Background: Splenic marginal zone lymphomas (MZL) in dogs arise from the marginal zone of B-cell follicles and can progress slowly.Objectives: To describe clinical features, treatment, and outcome of dogs with splenic MZL.Animals: Five dogs with naturally occurring MZL.Methods: Clinical, laboratory, and follow-up data were retrospectively reviewed. Diagnosis was based on clinical, histopathological, and immunophenotypic features.Results: All dogs had stage IV disease; among them, 2 were symptomatic (substage "b") because of splenic rupture. Four dogs underwent splenectomy and adjuvant doxorubicin, and 1 dog underwent surgery only. Three out of the 4 dogs treated with surgery and chemotherapy died of causes unrelated to lymphoma, after 760, 939, and 1,825 days, whereas the remaining dog was alive and in complete remission after 445 days. The dog not receiving any adjuvant treatment had recurrence of the tumor after 180 days.Conclusions and Clinical Importance: Splenic MZL appears indolent and can benefit from splenectomy, with or without systemic chemotherapy

D. Stefanello

E. Zini

G. Avallone

L. Marconato

M. Caniatti

M.E. Gelain

P. Roccabianca

P. Valenti

S. Comazzi

Background: Splenic marginal zone lymphomas (MZL) in dogs arise from the marginal zone of B-cell follicles and can progress slowly.Objectives: To describe clinical features, treatment, and outcome of dogs with splenic MZL.Animals: Five dogs with naturally occurring MZL.Methods: Clinical, laboratory, and follow-up data were retrospectively reviewed. Diagnosis was based on clinical, histopathological, and immunophenotypic features.Results: All dogs had stage IV disease; among them, 2 were symptomatic (substage "b") because of splenic rupture. Four dogs underwent splenectomy and adjuvant doxorubicin, and 1 dog underwent surgery only. Three out of the 4 dogs treated with surgery and chemotherapy died of causes unrelated to lymphoma, after 760, 939, and 1,825 days, whereas the remaining dog was alive and in complete remission after 445 days. The dog not receiving any adjuvant treatment had recurrence of the tumor after 180 days.Conclusions and Clinical Importance: Splenic MZL appears indolent and can benefit from splenectomy, with or without systemic chemotherapy. Copyrigh

M. E. Gelain

AVALLONE, GIANCARLO

Archivio istituzionale della ricerca - Alma Mater Studiorum Università di Bologna

English

Splenic marginal zone lymphoma in 5 dogs (2001-2008)

Splenic MZL appears indolent and can benefit from splenectomy, with or without systemic chemotherapy

