p/19q codeleted tumours. In response to these results, the CATNON intergroup study was initiated to determine whether temozolomide given concurrently with radiotherapy or temozolomide given adjuvant to radiotherapy would improve survival. An interim analysis done shortly before the end of enrolment showed superior overall survival in patients receiving 12 cycles of adjuvant temozolomide following 59•4 Gy of radio therapy compared with those not receiving 12 cycles of adjuvant temozo lomide. 4 At that time, no signal of efficacy or futility was observed for the concurrent temozolomide comparison.

Mutations in IDH1 and IDH2 were first identified in 2008 and subsequently reported to represent early events in gliomagenesis, occurring in up to 70-80% of all diffuse WHO grade II and III gliomas. [5][6][7] The presence of IDH1 and IDH2 mutations was found to have a major prognostic effect on overall survival of patients with diffuse glioma and has been related to therapy response. [8][9][10] The 2016 revision of the WHO classification of CNS tumours classified diffuse glioma according to IDH1 and IDH2 mutation status, with anaplastic astrocytoma now being classified as either IDH1 and IDH2 mutant or wildtype. 11 A second interim analysis of CATNON was planned to report on the efficacy or futility of concurrent temozolomide compared with not receiving concurrent temozolomide. We report the results of this second interim analysis and an analysis based on IDH1 and IDH2 mutation status, which aligns with the updated WHO 2016 brain tumour classification. We also present analyses by MGMT promoter methylation status in IDH1 and IDH2 wildtype tumours. 12

Methods

Study design and participants

The study design has previously been reported in detail. 4 In short, this randomised, openlabel, phase 3 study with a twobytwo factorial design was done in 137 institutions across Australia, Europe, and North America (appendix pp 3-7). Patients with newly diagnosed anaplastic glioma without 1p/19q codeletion, aged 18 years or older, with a WHO performance status of 0-2, with adequate haematological, renal, and liver function, and without known HIV, chronic hepatitis B or hepatitis C virus infection were eligible. Central confirmation of pathology and assessment of 1p/19q status and MGMT promoter methylation were required before randomisation, but dedicated centres with confirmed testing procedures were allowed to enrol patients based on local histological diagnosis and 1p/19q assessment.

All institutions obtained ethics approval from their institutional review boards or ethics review committees before enrolment started. All patients gave written informed consent according to local, national, and international guidelines before study enrolment. The latest (13th) version of the protocol can be accessed online.

Randomisation and masking

The randomisation schedule was generated centrally via the European Organization of Research and Treatment of Cancer (EORTC) webbased ORTA system, which was accessed by local investigators. Patients were stratified by institution, WHO performance status score (>0 vs 0),

Research in context

Evidence before this study When this study was planned, anaplastic oligodendrogliomas were considered to be responsive to chemotherapy. However, patients with these tumours had no survival benefit when treated with adjuvant procarbazine, lomustine, and vincristine chemotherapy following radiotherapy. Additionally, patients with 1p/19q non-co-deleted tumours had a much worse prognosis than those with 1p/19q co-deleted tumours. In patients with glioblastoma, a tumour thought to be relatively chemotherapy resistant, radiotherapy combined with concurrent and adjuvant temozolomide improved outcomes. No further systematic literature review was done. In the randomised, open-label, phase 3 CATNON trial, we aimed to investigate the addition of concurrent and adjuvant temozolomide chemotherapy to standard adjuvant radiotherapy in adults with 1p/19q non-co-deleted anaplastic glioma. The first planned interim analysis of CATNON (published in 2017) showed that the addition of 12 cycles of adjuvant temozolomide chemotherapy after radiotherapy improved overall survival. At that time, the question of the value of temozolomide given concurrently with radiotherapy could not be answered. Furthermore, no data were yet available on the association of the mutational status of IDH1 and IDH2 with outcome. These driver mutations are present in a large subset of anaplastic astrocytoma and are now diagnostic markers in the WHO 2016 brain tumour classification.

Added value of this study

In a second preplanned interim analysis, we found that temozolomide given simultaneously with radiotherapy does not improve overall survival compared with not giving temozolomide. Even more importantly, we found that the clinical benefit of adding adjuvant temozolomide to radiotherapy is limited to patients with IDH1 or IDH2 mutant tumours.

Implications of all the available evidence

For patients with IDH1 or IDH2 mutant anaplastic astrocytoma, the standard of care consists of 59•4 Gy radiotherapy in 33 fractions followed by 12 cycles of adjuvant temozolomide chemotherapy. Further molecular analysis will be required to establish the role of MGMT promoter methylation in IDH1 and IDH2 wild-type tumours. Further clinical follow-up is needed to fully evaluate the role of concurrent temozolomide in IDH1 or IDH2 mutant tumours, if any. age (>50 years vs ≤50 years), 1p loss of heterozygosity (yes vs no), the presence of oligodendroglial elements on microscopy (yes vs no), and MGMT promoter methyl ation status (methylated vs unmethylated vs undetermined or invalid). Patients were assigned (1:1:1:1) using the minimisation technique, to radiotherapy alone, radio therapy with concurrent temozolomide, radiotherapy with adjuvant temozolomide, or radiotherapy with both concurrent and adjuvant temozolomide. Patients and investigators were not masked to treatment.

Procedures

Radiotherapy (threedimensional conformal radiotherapy or intensitymodulated radiotherapy), given to patients in all groups, consisted of 59•4 Gy given in 33 fractions to involved fields. Patients assigned to concurrent temozolomide treatment received daily 75 mg/m² oral temozolomide during the entire radiotherapy period. Patients assigned to adjuvant temozolomide chemo therapy commenced treatment 4 weeks after the end of radiotherapy. 12 cycles of temozolomide were given on days 1-5 every 28 days; for the first cycle 150 mg/m² was given orally per day and, in case of no or minimal toxicity in cycle one, the dose was escalated to 200 mg/m² daily in subsequent cycles. Dose reductions and delays were per protocol as previously described. 1,4 Patients were followed up during concurrent temozolomide with weekly assess ment of haematology, and during radiotherapy and adjuvant temozolomide treatment every 4 weeks, with assessment of haematology, liver, and renal function, and electrolytes. Adverse events were monitored using the International Common Terminology Criteria for Adverse Events (CTCAE; version 3.0). After the end of treat ment, followup including MRI and neurological examination was obtained every 3 months until progression. Progressionfree survival was assessed locally using Macdonald criteria; 13 the protocol gave guidelines for handling of suspected pseudoprogression.

