Hepatic infiltration helps predict outcome in dogs with multicentric large B cell lymphoma

Revue vétérinaire clinique (2019) 54, 79—86

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ORIGINAL ARTICLE

Hepatic infiltration helps predict outcome in dogs with multicentric large B cell lymphoma夽,夽夽 Lymphome multicentrique B à grandes cellules chez le chien : impact pronostique d’une infiltration hépatique P. Denoeux a,∗, G. Chamel a, D. Sayag b, T. Denoeux c, F. Ponce a a

Univ Lyon, VetAgro Sup, Campus vétérinaire de Lyon, UR ICE, service de cancérologie, 69280 Marcy L’Étoile, France b Oncology unit, centre hospitalier vétérinaire Advetia, 9, avenue Louis-Bréguet, 78140 Vélizy-Villacoublay, France c UMR 7253 Heudiasyc, université de technologie de Compiègne, CNRS, Compiègne, France Received 25 June 2019; accepted 28 August 2019 Available online 15 November 2019

KEYWORDS Liver; Lymphoma; Large B cell; Prognosis; Proportional Hazards Models

Summary Background. — The five-level WHO staging system is currently the standard for canine lymphomas. In humans, staging for Non-Hodgkin lymphomas has recently evolved to the Lugano classification. In this four-level classification, the splenic infiltration does not induce stage migration from stage III, while hepatic involvement is considered as a more advanced stage together with bone marrow or non-lymphoid organs infiltration. Objective The objective of this pilot study is to evaluate the accuracy of the Lugano staging system, compared to the WHO, in predicting first remission duration (FRD) in dogs. Material and methods. — Thirty dogs with multicentric large B cell lymphoma, staged according to both systems, and treated with a standard chemotherapy protocol were retrospectively included. Kaplan-Meier curves were established for all stages and compared using a Log Rank test.

夽 Crédits de formation continue. - La lecture de cet article ouvre droit à 0,05 CFC. La déclaration de lecture, individuelle et volontaire, est à effectuer auprès du CNVFCC (cf. sommaire). 夽夽 Preliminary results were presented as an abstract at the ESVONC congress, Gran Canaria, Spain, May 2018. ∗ Corresponding author. E-mail address: [email protected] (P. Denoeux).

https://doi.org/10.1016/j.anicom.2019.08.001 2214-5672/© 2019 AFVAC. Published by Elsevier Masson SAS. All rights reserved.

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P. Denoeux et al. Results. — The median FRD was 157 days. Factors significantly associated with shorter FRD in the univariate analysis were substage b (P = 0.024), WHO stage V (P = 0.008) and Lugano stage IV (P = 0.002). Two multivariate Cox models were then compared: the model including substage together with both WHO and Lugano staging was statistically superior to predict FRD as compared to the model including only substage and WHO staging (P = 0.014). Conclusion. — Taking hepatic involvement into account allows better risk stratification for canine LBCL. © 2019 AFVAC. Published by Elsevier Masson SAS. All rights reserved.

MOTS CLÉS Foie ; Lymphome ; Grandes cellules B ; Pronostique ; Modèles à risque proportionnel

Résumé Contexte. — Le stade des lymphomes multicentriques canins est actuellement déterminé selon la classification OMS à 5 stades. Chez l’homme, une nouvelle classification à 4 stades, Lugano, a récemment été établie. Dans la classification Lugano, contrairement à celle de l’OMS, la présence d’une infiltration hépatique est considérée au même titre qu’une infiltration médullaire dans le stade le plus avancé (stade IV), alors que les chiens ayant une infiltration splénique restent en stade III. L’objectif de cette étude pilote, rétrospective, est de comparer, chez le chien, la pertinence pronostique de ces deux systèmes, en prenant comme critère d’évaluation la durée de première rémission. Matériel et méthodes. — Trente chiens diagnostiqués avec un lymphome multicentrique à grandes cellules B et traités selon un protocole standardisé ont été inclus. Le stade a été déterminé pour chaque chien, selon les systèmes OMS et Lugano. Les courbes de Kaplan-Meier ont été établies pour chaque stade et comparées avec un test log-rank, pour déterminer quel système est le mieux corrélé à la durée de première rémission. Résultats. — La durée médiane de la première rémission était de 157 jours. Les facteurs pronostiques négatifs identifiés sont un sous-stade b (p = 0,024), un stade OMS V (p = 0,008) et un stade Lugano IV (p = 0,002). Deux modèles multivariés ont ensuite été comparés: l’un incluant le sousstade et le stade OMS, et l’autre incluant également le stade Lugano. Le modèle intégrant les deux systèmes permet une meilleure évaluation du pronostic (p = 0,014). Conclusion. — La prise en compte de la présence d’une infiltration hépatique dans la détermination du stade clinique pourrait permettre une meilleure évaluation du pronostic. © 2019 AFVAC. Publi´ e par Elsevier Masson SAS. Tous droits r´ eserv´ es.