Stefanello, Damiano

Valenti, P

Zini, Eric

Comazzi, Stefano

Gelain, M E

Roccabianca, Paola

Avallone, Giancarlo

Caniatti, M

Marconato, L

ZORA

AIR Universita degli studi di Milano

Splenic Marginal Zone Lymphoma in 5 Dogs (2001 –2008)D. Stefanello, P. Valenti, E. Zini, S. Comazzi, M.E. Gelain, P. Roccabianca, G. Avallone,M. Caniatti, and L. MarconatoBackground: Splenic marginal zone lymphomas (MZL) in dogs arise from the marginal zone of B-cell follicles and canprogress slowly.Objectives: To describe clinical features, treatment, and outcome of dogs with splenic MZL.Animals: Five dogs with naturally occurring MZL.Methods: Clinical, laboratory, and follow-up data were retrospectively reviewed. Diagnosis was based on clinical, histo-pathological, and immunophenotypic features.Results: All dogs had stage IV disease; among them, 2 were symptomatic (substage ‘‘b’’) because of splenic rupture. Fourdogs underwent splenectomy and adjuvant doxorubicin, and 1 dog underwent surgery only. Three out of the 4 dogs treated withsurgery and chemotherapy died of causes unrelated to lymphoma, after 760, 939, and 1,825 days, whereas the remaining dogwas alive and in complete remission after 445 days. The dog not receiving any adjuvant treatment had recurrence of the tumorafter 180 days.Conclusions and Clinical Importance: Splenic MZL appears indolent and can beneﬁt from splenectomy, with or withoutsystemic chemotherapy.Key words: Canine; Indolent lymphoma; Spleen.The marginal zone is an anatomically distinct B-cellcompartment surrounding the lymphocytic coronaof the mantle zone in lymph node(s) (LNs), spleen, andmucosa-associated lymphoid tissues (MALT).1 Lympho-mas arising from this particular location withinlymphoid follicles are named marginal zone lymphoma(MZL).The World Health Organization (WHO) histologicalclassiﬁcation (2008) further divided human MZL into 3subtypes, including that of MALT, spleen, and LNs2; theclinical course is different for each of them. The WHOclassiﬁcation of hematopoietic and lymphoid tumorsof domestic animals has also divided them into sub-types.1,3,4 Because of the limited number of reportedsplenic MZL cases, a precise recommendation for ther-apy is not yet available. The goal of this report was todescribe clinical features, treatment, and outcome ofdogs with splenic MZL.Materials and MethodsCriteria for Selection of CasesMedical records were reviewed to identify dogs with a histologicaldiagnosis of MZL presented to the authors’ institutions between 2001and 2008. To be enrolled in the study, a complete staging work-up hadto be available, including signalment, history, presence or absence ofclinical signs, hematological proﬁle, calcemia, thoracic radiographs,abdominal ultrasound, cytology and immunophenotype of enlargedLN(s), spleen or liver, cytological and immunophenotypical bonemarrow (BM) evaluation, with or without histopathological examin-ation of the involved tissue(s). Clonality assessment was performed onspleen samples to conﬁrm the neoplastic origin of the disease.Data pertaining to number and type of therapies given, response,and outcome were retrieved.Morphologic Features and ImmunophenotypeHistological slides, including sections used for immunopheno-typing, were reviewed to conﬁrm the diagnosis. The diagnosis wasmade according to WHO histological classiﬁcation.3–5 Immuno-phenotyping had to demonstrate the B-cell nature of neoplasticlymphocytes.Polymerase chain reaction (PCR) for antigen receptor rearrange-ment (PARR) was performed on formalin ﬁxed-parafﬁn embeddedsamples. DNA was isolated with a commercially available kit in ac-cordance with the manufacturer’s protocol.a Nucleic acid wasspectrophotometrically measured with anND-100 SpectrophotometerbFrom the Department of Veterinary Clinical Sciences, School ofVeterinary Medicine, University of Milan, Milan, Italy (Stefanello,Valenti); the Clinic for Small Animal Internal Medicine, VetsuisseFaculty, University of Zu¨rich, Zu¨rich, Switzerland (Zini); the De-partment of Animal Pathology, Hygiene and Public Health, School ofVeterinary Medicine, University of Milan, Milan, Italy (Comazzi,Gelain, Roccabianca, Avallone, Caniatti); and the Clinica Veterin-aria L’Arca, Naples, Italy (Marconato). Dr Marconato and DrValenti are presently afﬁliated with the Animal Oncology and Imag-ing Center, Rothusstrasse 2,6331, Hu¨nenberg, Switzerland. Findingsof this study were presented in part at the Annual ESVONCCongress,Turin, Italy, 2010.Contributed equally to the work.Corresponding author: L. Marconato, DVM, DECVIM-CA (On-cology), Animal Oncology and Imaging Center, Rothusstrasse 2, CH-6331, Hu¨nenberg, Switzerland; e-mail: marconato@aoicenter.ch.Submitted September 21, 2009; Revised August 26, 2010;Accepted September 28, 2010.Copyright r 2010 by the American College of Veterinary InternalMedicine10.1111/j.1939-1676.2010.0639.xAbbreviations:BM