For biomarker analysis, 5-10 μm thick tissue sections were macrodissected for areas with highest tumour content (marked by a dedicated neuropathologist [JMK] on a consecutive haematoxylin and eosinstained section). DNA was then isolated using the formalinfixed paraffin embedded (FFPE) Allprep DNA/RNA FFPE kit (Qiagen, Venlo, Netherlands). [START_REF] Wijnenga | The impact of surgery in molecularly defined lowgrade glioma: an integrated clinical, radiological, and molecular analysis[END_REF] Mutations in IDH1 and IDH2 were assessed with a gliomadedicated capturebased next generation sequencing panel (Agilent sureselect, Agilent, Santa Clara, CA, USA; ThermoFisher Scientific, Waltham, MA, USA). [START_REF] Sahm | Nextgeneration sequencing in routine brain tumor diagnostics enables an integrated diagnosis and identifies actionable targets[END_REF][START_REF] Dubbink | Molecular classification of anaplastic oligodendroglioma using nextgeneration sequencing: a report of the prospective randomized EORTC Brain Tumor Group 26951 phase III trial[END_REF] For patients with insufficient tumour material available, a second smaller gliomadedicated sequencing panel that requires less material was used. [START_REF] Dubbink | Molecular classification of anaplastic oligodendroglioma using nextgeneration sequencing: a report of the prospective randomized EORTC Brain Tumor Group 26951 phase III trial[END_REF][START_REF] Dubbink | Diagnostic detection of allelic losses and imbalances by nextgeneration sequencing: 1p/19q codeletion analysis of gliomas[END_REF] MGMT promoter methylation analysis was done following bisulphite treatment: before random isation using methylationspecific PCR (MSPCR) and at the time of biomarker analysis using the Infinium MethylationEPIC BeadChip Kit (EPIC arrays; Illumina, San Diego, CA, USA) and the MGMTSTP27 algorithm. [START_REF] Bady | MGMT methylation analysis of glioblastoma on the Infinium methylation BeadChip identifies two distinct CpG regions associated with gene silencing and outcome, yielding a prediction model for comparisons across datasets, tumor grades, and CIMPstatus[END_REF][START_REF] Bady | Sensitivity analysis of the MGMTSTP27 model and impact of genetic and epigenetic context to predict the MGMT methylation status in gliomas and other tumors[END_REF][START_REF] Van Den Bent | MGMTSTP27 methylation status as predictive marker for response to PCV in anaplastic oligodendrogliomas and oligoastrocytomas. A report from EORTC study 26951[END_REF] For the molecular analysis, 1p/19q status was confirmed using the copy number alterations extracted from the methylation data. [START_REF] Capper | DNA methylationbased classification of central nervous system tumours[END_REF]

Outcomes

The primary endpoint was overall survival adjusted for stratification factors at randomisation. Secondary endpoints were progressionfree survival, neurological deteriorationfree survival, quality of life, safety, and development of cognitive deterioration. Overall survival was defined as time from randomisation to death from any cause, and progressionfree survival was defined as time from randomisation to disease progression or death from any cause. Analyses of quality of life, neurological deteriorationfree survival, and development of cognitive deterioration are subject to further analysis and results

are not yet available.

Statistical analysis

Two questions were addressed in this study: comparison of patients receiving radiotherapy alone or radiotherapy and adjuvant temozolomide with those receiving radio therapy and concurrent temozolomide or radiotherapy and concurrent temozolomide followed by adjuvant temozolomide; and comparison of patients receiving radio therapy alone and radiotherapy and concurrent temozolomide with those receiving radiotherapy alone followed by adjuvant temozolomide or radiotherapy and concurrent temozolomide followed by adjuvant temozolomide. For both questions, a hazard ratio (HR) of 0•775 (ie, a death risk reduction of 22•5%) was assumed based on a twosided logrank test at an overall significance level of 5% (median overall survival of 24 months in patients receiving radiotherapy only, 31 months in patients treated with concurrent temo zolomide or adjuvant temozolomide, and 40 months in patients treated with concurrent and adjuvant temozo lomide). To achieve 83% power to show the targeted difference for the two questions, 534 overall survival events were needed for the final analysis, and 748 patients had to be recruited. [START_REF] Kim | Design and analysis of group sequential tests based on the type I error spending rate function[END_REF] The first interim analysis for efficacy based on the ρ family (ρ=2) stopping boundaries was initially planned when 41% of the overall survival events had been observed. This analysis was done when a slightly larger number of overall survival events was observed at modified significance level 0•00855 (instead of 0•0084). The analyses were significant for the adjuvant temozolomide question, but not for the concurrent temozolomide one. 4 Further to the recommendation of the independent data monitoring committee (IDMC), the protocol was amended (version 13) on June 28, 2017, to include a second interim analysis to assess the concurrent temozolomide question with more mature data and to provide more details on the testing for IDH1 and IDH2 and other biological correlates, including MGMT.

The second interim analysis presented here proposed to stop either for futility (reject H1) or efficacy (reject H0) after twothirds of total planned events were observed (356 of 534) using an efficacy stopping boundary from the ρ family (ρ=2) and an O'BrienFleming futility stopping boundary. [START_REF] O'brien | A multiple testing procedure for clinical trials[END_REF] Futility for the concurrent temozolomide question would be declared if the observed HR was higher than 0•882 and efficacy if less than 0•778 (p<0•009). The HR 0•882 corresponds to an overall survival rate at 2 years of 71•9% in the control group versus 74•7% in the experimental group (difference <3%). The final analysis after 534 deaths would be done at a significance level of 0•044.

All efficacy (progressionfree survival and overall survival) analyses were done in the intentiontotreat population, which was defined as all patients randomly assigned to a treatment group. We used the KaplanMeier technique for the univariable estimates to calculate the HRs and 95% CIs for overall survival and progression free survival. For patients alive at the time of analysis, overall survival was censored at last followup visit date. For patients alive and without progression at the time of analysis, progressionfree survival was censored at the last followup visit date. In the primary analyses of overall survival and progressionfree survival adjusted for stratification factors (WHO performance status, age, the presence of 1p loss of heterozygosity only, the presence of oligodendroglial elements, and MGMT promoter methyl ation status at time of randomisation), a Cox proportionalhazards model was fitted with a question indicator for each question the trial was intended to answer. We did supportive analyses using Cox models with both questions adjusted by the stratification factors and MGMT methylation status after randomisation. The analysis of the adjuvant temozolomide question was repeated at retrospective 5% significance.

For both overall survival and progressionfree survival, Cox analyses proportionalhazards assumptions were checked with a Kolmogorovtype supremum test based on the ASSESS statement of SAS PHREG procedure at a conservative 1% significance level. The tests were computed with all variables in the models (treatment, age, WHO performance status, presence of 1p loss only, presence of oligodendroglioma components, and MGMT methylation status at randomisation). If the proportionalhazards assumptions for a variable were violated, the variable was removed from the models (eg, linear predictors) and used as a stratification factor in the Cox models. These analyses were considered as sensitivity analyses.

In response to emerging molecular data, the CATNON study protocol was amended (version 8) on June 27, 2011, to include analyses based on the mutation status of IDH1 and IDH2 genes. This amendment also allowed intensity modulated radiotherapy. For the analysis of the IDH1 and IDH2 mutation status, we did univariate (prognostic) and interaction (predictive) exploratory approach analyses of overall survival and progressionfree survival (with Cox models including marker by treatment interaction terms).

Agreement between results of different MGMT assays was assessed using concordance percentage and κ statistics with 95% CI. We measured relative dose intensity of temozolomide in patients with sufficient treatment information. Values were calculated as the sum of doses delivered per administration divided by the total planned dose for the total planned time of delivery. In the exploratory analyses, p values of less than 0•05 were considered to be significant (appendix p 30).

All clinical study data were captured locally on study specific case record forms and submitted to the EORTC Data Center, where they were entered in a central study database. We used SAS (version 9.4) for all analyses.

This study is registered with ClinicalTrials.gov, NCT00626990.

Role of the funding source

The funder had no role in the study design, data collection, data analysis, data interpretation, or writing of the report.