Abbreviations

NHL LBCL WHO CHOP

Non-Hodgkin lymphoma Large B-cell lymphoma World Health Organization Cyclophosphamide, Hydroxydaunorubicine, Oncovin, Prednisolone LCOP l-Asparaginase, Cyclophosphamide, Oncovin, Prednisolone CBC Complete Blood Count BM Bone Marrow PARR PCR for Antigen Receptor Rearrangement FDG Fluorodeoxyglucose PET Positron Emission Tomography CT Computed Tomography FRD First Remission Duration OS Overall Survival CR, PR, PD Complete Response, Partial Response, Progressive Disease

Introduction There is growing evidence that canine and human NonHodgkin lymphomas (NHL) share biologic, genetic and pathologic features, making of each species a model of interest for the other. Diffuse Large B-Cell Lymphomas (DLBCL) are the most frequent forms, representing approximately 48.3% of canine lymphomas and 31% of human Non-Hodgkin lymphomas [1—5]. Lymphadenectomy associated with immunohistochemistry, with a concordant clinical suspicion, is the gold standard for accurate diagnosis of lymphomas. In dogs however, this necessitates surgery with general anaesthesia, and is uncommonly performed in clinical situations. Many authors have recently recognized cytopathological analysis supported by immunocytochemistry and/or flow cytometry as an acceptable alternative for classification of canine highgrade lymphoma [6—11] as for human NHL [12—14]. In humans, the most adapted chemotherapy protocol depends on the genetic profile of the DLBCL. CHOP chemotherapy protocol, in addition to anti-CD20 antibody

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Table 1 World Health Organization staging system for dogs with lymphoma (1980).

Table 2 Revised staging system for Primary Nodal Lymphomas — Lugano (2014).

Stage

Definition

Stage

I II

Single lymph-node involvement Regional lymph nodes, on 1 side of the diaphragm Generalized lymph nodes, on both sides of the diaphragm Liver or spleen involvement or both; can also include stages I—III Blood, bone marrow, or other organ system involvement, or all (e.g., intestine, lung, neoplastic effusion); can also include stages I—IV

Limited I II

III IV V

and radiation therapy if needed, is one of the possible firstline treatments. The aim of the treatment is cure [5,15]. In dogs, CHOP protocol is often used as a sole treatment, and considered as palliative. The main objectives are achievement of a clinical remission, improvement of the quality of life and prolongation of survival. In both species, the use of standardized criteria for clinical staging carries a fundamental role, as it allows comparisons among studies, suggests prognosis, and helps in making accurate treatment decision. In dogs, clinical staging still follows the WHO five-level classification published in 1981 [16] (Table 1). The two substages ‘‘a’’ or ‘‘b’’ further subdivide patients according to the absence or presence of general clinical signs. Until recently, one of the major difficulties encountered in veterinary oncology was the lack of standardization of the staging tests used. In recent years, some efforts have been made toward more rigorous standardization, encouraging the systematic inclusion of a complete blood count (CBC) with blood smear, thoracic and abdominal imaging, obtaining splenic and hepatic cytology samples regardless of the ultrasonographic appearance, and bone marrow (BM) cytology [17—19]. The addition of newer and more sensitive staging tests like computed tomography, bronchoalveolar lavage, flow cytometry or PCR for Antigen Receptor Rearrangements (PARR) raises questions as it could identify previously undetectable infiltrations and lead to stage migration. However, the widespread use of those techniques is still limited by their cost and availability, but also by the lack of information regarding the impact on prognosis or therapeutic management of positive cases. In human, staging systems of lymphomas have been refined over the years to adapt to the introduction of newer and more sensitive staging modalities. The Ann Arbor staging classification, published in 1971, was the standard for assessment of lymphoma during decades. In this system, patients are subdivided into four stages according to the number of sites of involvement, their relation to the diaphragm and the presence of extra-nodal disease. Subclassification A and B is based on the presence of fever or weight loss. The Ann Arbor clinical stage is a known prognostic factor in human. However, its accuracy in identifying prognostic subgroups of patients is better for Hodgkin lymphomas than it is for Non-Hodgkin lymphomas.