bone marrowCR complete remissionLN(s) lymph node(s)MALT mucosa-associated lymphoid tissuesMMC medium macronucleated cellMZL marginal zone lymphomaPD progressive diseasePR partial remissionWHO World Health OrganizationJ Vet Intern Med 2011;25:90–93and PCR for GAPDH sequences was performed to evaluate thepresence of ampliﬁable DNA. Ampliﬁcation of immunoglobulin se-quence was performed as already described.6Treatment and Response AssessmentTreatment was provided by the attending clinician and was notstandardized. Dogs were treated according to disease stage andlocation with therapeutic options being available at the time ofdiagnosis.Outcome was recorded for each included case. Complete remis-sion (CR) was deﬁned as no clinical evidence of lymphoma. Partialremission (PR) was deﬁned as 50% or greater decrease in one di-mension of measurable tumor. Stable disease was deﬁned as lessthan a 50% decrease in 1 dimension but no more than a 10% in-crease in any dimension of all measurable tumors. Progressivedisease (PD) was deﬁned as a 10% or more increase in any dimen-sion of measurable tumor. Relapse was deﬁned as clinicalreappearance and cytological evidence of lymphoma in any ana-tomical site in dogs that have had CR, whereas relapse for animalswith PR was deﬁned as progression. Responses lasting o21 dayswere considered as PD.To evaluate response, reevaluation was conducted at the end ofthe adopted therapy, and monthly thereafter until relapse. All en-rolled dogs were monitored by the same clinician.ResultsDogsNine client-owned dogs with splenic MZL were iden-tiﬁed as potential cases for inclusion in the study.However, among them, only 5 had an available histo-pathological diagnosis of MZL and were enrolled. Theother 4 had a cytological diagnosis of medium macronu-cleated cell (MMC) and were therefore not included inthe study.Of the 5 dogs enrolled, 2 were pure-bred, 3 were mixed-bred. There were 4 males (3 intact, 1 castrated) and 1spayed female. Median age was 8 years (range, 7–10years) and median weight was 10 kg (range, 8.0–29.0 kg).All dogs had stage IV disease. Two dogs had signs relatedto hemoabdomen (‘‘b’’ substage).All dogs were treatment-naı¨ve at time of diagnosis.Anemia (hematocrit o35%) was observed in 2 dogsand none was hypercalcemic (Ca21 4 12mg/dL). Twodogs had hemoabdomen because of splenic rupture. Anabdominal mass was palpated during routine examina-tion in the remaining 3 dogs. In all dogs a focal, welldemarcated, hypoechoic mass was delineated in thespleen on ultrasonographic examination. In all dogs,both cytology and histology were available. Impressionsmears of the spleen were obtained after splenectomy,and cytology was consistent with a MMC diagnosis in allof them. Histological samples were diagnosed as MZLlymphomas, being characterized by focally extensivenodular splenic lesions often surrounded by hemorrhageor areas of severe extramedullary hematopoiesis (Figs1–4). Splenic neoplastic nodules examined in early caseswere composed of the characteristic lighter stained broadand continuous cuff of cells around small foci of darklystained residual small cells of mantle cell cuff with col-lapsed germinal centers. In more advanced cases, themultinodular pattern persisted but mantle cells were lostand nodules tended to coalesce. Neoplastic cells weremonomorphic, with variably evident cell boundaries, anabundant lightly eosinophilic cytoplasm, and round, 10–14 mm nuclei with 1 distinct nucleolus. Mitoses were rareto absent in all cases. Cells expressed consistently CD79-aplha and CD20 and were CD3-epsilon negative.B-cell clonality was detected in 3 splenic cases. In 2cases, formalin-ﬁxed parafﬁn embedded tissue yieldedvery low DNA amounts, thereby precluding the possibil-ity to assess clonality on them.Treatment and OutcomeAmong the 5 dogs, 4 underwent splenectomy and re-ceived chemotherapy (doxorubicin as single agent)thereafter, and 1 was splenectomized with no furthertreatment. Three out of the 4 dogs treated with surgeryand adjuvant chemotherapy died of causes unrelated tolymphoma, after 760, 939, and 1,825 days, whereas theFig 1. Histology of the spleen. Effacement of normal architectureby coalescing nodules composed of a prevalence of marginal cellsand characterized by loss of mantle cells. Hematoxylin and eosin,bar5 170mm.Fig 2. Histology of the spleen. Coalescing areas of neoplastic cellswith abundant eosinophilic cytoplasm and complete loss of normalsplenic architecture, bar5 160mm.91Marginal Lymphomas in Dogsremaining dog was alive and in CR at the end of thestudy, after 445 days.The dog not receiving any adjuvant treatment experi-enced tumor relapse after 180 days and was euthanized.Relapse occurred in the peripheral LN(s) and was con-ﬁrmed by means of cytology.DiscussionMZL is a well-known clinicopathological entity in hu-man medicine and