Results

Between Dec 4, 2007, and Sept 11, 2015, 1407 patients were registered and 751 patients were randomly assigned to radiotherapy alone (n=189), radiotherapy with concurrent temozolomide (n=188), radiotherapy and adjuvant temozo lomide (n=186), and radiotherapy with concurrent temo zolomide and adjuvant temozo lomide (n=188). The second interim analysis includes all data up to Feb 3, 2018 (second clinical cutoff date). All subsequent entries were censored at that date. The database was locked on Aug 2, 2018. At that date, the median followup was 55•7 months (IQR 41•0-77•3), 395 (53%) patients were still alive, and 267 (36%) patients were alive without progression. Figure 1 provides the CONSORT diagram of patients per study group and the IDH1 and IDH2 mutation status per study group. For the planned analyses of the prognostic and predictive value of IDH1 and IDH2 mutations, the clinical data were updated up to Feb 3, 2019, with another database lock on May 7, 2019. At that date, median followup was 66•7 months (IQR 50•1-88•6) and 384 (51%) patients were still alive. With an additional six patients enrolled after the first IDMC report (Oct 6, 2015), the treatment groups remained well balanced for baseline clinical characteristics (appendix p 8). The table provides the randomisation outcome, treatment details and temozolomide treatment intensity, reasons for stopping treatment, and survival status at the time of analysis.

Futility of concurrent temozolomide was declared (median overall survival was 66 With 484 patients having progressed or died (appendix p 13), progressionfree survival was not significantly different with versus without concurrent temozolomide (appendix p 12). Progressionfree survival was significantly improved after adjuvant temozolomide versus without adjuvant temozolomide (appendix p 12). Tests for inter action between the concurrent part of the treatment and the adjuvant part of the treatment remained negative (p=0•71; appendix p 27). For all Cox analyses, the proportionalhazards assumptions were violated for age. The results of the sensitivity analyses with age as a stratification factor in the Cox models are presented in the appendix (pp 31-62). Sensitivity analyses confirmed the results of the primary analyses for progressionfree survival and overall survival for the two therapeutic questions.

The number of progressionfree survival events per group and treatments given at progression are shown in the appendix (p 13). In the group of patients assigned to radiotherapy only, 118 (81%) of 145 patients who progressed had received some type of further treatment. This treatment was chemotherapy in 111 (77%) of 145 patients, 97 (67%) of whom received temozolomide.

Grade 3 and 4 adverse events were rare, except for haematological adverse events (appendix pp 16-21). 55 (15%) of 368 patients in both adjuvant temozolomide groups and 16 (9%) of 185 patients in the concurrent temozo lomide group had grade 3 or 4 haematological adverse events, in particular thrombocytopenia (36 [10%] of 368 patients in the adjuvant temozolomide groups and 14 [8%] of 185 patients in the concurrent temozolomide group) and neutropenia (24 [7%] and five [3%]). Treatment was stopped for adverse events in seven (4%) of 188 patients receiving concurrent temozolomide, 14 (8%) Sufficient FFPE tumour tissue was available to deter mine IDH1 and IDH2 mutation status in 671 patients. 11 IDH1 or IDH2 mutant tumours were found to be 1p/19q codeleted after copy number analysis of EPIC array data, and were subsequently excluded from this analysis (three in the radiotherapy only group, three in the concurrent temozolomide group, one in the adjuvant temozolomide group, and four in the concurrent and adjuvant temozolomide group). In the 740 patients with 1p/19q intact, the numbers of patients with a IDH1 or IDH2 mutant tumour were well balanced between the four groups and varied between 107 (58%) of 186 patients in the radiotherapy only group and 113 (61%) of 184 patients in the radiotherapy with concurrent and adjuvant temozo lomide group (appendix p 14). The IDH1 and IDH2 mutation status was available in a total of 660 anaplastic astrocytoma tumours: 216 (33%) were wildtype IDH1 and IDH2 and 444 (67%) were mutant IDH1 or IDH2. At the time of analysis, 32 (15%) of the 216 patients with IDH1 and IDH2 wildtype tumours and 292 (66%) of the 444 patients with IDH1 or IDH2 mutant tumours were still alive. In the 740 patients without 1p/19q codeletion, MGMT promoter methylation status could be assessed in 663 (90%) patients using the MGMTSTP27 algorithm. Originally, 185 (25%) patients could not have their MGMT status assessed by the MSPCR method, so we used the MGMTSTP27 algorithm. Of the IDH1 and IDH2 wild type tumours, 133 (62%) of 216 had unmethylated MGMT and 78 (36%) had methylated MGMT; no results were obtained in five tumours. In the IDH1 and IDH2 wildtype tumours, MGMT status as determined with MSPCR correlated moderately with the MGMTSTP27 algorithm (percentage concordance 85%, κ statistics 0•67 [95% CI 0•55-0•80]). 117 (88%) of the 133 patients with unmethylated MGMT tumours and 63 (81%) of the 15) 2 ( 13) 2 ( 16) 1 ( 13) 1 ( 17) 1 (13) 0 ( 18 78 patients with methylated MGMT tumours had died at the time of the analysis. Survival of these patients according to treatment is presented in the appendix (p 14). Overall survival by MGMT methyl ation status with and without concurrent temozolomide, with and without adjuvant temozolomide, and with and without any temozolomide are shown in the appendix (pp 28-29).

Discussion

The initial report on the CATNON trial on newly diagnosed anaplastic astrocytoma without 1p/19q codeletion showed a benefit from the addition of 12 cycles of adjuvant temozolomide and defined the new standard of care in this disease. 4 In this second interim analysis, we show no benefit from giving daily temozolomide concurrently with radiotherapy. Importantly, we are now able to report the study incorporating the IDH1 and IDH2 mutation status, according to the revised WHO 2016 brain tumour classification. 11 This analysis reveals lack of clinical benefit from either concurrent or adjuvant temozolomide in the IDH1 and IDH2 wildtype tumours, and clinical benefit from the adjuvant temozolomide only in patients with IDH1 or IDH2 mutant tumours. With the current number of events analysed, there is no clinically relevant benefit of temozolomide given concurrently with radiotherapy to patients with IDH1 or IDH2 mutant anaplastic astro cytoma. The lack of a clear effect is further emphasised if one considers patients who were treated with adjuvant temozolomide. However, in the subgroup of patients with IDH1 or IDH2 mutant tumours who did receive temozolomide concurrently with radiotherapy, adding adjuvant temozolomide still significantly improved overall survival. Taken together, the CATNON trial shows that the benefit from temozolomide is derived from the adjuvant phase and is limited to patients with IDH1 or IDH2 mutant anaplastic astrocytoma. Previous trials examining adjuvant procarbazine, lomustine, and vincristine in anaplastic oligo dendro glioma and lowgrade glioma identified either IDH1 and IDH2 mutation status or MGMT promoter methylation as predictive factors for benefit from alkylating chemo therapy. 10,[START_REF] Van Den Bent | MGMTSTP27 methylation status as predictive marker for response to PCV in anaplastic oligodendrogliomas and oligoastrocytomas. A report from EORTC study 26951[END_REF][START_REF] Buckner | Radiation plus procarbazine, CCNU, and vincristine in lowgrade glioma[END_REF] At the time that this study was initiated, it was well established that MGMT promoter methylation was a predictor of sensitivity to alkylating chemotherapy. Subsequent studies found that the vast majority (85-90%) of IDH1 or IDH2 mutant tumours also had a methylated MGMT promoter. 12 In this trial, we used two methodologies to determine the MGMT status: MSPCR and genome wide methylation analysis. In the prespecified multi variable analysis of the primary endpoint, MGMT status as determined by MSPCR was of independent signifi cance, but this potentially also reflects its relation to IDH1 and IDH2 mutation status; moreover, methylation status could not be determined in about a quarter of patients. We explored the association of MGMT promoter methylation status with outcome in the group of 216 patients with IDH1 and IDH2 wildtype tumours, and found no evidence of clinical benefit after concurrent temozolomide or after adjuvant temozolomide in patients with MGMT methyl ated, IDH1 and IDH2 wildtype tumours. This lack of efficacy is unexpected in view of the significant benefit of the addition of temozolomide to radiotherapy in patients with glioblastomas with MGMT promoter methylation, which are predominantly wildtype for IDH1 and IDH2. [START_REF] Hegi | MGMT gene silencing and benefit from temozolomide in glioblastoma[END_REF][START_REF] Perry | A phase III randomized controlled trial of shortcourse radiotherapy with or without concomitant and adjuvant temozolomide in elderly patients with glioblastoma (CCTG CE.6, EORTC 2606222061, TROG 08.02, NCT00482677)[END_REF] One potential explanation for this might be the limited number of patients with IDH1 and IDH2 wild type tumours in this study. Additionally, IDH1 and IDH2 wildtype astrocytomas are not a single entity, and only a subgroup of such tumours show a molecular background reflecting glioblastoma. [START_REF] Brat | cIMPACTNOW update 3: recommended diagnostic criteria for "Diffuse astrocytic glioma, IDHwildtype, with molecular features of glioblastoma, WHO grade IV[END_REF][START_REF] Tesileanu | Survival of diffuse astrocytic glioma, IDH1/2wildtype, with molecular features of glioblastoma, WHO grade IV: a confirmation of the cIMPACTNOW criteria[END_REF] A further report on the CATNON study will detail the association of MGMT status with outcome in the IDH1 and IDH2 wildtype tumours, including the subgroup of anaplastic glioma with molecular features of glioblastoma.