Advanced III

IV

Definition One node or a group of adjacent nodes Two or more nodal groups on the same side of the diaphragm Nodes on both sides of the diaphragm, nodes above the diaphragm with spleen involvement Additional noncontiguous extralymphatic involvement

Extent of disease is determined by PET/CT for FDG-avid lymphomas and CT for non-avid histologies.

Therefore, for the latter, a more predictive classification was needed. From 2007 onwards, with the establishment of the Deauville criteria, interest has grown for the 18-Ffluorodeoxyglucose (FDG)-positron emission tomography (PET) [20,21]. The avidity for FDG is variable depending of the tumour histological subtype. Encompassing this use of functional imaging, the new Lugano classification finally emerged in 2014 [22] (Table 2). For the assessment of FDG-avid lymphomas, FDG-PET coupled with computed tomography (CT) is now the modality of choice, and BM biopsy is not required. For non-FDG-avid lymphomas, however, CT remains the standard imaging modality. Finally, the presence of general clinical signs at diagnosis (‘‘B symptoms’’) is not taken into account anymore for human NHL. Of particular interest in both Ann Arbor and Lugano classifications, splenic infiltration does not induce stage migration. By contrast, hepatic involvement is considered as a more advanced stage together with BM or non-lymphoid organs infiltration. The main objective of this retrospective pilot cohort study was to compare the accuracy of both Lugano and WHO staging system in predicting first remission duration (FRD) in dogs, and particularly to determine if hepatic infiltration should trigger a stage migration as in human. Initially, we hypothesized that liver infiltration was associated with a shorter FRD.

Material and methods Study population The database of the clinical oncology unit of our institution was retrospectively searched from January 2003 to December 2017 for dogs with multicentric large B cell lymphoma (LBCL). Inclusion criteria were (1) multicentric lymphoma, with a morphological diagnosis of LBCL established either via cytopathological analysis in combination with flow cytometry, or with histopathological analysis and immunohistochemistry [8—11,23] (2) a minimal staging including

82 complete blood count with blood smear, three-view thoracic radiographs, abdominal ultrasound with systematic fine-needle aspiration of the liver and spleen regardless of the sonographic appearance, and (3) follow-up data available until death or first relapse. All cytological slides were viewed by a single boardcertified clinical pathologist. All dogs were naïve for cytotoxic treatments at the time of diagnosis. Treatment with corticosteroids before diagnosis was not an exclusion criterion.

Collected data Clinical stage was determined for each case according to the five-level WHO staging system. Then, dogs were classified according to the four-level system used in the Lugano classification. Data regarding the breed, sex, age, clinical signs at diagnosis and calcaemia when available were collected. Dogs were classified into substage a or b depending of the presence of general clinical signs, and those clinical signs were recorded. All dogs received a standardized LCOP protocol as a first-line protocol, with a five-week induction phase (lAsparaginase 400 UI/kg IM week 1, vincristine 0,75 mg/m2 IV and cyclophosphamide 250 mg/m2 PO weeks 2 and 5 and vincristine 0,75 mg/m2 IV weeks 3 and 4) followed by a maintenance phase consisting in vincristine and oral cyclophosphamide administration every three weeks. Dose delays, reductions and supportive treatment for adverse events were carried out at the discretion of the attending clinician. Response to treatment was assessed at each visit according to the standardized evaluation criteria for peripheral nodal lymphoma [24]: complete response (CR) was defined as the disappearance of all evidence of disease, partial response (PR) represented a decrease of at least 30% in the mean sum of target lesions, and progressive disease (PD) an increase of at least 20% of this sum. Imaging reassessment was performed if deemed necessary by the attending clinician. Relapse was always confirmed by cytological analysis.