clinical outcome mostly depends onanatomical site of involvement.7 In veterinary medicine,the recognition of MZL as a distinct disease has been re-cent,1,3 therefore data on natural history and treatmentresponse have been limited. Our goal was to describeclinical characteristics, treatment, and outcome of dogswith splenic MZL, to understand whether they behavelike the indolent ones, as previously stated,3 or should beconsidered as clinically aggressive. By deﬁnition, indo-lent lymphomas grow slowly or remain stable for longperiods of time, and are histologically of low to interme-diate grade. Indolent lymphomas are traditionallymoderately to fairly chemo- and radio-resistant; there-fore a conservative treatment is usually suggested,meaning metronomic strategy instead of a dose-intenseapproach (aiming at administrating the chemotherapeu-tic drugs as high as possible), to keep quality of life andavoid unnecessary toxicity. If lymphoma is asymptomat-ic, deferring treatment may represent an option.MZL is the lymphoma type most frequently arising inthe spleen in both humans and dogs. Splenic MZL ischaracterized by uniform involvement of follicular areas,leading to generalized splenomegaly in human pa-tients,8,9 which usually becomes symptomatic if massiveor if associated with peripheral cytopenias. BM andblood are frequently involved (in up to 90% of cases)without worsening prognosis.10 Because of the relativelybenign clinical course, treatment is required in selectedcases only, such as those exhibiting symptomatic spleno-megaly and/or cytopenia, with splenectomy being thetreatment of choice.10,11 Follow-up chemotherapy(mainly consisting of alkylating agents or purine ana-logues) can play a role in the presence of poor prognosticfactors, positive staging work-up, ‘‘b’’ substage and/orcytologic presence of large cells (420%).12In our series of dogs, the hallmark of splenic MZL wasa focal hypoechoic mass, as previously described,3 whichonly became symptomatic in case of rupture, leading tosecondary hemoabdomen. Early in the disease, splenicMZL was identiﬁed accidentally during palpation. Thisis in contrast with high-grade lymphomas, in which gen-eralized splenomegaly, rather than an isolated mass, isusually observed.13 Unlike human patients, in which BMinvolvement is very common, in this series none of thedogs had BM inﬁltration at presentation. Based on ourresults, splenic MZL may have an indolent biologic be-havior, and this is in agreement with a previous study.3Diagnosis is usually made when dogs become symptom-atic, as in case of splenic rupture, or by abdominalultrasound performed for other reasons. Here, treatmentof splenic MZL varied: 4 dogs underwent splenectomyand adjuvant chemotherapy, whereas 1 dog underwentsurgery only. The 3 longest survivals were recorded indogs treated by means of surgery and adjuvant chemo-therapy, possibly suggesting a beneﬁt for the combinedtherapy. Lymphoma cases are usually treated pharmaco-logically in dogs, and the role of splenectomy has beenlimited to palliate clinical signs related to hypersplenism,remove spleens at risk for rupture or eliminate the focusof residual disease in dogs having successfully respondedto chemotherapy in other sites.14 Based on our results, wehypothesize that surgery may also play an important rolein the management of splenic MZL, possibly improvingoutcome. Whether adjuvant chemotherapy is necessary inan adjuvant (postsurgical) setting has not been evaluatedbefore. Our ﬁndings suggest that systemic monochemo-therapy can be useful in preventing relapse, possiblyeradicatingminimal residual disease. However, 3 dogs withsplenic MZL were alive between 7 and 19 months aftersplenectomy only.3 This ﬁnding has important clinicalFig 3. Histology of the spleen. High magniﬁcation of a nodule ofFigure 1 characterized by compression of residual splenic paren-chyma by marginal zone expansion, bar5 45 mm.Fig 4. Histology of the spleen. Substitution of a normal follicle bya monomorphic population of cells with central round nucleus char-acterized by chromatin peripheralization and evidence of a centralprominent nucleolus, bar5 25mm.92 Stefanello et alimplication, as conservative (surgical) treatment could leadto prolonged survival, thereby outlining the importance ofobtaining a deﬁnitive histological diagnosis before discuss-ing prognosis with the owners. Indeed, the generaldiagnosis of ‘‘lymphoma’’ is largely inaccurate, as differ-ent types of lymphoma have different biological behaviorand harbor different prognosis, not necessarily being poor,as demonstrated by this series of dogs.The detection of clonality by means of molecularmethods may further improve the possibility to achievea deﬁnitive diagnosis. In our study, 3 out of 5 splenicsamples showed IgH clonal rearrangement, while on 2cases no PCR products were detected. The lack of