The most relevant limitation of these analyses is that the study was powered to ask the concurrent and adjuvant temozolomide question in a twobytwo factorial design trial, and not for further subgroup analysis. A second limitation is the thusfar short followup and the low number of survival events in patients with IDH1 or IDH2 mutant tumours. This limited followup might mask more subtle beneficial effects of concurrent temozolomide treatment in the IDH1 or IDH2 mutant tumours. Given the slight separation of the survival curves after concurrent temozolomide in IDH1 or IDH2 mutant tumours, with longer followup, an overall survival signal might emerge similar to observations in studies on lowgrade glioma and on anaplastic oligoden dro glioma. [START_REF] Buckner | Radiation plus procarbazine, CCNU, and vincristine in lowgrade glioma[END_REF][START_REF] Van Den Bent | Adjuvant procarbazine, lomustine, and vincristine chemotherapy in newly diagnosed anaplastic oligodendroglioma: longterm followup of EORTC brain tumor group study 26951[END_REF] Hence, a longerterm analysis is required, with the required number of survival events needed for the final analysis anticipated to occur in 2024-25. Nevertheless, the current data convincingly show that the adjuvant administration of temozolomide has a greater effect on overall survival than concurrent administration. There are two toxicity concerns with concurrent temozolomide (although admittedly data are scarce): first, concurrent temozolomide and radiotherapy might potentiate delayed neurotoxicity, and second, pseudoprogression occurs more often if radiotherapy is combined with chemo therapy. [START_REF] Taal | Incidence of early pseudo progression in a cohort of malignant glioma patients treated with chemoirradiation with temozolomide[END_REF] In this context, currently, our results argue for treating IDH1 or IDH2 mutant anaplastic astrocytoma with only 12 cycles of adjuvant temozolomide.

To conclude, the present analysis shows that the benefit from temozolomide in anaplastic astrocytoma is derived from the adjuvant phase of the treatment and is observed only in patients with IDH1 or IDH2 mutant tumours. Longer followup is needed, especially in IDH1 or IDH2 mutant tumours, as the increase in progression free survival could in time translate into an overall survival benefit. Further studies are ongoing to assess molecular factors associated with prognosis.

Contributors

MvdB, WW, MW, MAV, and WPM did the literature search. MvdB, WW, MW, MAV, WPM, TG, PF, and BGB designed the study. MvdB, CMST, WW, MS, AAB, PMC, SE, MAV, AKN, JFB, WPM, HW, OLC, SG, MG, LR, WT, RR, MW, CM, JR, RHE, FC, TL, SC, AG, EL, UH, PH, FD, IdH, KA, RBJ, HJD, JMK, PW, SN, VG, TG, PF, and BGB collected data. MvdB, CMST, TG, and PF analysed the data. MvdB, CMST, TG, and PF interpreted the data. All authors wrote and approved the final version of the report. MvdB, TG, and CMST verified all the data. All authors had access to all data in the study and had final responsibility for the decision to submit for publication.

Declaration of interests

MJvdB reports grants from the Dutch Cancer Foundation, Brain Tumor Charity, Strijd van Salland, and Merck Sharp & Dohme (formerly Schering Plough), during the conduct of the study; and personal fees from Carthera, Nerviano, Bayer, Cellgene, Agios, AbbVie, Karyopharm, Boston Pharmaceuticals, and Genenta, outside the submitted work. PMC reports personal fees from Bristol Meyers Squibb, AbbVie, Merck Serono, Merck Sharp & Dohme, Vifor Pharma, DaNchi Sankyo, LEO Pharma, AstraZeneca, and Takeda, outside the submitted work. MAV reports grants from the US National Cancer Institute, during the conduct of the study, and personal fees from Tocagen and Celgene, outside the submitted work. AKN reports grants and personal fees from AstraZeneca, and Douglas Pharmaceuticals, and personal fees from Bayer Pharmaceuticals, Roche Pharmaceuticals, Boehringer Ingelheim, Merck Sharp & Dohme, Pharmabcine, Atara Biotherapeutics, and Trizell, outside the submitted work. MW reports grants and personal fees from AbbVie, Merck Sharp & Dohme, Merck (EMD); grants from Adastra, Novocure; and personal fees from Basilea, Bristol Myer Squibbs, Celgene, Nerviano, Orbus, Roche, and Tocagen, outside the submitted work. UH reports nonfinancial support from Medac, and personal fees from Novartis, DaiichiSankyo, Noxxon, AbbVie, Bayer, Janssen, and Karyopharm, outside the submitted work. HJD reports personal fees from AbbVie, outside the submitted work. BGB reports personal fees and nonfinancial support from Roche, outside the submitted work. All other authors declare no competing interests.

Data sharing

Clinical data will be shared upon full completion of the protocol.

BFigure 2 :

 2 Figure 2: Univariable analysis of overall survival in all patients regardless of IDH1 and IDH2 mutational status (A) Patients who received concurrent temozolomide versus those who did not. (B) Patients who received adjuvant temozolomide versus those who did not.

AFigure 3 :

 3 Figure 3: Univariable analysis of overall survival in patients with IDH1 and IDH2 wild-type tumours (A) Patients who received concurrent temozolomide versus those who did not. (B) Patients who received adjuvant temozolomide versus those who did not.

Figure 4 :

 4 Figure 4: Univariable analysis of overall survival in patients with IDH1 or IDH2 mutant tumours (A) Patients who received concurrent temozolomide versus those who did not. (B) Patients who received adjuvant temozolomide versus those who did not.

  •9 months [95% CI 45•7-82•3] with concurrent temozolomide vs 60•4 months [45•7-71•5] without concurrent temozolomide; HR 0•97 [99•1% CI 0•73-1•28], p=0•76), and the IDMC therefore recommended release of the study results (appendix p 11).