Statistical analysis Since all dogs did not have the same rescue treatment after relapse, FRD was considered more reliable than overall survival (OS) and was chosen as primary endpoint. FRD was defined as the length of time in days between the first observation of a complete clinical response and the clinical recurrence. OS was defined as the length of time in days between diagnosis and death or loss to follow-up. Dogs that failed to achieve CR at the end of the induction phase of the protocol were considered as having an FRD equal to zero. Kaplan-Meier product limit analysis was used for remission and survival analysis. A complete event was defined as lymphoma relapse (remission analysis) or death due to any cause (survival analysis). Kaplan-Meier log rank analysis was used to evaluate the influence of WHO stage, Lugano stage, substage and sex on FRD and OS. Confidence intervals for the survival function were determined using the log-transformed method.

P. Denoeux et al. Univariate logistic regression analysis was used to evaluate the influence of the above listed parameters on whether a dog reached CR. Cox regression was then used for multivariate analysis. Three statistical models were fitted: one including WHO stage, Lugano stage and substage (model 1), one including WHO stage and substage (model 2) and one including Lugano stage and substage (model 3). For each model, the global significance was assessed according to the three asymptotically equivalent methods commonly used: the log likelihood test, Wald test and Score test. Since the models 1 and 2 and models 1 and 3 are nested they were then easily compared using the likelihood ratio test. The use of a statistical test to compare non-nested models like models 2 and 3 is more challenging. However, the Akaike’s Information Criterion (AIC) can be used for model selection [25]. For each model, AIC was calculated. Then, differences were calculated as i = AICi − AICmin , where AICi is the AIC for the iith model and AICmin is the minimum of AIC among all the models. Models having i ≤ 2 have substantial support from the data, whereas those in which 4 ≤ i ≤ 7 have considerably less support. For all tests, a P-value < 0.05 was considered significant. Statistical analysis was performed with a software package (XLSTAT-Biomed 2018).

Results Clinical characteristics Search results revealed 34 dogs with a diagnosis of large B-cell lymphoma. Three dogs were excluded due to incomplete staging and one dog was excluded because of lack of adequate follow-up. Thirty dogs met the inclusion criteria. Sixteen breeds were represented. The most common were crossbreeds (6 dogs), Golden Retrievers (3), Bernese mountain dogs (3), German shepherds (3), Rottweilers (2) and French Bulldogs (2). The mean age at diagnosis was 8 years (5—13, SD = 2.52 years). There were ten intact males, five neutered males, ten intact females and five neutered females. Determination of ionized blood calcium level was carried out at diagnosis for 27 dogs (90%) and none of them had hypercalcaemia. Two dogs had bronchoalveolar lavage confirming lung infiltration. Twenty-three dogs (76%) had BM aspiration at diagnosis and cytological analysis concluded to medullary infiltration for five of them. Of the seven dogs without BM aspiration, one was considered in WHO stage V because of the presence of numerous blast cells on the blood smear. The others had neither circulating blasts cells nor cytopenias. For the 14 dogs in substage b, clinical signs at diagnosis were lethargy (n = 9, 60%), dysorexia (n = 6, 40%), weight loss (n = 6, 40%), respiratory signs like cough of breathlessness (n = 5, 33.3%), diarrhoea or vomiting (n = 3, 20%) and polyuropolydipsia (n = 1, 6.6%).

Treatment outcome The median follow up time was 8.7 months. Twenty dogs (66%) achieved CR. The median OS for all dogs was 263 days, which is similar to previously published data [6]. For dogs

Canine large B cell lymphoma: liver infiltration helps predict outcome

83

Table 3 Clinicopathological characteristics of 30 dogs with large B cell lymphoma. Characteristics Age (years) ≥8 <8 Sex Male Female Neutering status Entire Neutered Substage A B Anemia Hb < 12 g/L Hb ≥ 12 g/L

n (%)

FRD P-value

OS P-value

16 (53) 14 (46)

0.049

0.322

15 (50) 15 (50)

0.132

0.287

20 (66) 10 (34)

0.152

0.649

16 (53) 14 (46)

0.024

0.108

4 (13) 26 (87)

0.813

0.996

achieving CR, the median OS was 323 days and the median FRD was 157 days. For dogs that failed to achieve CR, the median OS was 159 days. By univariate analysis, substage b was the only clinical characteristic that significantly correlated with poor FRD. No clinical characteristic correlated with OS (Table 3).