visibleimmunoglobulin rearrangement, however, could be in-terpreted as a false negative result. Indeed, as alreadyreported,15 there are several causes of PARR false nega-tive results, such as exhibition of V- or J-region genesegments not being ampliﬁed by the primers used and in-adequate tissue quality for PCR analysis. This couldexplain the negative results obtained in 2 samples, inwhich the lymphoid neoplasm was histopathologicallyconﬁrmed. The nucleic acid extracts from formalin-ﬁxedtissues are not always suitable for ampliﬁcation, beingattributable to scarce or degraded DNA, or presence ofremnants of formalin or xylene, which contain inhibitorsof extraction procedures or ampliﬁcation reaction. Thus,as already reported,3 the accurate interpretation of clon-ality results requires histological tissue examination aswell as immunophenotyping.This study has several limitations, including low num-ber of cases and retrospective nature, which precluded tohave a homogeneous population in terms of treatment.Furthermore, since histopathological evaluation was aninclusion criteria, cases for which only cytology wasavailable were excluded.In people, MZL arising from different anatomic siteshave different biologic behavior, with nodal lymphomashaving a more aggressive clinical course than splenicones, thereby requiring a more aggressive therapeuticstrategy.16 Future works should be aimed at evaluating ifthe same holds true for dogs.In conclusion, splenic MZL seems to be an indolentdisease and may beneﬁt from splenectomy, possibly fol-lowed by systemic chemotherapy. Future clinical trialsshould focus on the prognostic role of adjuvant chemo-therapy. Best treatment options for this poorly describeddisease remain to be deﬁned in dogs.FootnotesaRecoverAll Total Nucleic Acid Isolation Kit, Ambion, Austin, TXbNanoDrop Technologies Inc, Wilmington, DEReferences1. Valli VE. Marginal zone and MALT lymphoma. In: ValliVE, ed. Veterinary Comparative Hematopathology. Ames, IA:Blackwell Publishing; 2007:168–189.2. Fournel-Fleury C, Magnol JP, Bricaire P, et al. Cytohisto-logical and immunological classiﬁcation of canine malignantlymphomas: Comparison with human non-Hodgkin’s lymphomas.J Comp Pathol 1997;117:35–59.3. Swerdlow SH, Campo E, Harris NL, et al. WHO Classiﬁca-tion of Tumours of Haematopoietic and Lymphoid Tissues. Lyon,France: IARC Press; 2008.4. Valli VE, Vernau W, de Lorimier LP, et al. Canine indolentnodular lymphoma. Vet Pathol 2006;43:241–256.5. Valli VE, Jacobs RM, Parodi AL, et al. WHO HistologicalClassiﬁcation of Hematopoietic Tumors of Domestic Animals, VolVIII, 2nd ed. Washington, DC: Armed Forces Institute of Pathol-ogy; 2002.6. Burnett RC, Vernau W, Modiano JF, et al. Diagnosis of ca-nine lymphoid neoplasia using clonal rearrangements of antigenreceptor genes. Vet Pathol 2003;40:32–41.7. Berger F, Felman P, Thieblemont C, et al. Non-MALT mar-ginal zone B-cell lymphomas: A description of clinical presentationand outcome in 124 patients. Blood 2000;15:1950–1956.8. Troussard X, Valensi F, Duchayne E, et al. Splenic lym-phoma with villous lymphocytes: Clinical presentation, biology andprognostic factors in a series of 100 patients. Groupe francaisd’hematologie cellulaire (GFHC). Br J Haematol 1996;93:731–736.9. Thieblemont C, Felman P, Callet-Bauchu E, et al. Splenicmarginal-zone lymphoma: A distinct clinical and pathological en-tity. Lancet Oncol 2003;4:95–103.10. Thieblemont C, Felman P, Berger F, et al. Treatment ofsplenic marginal zone B-cell lymphoma: An analysis of 81 patients.Clin Lymphoma 2002;3:41–47.11. Mulligan SP, Matutes E, Dearden C, Catovsky D. Spleniclymphoma with villous lymphocytes. Natural history and responseto therapy in 50 cases. Br J Haematol 1991;78:206–209.12. Lefre`re F, Hermine O, Belanger C, et al. Fludarabine: Aneffective treatment in patients with splenic lymphoma with villouslymphocytes. Leukemia 2000;14:573–575.13. Wrigley RH, Konde LJ, Park RD, Lebel JL. Ultrasono-graphic features of splenic lymphosarcoma in dogs: 12 cases (1980–1986). J Am Vet Med Assoc 1988;193:1565–1568.14. Vail DM, Young KM. Canine lymphoma and lymphoid leu-kemia. In: Withrow SJ, MacEwen EG, eds. Small Animal ClinicalOncology, 4th ed. Philadelphia, PA: WB Saunders Co; 2007:699–733.15. Avery PR, Avery AC. Molecular methods to distinguish re-active and neoplastic lymphocyte expansions and their importancein transitional neoplastic states. Vet Clin Pathol 2004;33:196–207.16. Zucca E, Bertoni F, Stathis A, Cavalli F. Marginal zonelymphomas. Hematol Oncol Clin North Am 2008;22:883–901.93Marginal Lymphomas in Dogs

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Splenic marginal zone lymphoma in 5 dogs (2001-2008)

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Archivio istituzionale della ricerca - Alma Mater Studiorum Università di Bologna

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