	The analysis for adjuvant temozolomide question
	remained significant (median overall survival 82•3 months
	[95% CI 67•2-116•6] with adjuvant temozolomide vs
	46•9 months [37•9-56•9] without adjuvant temozolomide;
	HR 0•64 [95% CI 0•52-0•79], p<0•0001; appendix p 11).
	The sup portive univariable analyses provided similar con
	clusions (figure 2A, 2B; appendix p 12). With 356 patients
	having died (table), overall survival was not significantly
	different with versus without concurrent temozolomide
	(5year overall survival 52•7% [95% CI 46•9-58•1]
	vs 50•2% [44•4-55•7]; HR 0•93 [95% CI 0•75-1•14],
	p=0•46; figure 2A; appendix p 12). Overall survival was,
	however, significantly different with versus without
	adjuvant temozolomide (5year overall survival 58•5%
	[95% CI 52•8-63•8] vs 44•3% [38•6-49•9; HR 0•67
	[95% CI 0•55-0•83], p<0•0001; figure 2B; appendix p 12).

Table : Overview of patients per group, number of events at the time of the second IDMC report, and treatment

 :

		A
		100
		90
		80
		70
	Overall survival (%)	40 50 60
		30
		20	Concurrent temozolomide
		10	No concurrent temozolomide HR 0•93 (95% CI 0•75-1•14); p=0•46
		0
		0	1	2	3	4	5	6	7	8	9	10	11
	Median overall survival was 19•9 months (95% CI 16•8-22•7) for patients with IDH1 and IDH2 wildtype tumours and 98•4 months (85•2-116•6) for patients with IDH1 or IDH2 mutant tumours (HR 0•14 [95% CI 0•12-0•18], p<0•0001; appendix p 25). In patients with IDH1 and IDH2 wildtype tumours, neither con current nor adjuvant temozolomide improved overall survival compared with radiotherapy alone (86 patients who received concurrent temozolomide and 98 patients who did not receive concurrent temozolomide died; 91 people who received adjuvant temozolomide and 93 patients who did not receive adjuvant temozolomide died; figure 3). In patients with IDH1 or IDH2 mutant tumours, adjuvant temo zolomide improved overall survival compared with no adjuvant temozolomide, but (number censored) Concurrent temozolomide No concurrent temozolomide 376 (0) 375 (0) 332 (5) 332 (7) 259 (21) 263 (18) 197 (53) 186 (49) 130 (94) temozo lomide compared with no concur rent temo zolomide (85 patients who received concurrent temo zolomide and 67 patients who did not receive concurrent temozolomide died; 59 people who received adjuvant temozolomide and 93 patients who did not receive adjuvant temozolomide died; figure 4). With tests for interaction, IDH1 and IDH2 mutation status was highly significant for benefit to adjuvant temo zolomide (p=0•001), but not concurrent temozolomide (p=0•29). When considering IDH1 or IDH2 mutant tumours treated with adjuvant temozo lomide, the addition of concurrent temozolomide did not improve overall survival (HR 0•82 [95% CI 0•49-1•36], p=0•44; appendix p 24); 5year survival was 80•5% (95% CI 126 (92) 87 (131) 84 (125) 53 (158) 54 (145) 28 (179) 30 (166) 15 (191) 20 (172) 6 (200) 9 (182) 0 (205) 0 (190) •• •• Number at risk (number censored) Adjuvant temozolomide 0 1 2 3 4 6 5 7 8 9 10 11 374 (0) 339 (7) 272 (18) 206 (51) 143 (98) 94 (141) 58 (171) 30 (196) 20 (204) 12 (212) 0 (222) 71•3Number at risk No adjuvant temozolomide 377 (0) 325 (5) 250 (21) 177 (51) 113 (88) 77 (115) 49 (132) 28 (149) 15 (159) 3 (170) 0
	no overall survival benefit was observed after concurrent

-87•0) in patients who did not receive concurrent temozolomide versus 82•8% (73•7-89•0) in patients

www.thelancet.com/oncology Vol 22 June 2021

www.thelancet.com/oncology Vol 22 June 2021

Acknowledgments

This study was funded by Merck Sharp & Dohme (formerly Schering Plough) through an educational grant and provision of temozolomide. This work was supported by the NRG (grants U10CA180868 and For the data sharing policy see http://www.eortc.org/app/ uploads/2017/03/pol008.pdf U10CA180822), Cancer Research UK grant CRUK/07/028, and Cancer Australia (project grants 1026842 and 1078655). The molecular studies were supported by grant GN000577 from The Brain Tumour Charity, grant 10685 from the Dutch Cancer Society, and support from the Vereniging Heino 'Strijd van Salland'. We thank our patients and their relatives for their willingness to participate to this study. We also thank all sites and their staff for contributing to this study. The study protocol was prepared and the study database was developed, housed, and analysed by the EORTC. We acknowledge the support of this study by the staff at the EORTC Headquarters in Brussels, Belgium, the NRG Oncology (formerly the Radiation Therapy Oncology Group) staff at the American College of Radiology; the staff at the Australian National Health and Medical Research Council (NHMRC) Clinical Trials Centre (COGNO Coordinating Centre); and the staff at MRC Clinical Trials Unit, London UK.

Funding Merck Sharpe & Dohme.


W J Loughry
email: jloughry@valdosta.edu

Richard W Truman

Colleen M Mcdonough

Marie-Ka Tilak

Stéphane Garnier

Frédéric Delsuc

IS LEPROSY SPREADING AMONG NINE-BANDED ARMADILLOS IN THE SOUTHEASTERN UNITED STATES?

Keywords: armadillo, Dasypus novemcinctus, leprosy, Mycobacterium leprae, population genetics

In the United States, nine-banded armadillo (Dasypus novemcinctus) populations are derived from two sources: (1) a continuous range expansion from Mexico led to western populations, some of which -particularly along the western Gulf Coast and west side of the Mississippi River delta -exhibit persistently high levels of leprosy infection, and (2) a small group of animals released from captivity in Florida gave rise to eastern populations that were all considered leprosy-free. Given that western and eastern populations have now merged, an important question becomes, to what extent is leprosy spreading into formerly uninfected populations? To answer this question we sampled 500 animals from populations in Mississippi, Alabama, and Georgia. Analyses of nuclear microsatellite DNA markers confirmed the historical link between source populations from Texas and Florida, but did not permit resolution of the extent to which these intermediate populations represented eastern versus western gene pools. Prevalence of leprosy was determined by screening blood samples for the presence of antibodies against Mycobacterium leprae, and via PCR amplification of armadillo tissues to detect M. leprae DNA. The proportion of infected individuals within each population varied from 0-10%. Although rare, a number of positive individuals were identified in eastern sites previously considered uninfected. This indicates leprosy may be spreading eastward and calls into question hypotheses proposing leprosy infection is confined because of ecological constraints to areas west of the Mississippi River.

INTRODUCTION

Aside from humans, nine-banded armadillos (Dasypus novemcinctus) are the only other free-ranging vertebrates known to harbor naturally-occurring infections of Mycobacterium leprae, the causative agent in producing leprosy. Within the United States (U.S.), armadillo populations are derived from two sources. First, armadillos crossed the Rio Grande into southeast Texas sometime in the 1820s, with populations spreading rapidly north and east since [START_REF] Taulman | Recent range expansion and distributional limits of the nine-banded armadillo (Dasypus novemcinctus) in the United States[END_REF]. Second, releases of a limited number of captive animals occurred in south-central Florida in the 1920s [START_REF] Humphrey | Zoogeography of the nine-banded armadillo (Dasypus novemcinctus) in the United States[END_REF]. Descendents of these animals established populations which subsequently spread north and west. Western and eastern populations have now merged, probably in eastern Mississippi or western Alabama in the 1980s [START_REF] Taulman | Recent range expansion and distributional limits of the nine-banded armadillo (Dasypus novemcinctus) in the United States[END_REF], to form a continuous distribution across much of the southern U.S.