Staging systems According to the WHO staging system, none of the dogs were in stage I or II, three dogs were in stage III, twenty in stage IV and seven in stage V. Considering the low number of dogs in stage III, for the purpose of statistical analysis, we chose to aggregate WHO stages III and IV together and compare them to stage V. This allowed us to evaluate the prognostic significance of medullary or other non-lymphoid organs infiltration. According to the Lugano classification, nine dogs were in stage III and twenty-one dogs in stage IV. No dogs were in stage I or II. Logistic regression failed to demonstrate any significant correlation between the probability to achieve CR and the WHO stage, Lugano stage or substage. FRD was significantly shorter for dogs with WHO stage V compared with WHO stages III and IV (P-value = 0.008) and for dogs with Lugano stage IV compared with Lugano stage III (P-value = 0.002) (Fig. 1). Thus, according to these results, both systems are able to stratify dogs into at least two different risk subgroups for FRD. Furthermore, bone marrow infiltration (WHO stage V) appears to be a negative prognostic factor. No classification had prognostic value regarding OS. Including clinical characteristics that were prognostic in univariate analysis, we fitted three multivariate Cox models. Model 1 including WHO stage, Lugano stage and substage, Model 2 including WHO stage and substage and Model 3 including Lugano stage and substage. The proportional hazards assumption was tested for all covariates and no violation was found. All three regressions were significant

Figure 1. Kaplan-Meier estimates of first remission duration according to substage a or b.

according to the log likelihood test, Wald test and Score test (Table 4). The likelihood ratio test was then used to compare the nested models (Models 1 and 2 and Models 1 and 3) and to find out which of them best correlates with outcome (Fig. 2). The P-value was 0.014 for Models 1 and 2. Therefore, the Cox-model including the Lugano stage together with the WHO stage and substage is statistically superior to the Cox-model including WHO stage and substage only. In other words, the use of Lugano classification in conjunction with WHO staging appears to further improve the capacity to discriminate risk subgroups. By contrast, the P-value was 0.15 for Models 1 and 3. Model 1 is not statistically superior to Model 3. This suggests that WHO stage does not confer additional information when Lugano stage is taken as explicatory variable. AIC were then calculated for each model (Table 4). Model 1 has the lower AIC and is therefore the preferred model. The AIC of model 3 is very close. When calculating the delta AIC, Model 3 appears to fit the data significantly better than Model 2. To further determine the prognostic value of hepatic infiltration independently of medullary infiltration, all dogs in WHO stage IV were then classified into stage IV1 (absence of hepatic infiltration) and IV2 (hepatic infiltration). KaplanMeier curves were compared pairwise with application of the Bonferroni correction. The results highlight that the FRD is significantly shorter for dogs in WHO stage IV2 with hepatic infiltration as compared to dogs in WHO stage IV1 (Fig. 3).

Discussion Reliable prognostic indicators are important for assisting owner and clinician in their treatment decisions. The

84 Table 4

P. Denoeux et al. Cox regression data and Aikake’s criterion for Models 1, 2 and 3.

Covariates WHO stage, HR [95%CI] Lugano Stage, HR [95%CI] Substage, HR [95%CI] Tests Log (likelihood), Khi2 (Pr > Khi2 ) Wald test, Khi2 (Pr > Khi2 ) Score test, Khi2 (Pr > Khi2 ) Aikake’s criterion AIC

Model 1

Model 2

Model 3

0.485 [0.159—1.478] 3.817 [1.241—1.742]

0.373 [0.122—1.141] —

— 4.107 [1.367—12.341]

1.646 [0.689—3.931]

2.029 [0.870—4.736]

1.808 [0.774—4.224]

11.17 (0.004)

7.14 (0.028)

11.6 (0.003)

12.78 (0.005)

8.23 (0.016)

9.38 (0.005)

11.06 (0.011)

7.45 (0.024)

10.73 (0.009)

121.4706

125.5592 1-2 = 4.08857875

121.5152 1-3 = 0.04463735

HR: hazard ratio.