This biogeographical history appears linked with patterns of leprosy infection. Early histopathological studies and later serological analyses both identified infected animals at multiple locations in Louisiana and Texas, in populations clearly derived from western sources (review in Truman, 2008). In contrast, virtually no infected animals were found east of the Mississippi River [START_REF] Howerth | Survey for leprosy in nine-banded armadillos (Dasypus novemcinctus) from the southeastern United States[END_REF]Truman, 2008), perhaps due to the absence of the disease among the small number of founding individuals from which these populations were derived. Now that eastern and western populations have merged, it seems possible that leprosy could spread eastward into previously uninfected populations.

However, not all western populations of armadillos exhibit leprosy infection. Mirroring the pattern of human infection, most cases of leprosy in armadillos appear confined to a region along the western Gulf Coast and west side of the Mississippi River (see Fig. 1 in Truman, 4 2005). [START_REF] Truman | Environmental associations for Mycobacterium leprae[END_REF]Truman ( , 2005) ) argued this might be due to certain ecological features that restrict leprosy to these areas. The exact nature of these features remains unknown, but might include:

(1) appropriate low-lying, humid soils to facilitate survival of M. leprae outside a host, (2) high densities of armadillos, and/or (3) other environmental agents that affect either disease transmission or susceptibility. If true, this "ecological constraints" hypothesis predicts no eastward spread of leprosy because the necessary conditions do not typically exist east of the Mississippi River. In contrast, "epidemic" models (e.g., [START_REF] Scholl | Simulation of naturally occurring leprosy transmission in free-living armadillo populations[END_REF] predict a steady spread of leprosy as infected animals disperse eastward.

The purpose of the present study was to sample armadillo populations at several locations east of the Mississippi River in order to determine whether leprosy has spread in that direction.

These results are then discussed in light of the ecological constraints versus epidemic models of leprosy distribution.

MATERIALS AND METHODS

Field work

Samples were collected from May-July 2005 at the five locations shown in Fig. 1, with follow-up sampling at two sites (YZ and RS) during the summer of 2006. Sites were chosen on the basis of the following: (1) Yazoo National Wildlife Refuge (YZ; 33º 05'/90º 59') had a soil profile and proximity to infected populations across the Mississippi River such that both ecological constraints and epidemic hypotheses predicted high levels of leprosy infection; (2) St.

Catherine's Creek National Wildlife Refuge (SC; 31º 22'/91º 26'), like Yazoo, was in close proximity to populations exhibiting high levels of infection (Truman, 2005(Truman, , 2008)), but possessed a different soil profile. Thus, the ecological constraints hypothesis predicted little occurrence of leprosy, while the epidemic hypothesis predicted the opposite; (3) Stimpson Wildlife Sanctuary (ST; 31º 23'/87º 51') and (4) DeWayne Hayes Recreational Area (DH; 33º 36'/88º 28') were located at roughly the same latitudes as St. Catherine's Creek and Yazoo, respectively, but 241-338 km farther east. Soil conditions at both sites led to the prediction from the ecological constraints hypothesis that no leprosy should be present. However, the epidemic hypothesis predicted leprosy could occur, with prevalence dependent on the extent to which each population had contact with western-derived individuals. Samples from Riverside (RS; 32º 54'/88º 11') were pooled as part of the DeWayne Hayes data in 2005, but were collected and analyzed separately in 2006. Finally, (5) Pinebloom Plantation (PB; 31º 43'/84º 38') was located so far east and had a soil profile such that both hypotheses predicted no occurrence of leprosy there.

Basic methods for capturing live animals followed previously published protocols (e.g., [START_REF] Mcdonough | Impacts of land management practices on a population of nine-banded armadillos in northern Florida[END_REF]. Upon capture, two ear notches were collected: one was preserved in 100% ethanol for genetic screening, the other in 70% ethanol for PCR analyses to detect M. leprae DNA in putatively infected animals. In addition, we clipped the end of one toenail to obtain a small blood sample, collected onto Nobuto blood strips (Advantec, Dublin, California, USA).

We also sampled dead armadillos. These included all the animals from Pinebloom and Riverside (in 2006), 34 of the animals from DeWayne Hayes, and fresh road-kills from a variety of locations. In these cases, in addition to collecting ear and blood samples, we also collected the spleen from each animal, preserving it in 100% ethanol.

Genetic analyses

To further evaluate the epidemic hypothesis, ear samples were screened for genetic markers that might discriminate between western and eastern source populations and allow estimates of gene flow. Previously collected samples from Welder Wildlife Refuge (WR; 28º 07'/97º 22') and Tall Timbers Research Station (TT; 30º 38'/84º 13') were used as representatives of western and eastern gene pool sources respectively, while samples from the Tulane University Museum of Natural History (NO; 29º 50'/90º 00') were included as an additional intermediate population (Fig. 1).

Twenty individuals from each sampled population (n = 7; note Pinebloom was not included because of its close proximity to Tall Timbers and that samples from DeWayne Hayes and Riverside were pooled) were randomly selected and genotyped for five of the nuclear microsatellite loci (Dnov1, Dnov6, Dnov7, Dnov16 and Dnov24) previously identified by [START_REF] Prodöhl | Molecular documentation of polyembryony and the micro-spatial dispersion of clonal sibships in the nine-banded armadillo, Dasypus novemcinctus[END_REF]. Loci were PCR amplified using an end-labeled reverse primer with fluorescein. Two µl of PCR product from each individual was subsequently loaded onto an 8% acrylamide gel (Bio-Rad, Hercules, California, USA). DNA fingerprints were visualized with the FMBIO fluorescent imaging system (Hitachi). Allele numbers were determined with a fluorescently labeled ladder (Promega), using the FMBIO ANALYSIS 8.0 image analyzer program.

Intra-population genetic variation was estimated by observed (Ho) and expected (He) heterozygosities (unbiased estimate, Nei, 1978), using GENETIX 4.04 [START_REF] Belkhir | GENETIX 4.02, logiciel sous Windows TM pour la génétique des populations. Laboratoire Génome, Populations, Interactions[END_REF]. We estimated the number of alleles independent of the sample size (hereafter called allelic richness) using an adaptation of the rarefaction index of [START_REF] Hurlbert | The nonconcept of species diversity: a critique and alternative parameters[END_REF], as implemented in FSTAT 2.9.3 [START_REF] Goudet | FSTAT (vers. 1.2): a computer program to calculate F-statistics[END_REF]. Because one locus exhibited a serious Hardy-Weinberg disequilibrium in six populations (see Results), measures of intra-population genetic diversity were averaged over the four remaining loci.

We tested for linkage disequilibrium between all pairs of loci in each population using exact tests as implemented in GENEPOP 3.3 [START_REF] Raymond | GENEPOP (Version 1.2): population genetics software for exact tests and ecumenism[END_REF]. Departure from Hardy-Weinberg equilibrium was tested using permutation procedures of GENETIX for each locus in each population and across all loci. Deviations from Hardy-Weinberg proportions were quantified by the unbiased estimator of Wright's inbreeding coefficient (F IS ), calculated according to [START_REF] Weir | Estimating F-statistics for the analysis of population structure[END_REF].

Differentiation between all pairs of populations was assessed using a log-likelihood Gbased exact test (Goudet et al., 1996). Tests computed for each locus were combined in a global test by use of Fisher's method [START_REF] Manly | The statistics of natural selection on animal populations[END_REF]. Pairwise levels of differentiation (F ST ) were calculated using [START_REF] Weir | Estimating F-statistics for the analysis of population structure[END_REF] estimators. These analyses were conducted using GENEPOP. In addition, a neighbor-joining tree [START_REF] Saitou | The neighbour-joining method: a new method for reconstructing phylogenetic trees[END_REF] was constructed from chord distances (Dc) among populations [START_REF] Cavalli-Sforza | Phylogenetic analysis: models and estimation procedures[END_REF].

Leprosy screening

Some animals from Yazoo were captured and transferred to the animal colony maintained by the National Hansen's Disease Program at Louisiana State University (LSU). For these animals, whole blood was collected by venipuncture and the serum was harvested after centrifugation in serum separation tubes (SST BD-Vacutainer, Franklin Lakes, New Jersey, USA) and stored frozen until use. For all other animals, whole blood collected on Nobuto strips was air dried and held at room temperature before the serum was extracted by immersion in Phosphate Buffered Saline pH 7.2 for 3 hours. Both regular sera and Nobuto eluted whole blood showed equal proficiency among test animals in an earlier pilot study.

Serum or eluted whole blood was tested in an ELISA for IgM antibodies to the species specific PGL-1 antigen of M. leprae using the procedure of Truman et al. (1986) Tissues (ear or spleen) from animals deemed to be positive for PGL-1 IgM antibodies in the ELISA were examined with PCR to confirm presence of M. leprae DNA following the methods of [START_REF] Williams | The use of a specific DNA probe and polymerase chain reaction for the detection of M. leprae[END_REF]. Automated DNA sequencing was performed by the LSU Genelab facility to confirm identity as M. leprae.

Population data analyses

Using data collected in 2005, we compared the number of armadillos observed per hour of observation at each site to determine if prevalence was related to population density (Truman et al., 1991). Pair-wise Chi-square tests were used to compare leprosy prevalence between populations. We used previously published prevalence data (Truman, 2005) from two populations west of the Mississippi River, Tensas National Wildlife Refuge (TS; 31º 57'/91º 23'), Louisiana (18/77 animals leprosy-positive), and Desha County (DC; 33º 44'/91º 16'), Arkansas (9/42 animals positive; Fig. 1) to compare prevalence between eastern and western populations.

Because leprosy prevalence in these western populations was similar, we combined data from them for comparison with the eastern populations.

RESULTS

Population density

There were no obvious demographic differences among the sampled populations, but significantly more armadillos were observed per hour at Yazoo than at other sites where such data were available (Table 1, ANOVA F 3, 57 = 69.47, P < 0.0001; post-hoc pair-wise comparisons were significant for Yazoo versus each other site but there were no significant differences in pair-wise comparisons among the other 3 sites).

Intra-population genetic diversity

The loci Dnov1, Dnov6, Dnov7, Dnov16, and Dnov24 revealed 10, 8, 4, 4, and 5 alleles respectively. Indices of intra-population genetic diversity are presented in Table 2. Overall, intra-population genetic diversity declined slightly from west to east, as did allelic richness. We found no evidence for genotypic disequilibrium, as only four of the 70 tests performed (population-loci pair combinations) were statistically significant at the 0.05 level, and none remained significant after sequential Bonferroni correction (Table 3). Thus, the five loci can be considered statistically independent. However, we did find strong evidence for Hardy-Weinberg disequilibrium at one locus (Dnov16). Indeed, 11 of the 35 exact tests performed were significant at the 0.05 level, and six tests remained significant after Bonferroni correction (Table 3). These six tests corresponded to a strong heterozygote deficiency (F IS from 0.743 to 1.000) at locus Dnov16. This may be due to the presence of null alleles in high frequencies. Hence, locus Dnov16 was not included in subsequent population genetic analyses.

Population differentiation

All population pairs were significantly differentiated, even after Bonferroni correction. Levels of differentiation measured by F ST values ranged from 0.030 between populations NO and ST to 0.232 between populations TT and DH. However, the tree constructed from genetic distances between populations did not show a marked population structure (Fig. 2).

Interestingly, populations from Welder in Texas and Tall Timbers in Florida were relatively close to each other, even though they were geographically the most widely separated. Apart Accepté pour publication dans Journal of Wildlife Diseases le 2 Juillet 2008 10 from this peculiar population pair, other populations clustered according to their geographical proximity (Fig. 2).

Leprosy prevalence

A total of 500 armadillos were sampled (Table 1). No road-killed animals tested positive for leprosy, nor were any collected at Pinebloom or St. Catherine's, but each of the remaining three sites presented at least one leprosy-positive individual (Table 1). In all cases, infection was confirmed both serologically and with PCR. The prevalence presented for Yazoo is somewhat problematic because of fungal contamination on some of the Nobuto strips collected there.

Consequently, a second estimate was obtained from the 71 animals (38 adult males and 33 adult females) relocated to LSU. Five (7.04%) of these animals were positive.

Except for the small group of roadkilled animals, all sampled populations exhibited significantly lower levels of leprosy prevalence in comparison with reference populations from west of the Mississippi River (Table 4). Leprosy prevalence at Riverside in 2006 was significantly higher than that observed in the Pinebloom and St. Catherine's samples, but otherwise there were no significant differences in prevalence among the sampled populations (Table 4).

DISCUSSION

The last extensive sampling for leprosy among armadillos east of the Mississippi River occurred in the late 1980s and found no evidence of infection based on histopathological examination of nerve damage in ear tissues [START_REF] Howerth | Survey for leprosy in nine-banded armadillos (Dasypus novemcinctus) from the southeastern United States[END_REF]. Although this method is less sensitive, the large sample size in that study argues against the possibility that infected animals were present and not detected, leading us to conclude that, historically, leprosy was not present in eastern populations. Our study indicates this is no longer true and provides a valuable update of current infection patterns.

Our data also provide some support for both hypotheses of leprosy occurrence in ninebanded armadillos. First, consistent with the ecological constraints hypothesis, no infected animals were found at St. Catherine's even though, like Yazoo, this population is located close to populations across the Mississippi River that exhibit substantial levels of infection (Fig. 1).

Similarly, Yazoo, with its appropriate soil conditions, harbored a population with several infected individuals, although prevalence was significantly lower than in nearby populations to the west. In contrast, consistent with the epidemic hypothesis, both populations in eastern Mississippi/western Alabama contained at least one infected animal. Most remarkable were the data from Riverside, where prevalence mirrored that found 240 km to the west at Yazoo. This is the first report of any leprosy-infected armadillos occurring at substantial distances east of the Mississippi River and indicates leprosy is spreading eastward. Thus, if ecological constraints do influence the occurrence of leprosy, our results suggest they are not as restrictive as previously supposed [START_REF] Truman | Environmental associations for Mycobacterium leprae[END_REF](Truman, , 2005)).