Figure 2. system.

Kaplan-Meier estimates of first remission duration and overall survival according to Lugano classification and WHO staging

prognostic value of the WHO clinical stage for canine lymphoma is still a controversial issue. Some publications, focusing on canine lymphoma at large and not only on LBCL, reported a poorer outcome for stage V as compared with stages III-IV [26—28], while other reported no correlation between WHO stage and prognosis [17,18,29,30]. Our results support the hypothesis that dogs in WHO stage V have a shorter FRD. However, the lack of BM aspiration for 24% included here is one limitation, due to the retrospective design. In those cases, we based the staging assessment on

the identification of blast cells on blood smears. One study identified the presence of at least 10% neoplastic lymphocytes on blood smears as a significant variable for predicting the presence of BM involvement, with a sensitivity of 60% [31]. Therefore, cases without blast cell identified on the blood smear may actually be in stage V, and misclassified. To our knowledge, our pilot study is the first to suggest that hepatic infiltration in dogs with multicentric LBCL may be of prognostic value, as supported by the comparison of multivariate Cox models. While both Lugano and

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85

staging systems for limited stages. However, in the authors experience they are only exceptionally encountered at the time of diagnosis. Furthermore, we have been compelled to aggregate WHO stages III and IV for the purpose of statistical analysis. This prevented us to determine if dogs in WHO stage III have a better outcome than those in WHO stage IV in our cohort, and particularly to evaluate the prognostic significance of a splenic infiltration. However, this was not part of the main objectives of this study. Furthermore, it could have been interesting to distinguish the different lymphoma variants that are here grouped together under the term ‘‘LBCL’’. In a review of the recent literature, Sayag et al. (2017) [37] conclude that some reasonable evidence exists regarding a significant prognostic value of the different morphological variants. Lastly, biochemical parameters related to hepatic function were not assessed for all cases. Therefore, it is unknown if the worsening of the prognosis for dogs with hepatic involvement was related to an alteration of hepatic function consecutive to lymphoma infiltration.

Figure 3. Kaplan-Meier estimates of first remission duration according to a modified six-level WHO staging system.

WHO staging proved efficient, our data showed that Lugano classification in conjunction with WHO staging and substage could further improve their capacity to discriminate risk subgroups. When referring to the Akaike information criteria, Lugano classification seems to better correlate with FRD. Moreover, when classifying dogs according to a modified WHO staging system with subdivision of stage IV based on the absence (IV1 ) or presence of liver infiltration (IV2 ), the Kaplan-Meier product method highlights that the FRD is significantly shorter for dogs with hepatic infiltration (IV2 ) in comparison with dogs without infiltration (IV1 ). Regarding substages, our results are supportive of substage b being a negative prognostic factor, in accordance to the previous study of Jagielski et al. (2002) [26] and contrary to older publications [32—35]. In humans, suffixes ‘‘A’’ or ‘‘B’’ relative to the presence or absence of disease-related symptoms are now abandoned for NHL, since they were often neither recorded nor accurate, and were not associated with unfavourable outcome in the International Prognostic Index. In veterinary oncology, formal criteria to define these parameters are also lacking. A survey among veterinary oncologists concluded that mild to moderate gastro-intestinal, constitutional or respiratory signs was deemed sufficient for substage b designation [36]. The nature of the clinical signs recorded in our study fits with those categories. However, their severity was not recorded. The decision to consider a clinical sign as severe enough to figure in the animal record was at the attending clinician’s discretion, which could prevent inter-observer repeatability. Our results suggest that the establishment of a consensus on substage determination may be a worthwhile objective. Despite these promising results, our study suffers from several limitations. The first of them is the relatively low number of cases. Moreover, LBCL often are diagnosed at advanced stages. This results in an absence of WHO and Lugano stages I—II and a relative lack of WHO stage III. As a consequence, we could not appreciate the accuracy of both

Conclusion Further large-scale prospective studies are needed to reinforce our results. However, it is the authors’ opinion that the evaluation of liver infiltration through cytopathological analysis and a subsequent stage migration in case of positive results could be a cost-effective mean to improve prognostication in dogs with multicentric LBCL.

Disclosure of interest The authors declare that they have no competing interest.

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