Of course, the two hypotheses are not mutually exclusive, so both likely play a role. For example, environmental conditions in eastern populations might limit leprosy prevalence to levels much lower than those seen west of the Mississippi River, but infection could nonetheless persist due to dispersal of infected animals into these areas. Such a scenario is supported by data indicating armadillo populations are relatively fluid and that some individuals move considerable distances in short periods of time [START_REF] Taulman | Recent range expansion and distributional limits of the nine-banded armadillo (Dasypus novemcinctus) in the United States[END_REF][START_REF] Bond | Home range dynamics and den use of nine-banded armadillos on Cumberland Island, Georgia[END_REF][START_REF] Gammons | Radiotelemetry studies of armadillos in southwestern Georgia[END_REF]McDonough et al., 2007). Alternatively, waves of infection may have spread rapidly through the entire range of D. novemcinctus in the U.S., with pockets of infection persisting in local areas containing appropriate environmental conditions (C. Brooks, pers. comm.). Additional sampling will be required to evaluate these possibilities. Further sampling will be necessary to address other questions raised by our findings. For example, populations in central Mississippi need to be examined to determine whether there is a continuous distribution of infection across the state or if Stimpson and DeWayne Hayes/Riverside represent isolated eastern pockets of infected animals. Similarly, the prevalence of leprosy at Riverside suggests populations in central and eastern Alabama should be screened to ascertain if the disease has spread even farther to the east.

Unfortunately, genetic analyses were of limited value in understanding the patterns of infection we report. The recent founding of U.S. populations seems to have resulted in high levels of uniformity at both protein [START_REF] Ramsey | Protein variation in populations of Dasypus novemcinctus and comparisons to D. hybridus, D. sabanicola and Chaetophractus villosus[END_REF][START_REF] Moncrief | Absence of genic variation in a natural population of nine-banded armadillos, Dasypus novemcinctus (Dasypodidae)[END_REF][START_REF] Huchon | Armadillos exhibit less genetic polymorphism in North America than in South America: nuclear and mitochondrial data confirm a founder effect in Dasypus novemcinctus (Xenarthra)[END_REF] and molecular genetic levels [START_REF] Loughry | Polyembryony in armadillos[END_REF][START_REF] Huchon | Armadillos exhibit less genetic polymorphism in North America than in South America: nuclear and mitochondrial data confirm a founder effect in Dasypus novemcinctus (Xenarthra)[END_REF] that prevent easy discrimination among them. Ideally, analyses of gene flow could provide important insights.

For example, the epidemic hypothesis would be strengthened if we could show that animals at Stimpson and Riverside possess genetic markers shared by infected populations to the west.

However, the limited number of loci we examined did not permit such fine scale assessment. Nevertheless, our genetic data did retrieve the historical pattern of colonization in confirming that founding populations from Florida were derived from individuals of western origin in Texas. Moreover, the pattern of intra-population genetic diversity was consistent with the colonization history of the sampled area. It is also noteworthy that populations from Mississippi (Yazoo and St. Catherine's) and Louisiana (New Orleans) had a genetic diversity comparable to that of the reference source population from Texas (Welder). Two alternative scenarios might explain these observations. The first is that there has been no erosion of genetic Accepté pour publication dans Journal of Wildlife Diseases le 2 Juillet 2008 13 diversity following colonization towards the northeast from the region around Welder. The second envisions an initial decrease in genetic diversity following this colonization, but which was later counterbalanced by an increase in diversity due to the meeting of populations coming from the east after their introduction in Florida. Both additional sample sites and more molecular markers (such as those now available due to the sequencing of the D. novemcinctus genome, [START_REF] Chang | Sequencing the armadillo genome[END_REF] are required to evaluate these alternatives and allow more precise determination of gene flow among U.S. armadillo populations. FIGURE 2. Neighbor-joining radial tree inferred from Cavalli-Sforza and Edwards ' (1967) genetic distances between populations, as calculated from microsatellite data. Branch lengths are drawn proportionally to genetic distances between populations, expressed in units of expected numbers of mutations accumulated per locus.

ACKNOWLEDGMENTS

+ DC ------ ------ ------ ------ ------ ------ ------ ------ ------ YZ-all 15.20*** ------ ------ ------ ------ ------ ------ ------ ------ YZ-LSU 6.70** 0.002 ------ ------ ------ ------ ------ ----

  The synthetic neoglycoconjugate antigen (ND-O-BSA) was used in all studies and supplied by Dr. Patrick Brennan (Colorado State University, Fort Collins, Colorado, USA) through contract with the National Institute of Allergy and Infectious Disease. Samples were tested in triplicate at a 1:35 dilution and all positive or equivocal reactions reassessed 3 times to confirm consistency.

Funding

  for field collecting was provided by a National Geographic Society grant to WJL and CMM. Laboratory analyses were partly supported by a National Institutes of Health award to RWT. Agustín Abba provided superb help in all phases of the field work and Ian Hester was invaluable in processing the animals collected at Riverside in 2006. Jason Ross and colleagues collected a great many of the animals at DeWayne Hayes and Riverside, and Mitch Lockhart generously shared access to specimens collected at Pinebloom. Patrice Boily kindly provided samples from the New Orleans population. Thanks to them, and to the personnel at each of the collecting localities, for their enthusiastic support of this project. We also thank Paulo Prodöhl for technical information about microsatellite loci and Mitch Lockhart for his comments on an earlier version of this paper. This is contribution ISEM 2008-088 of the Institut des Sciences de l'Evolution de Montpellier.

  < 0.05, ** P < 0.01, *** P < 0.001 b Full names for all populations are given in Fig. 1. DH-all includes data from DeWayne Hayes and Riverside collected in 2005 and 2006; YZ-LSU refers to 71 animals relocated from Yazoo to the Hansen's Disease research colony at Louisiana State University. c Not calculated because two cells contained 0s.

FIGURE CAPTIONS FIGURE 1 .

 CAPTIONS1 FIGURE CAPTIONS

Figure 1 Figure 2

 12 Figure 1

TABLE 1 .

 1 Prevalence of leprosy infection and other features of the nine-banded armadillo populations sampled.

	Population

a All infected animals tested positive in both serological and PCR analyses.

TABLE 2 .

 2 Genetic polymorphism in seven populations of nine-banded armadillos. a

	Population	n	Allelic	Observed	Gene
			richness	heterozygosity (Ho)	diversity (He)
	DeWayne Hayes	20	3.77	0.63	0.57
	New Orleans	20	3.95	0.79	0.69
	St. Catherine's Creek	19	4.29	0.55	0.62
	Stimpson	20	3.89	0.54	0.63
	Tall Timbers	20	3.48	0.62	0.56
	Welder	20	4.33	0.64	0.68
	Yazoo	20	3.81	0.73	0.67

a Measures were averaged over four loci (Dnov16 excluded).

TABLE 3 .

 3 Departures from Hardy-Weinberg proportions (F IS ) for seven populations of nine-banded armadillos at each of the five loci examined and their combination.

	Population	Dnov1	Dnov6	Dnov7	Dnov16	Dnov24	All loci
	DeWayne Hayes	-0.356 a	0.069	0.043	0.887 b,c	-0.068	-0.100
	New Orleans	-0.197	-0.308 a	0.000	0.900 b,c	-0.083	-0.156 a
	St. Catherine's Creek	0.378 a	-0.041	0.357	0.743 b,c	-0.112	0.110
	Stimpson	-0.206	0.293 a	0.253	1.000 b,c	0.179	0.143
	Tall Timbers	-0.073	-0.136	-0.167	0.273	-0.088	-0.109
	Welder	-0.143	-0.216	0.526	0.876 b,c	0.089	0.056
	Yazoo	-0.261	-0.039	-0.023	1.000 b,c	-0.032	-0.094

a P < 0.05 b P < 0.001 c P < 0.05 after Bonferroni correction

TABLE 4 .

 4 Chi-square values a for pair-wise comparisons of leprosy prevalence (proportion of infected individuals) among sampled populations of nine-banded armadillos.

	Population b	TS + DC YZ-all YZ-LSU DH-all	RS	PB	SC	ST	RK
	TS