Canine Lymphoma Update: What’s New? Craig Clifford DVM, MS, DACVIM (Onco) Lymphoma: Etiology • Viral etiology proposed • No conclusive evidence in the dog • • • Breed Predilection Family Predilection Weak link to: • 2,4-D herbicide exposure • high tension lines • Inc incidence in industrial areas • Immune mediated disease Epizootiology • • Approximately 20% of canine neoplasms Incidence: • 23/1000 population • • No sex predilection Age: • 7-10 years • Breeds • Basset Hound, St, Bernard, Scottish terrier • golden retriever, rottweiler, BMD, Boxer Anatomic sites • Generalized nodal (84%) • Alimentary (6.9%) • Cutaneous (6.3%) • Mediastinal (2.2%) • Extranodal (2%) • CNS • Bone • Heart Clinical signs: Multicentric lymphoma • Generalized lymphadenopathy • Most common (85%) • Inappetence, lethargy & wt loss • ~15% • PU/PD via hypercalcemia • ~15% of cases • 40% in mediastinal LSA Alimentary lymphoma • Wt loss and lethargy • Vomiting/diarrhea • Blood may be present in vomitus or stool Cutaneous LSA: Clinical Signs • AKA: • Mycosis Fungoides (MF) • Epitheliotropic LSA (ELSA) • Wide variation in appearance • May be single or multiple • Flat plaques to nodular dz • • • +/- systemic involvement ~½ are pruritic Generally T cell phenotype Mycosis fungoides Pautrier’s microabscesses •Mycosis fungoides also affects the oral mucosa. •There are lymphocytes in surface epithelium. •The ultrastructure shows extremely bizarre nuclear conformations. ELSA - lomustine Clinical Signs: Mediastinal LSA • Respiratory signs • Mass • Effusion • Precaval syndrome • PU/PD • 40% due to Ca++ Hypercalcemia of malignancy • • • • Rehydrate (NaCL) Diurese with furosemide Glucocorticoid Bisphosphonate • • • • Etidronate Pamidronate Alendronate Zoledronate Clinical Signs: Extranodal • Site dependent • Anything is possible Diagnosis • Physical exam • Hematology/Chemistry • Histopathology/cytology • Imaging • Rads • Abd U/S • Bone marrow aspirate Lymphoma: Lymph Node Aspiration Most Frequent reason for mis-diagnosis is crush artifact Non-pathologist Diagnosis of lymphoma Cells are larger than a neutrophil Absence of plasma cells Variability in nucleolar size/number Slide courtesy of Chand Khanna Immunohisto/cytochemistry • Immunophenotyping • B-cell/T-cell • Proliferation indices: • PCNA • Ki67 • AgNOR • MDR • Submission to IHC services • $25.00/slide Novel Diagnosis/Management Flow Cytometry PCR • Use of Clonality of cancer for diagnosis of LSA • Blood/lymph node aspirates • Can distinguish reactive lymphocytosis vs. leukemia • NC St Laboratory ($70.00) • Turn around ~ 3-5 days FCM • Performed from: • Blood • Bone marrow • Lymph node • Panel of antibodies • Phenotype, MDR, etc • Outside lab • Overnight shipping • NC St Laboratory ($70.00) • Turn around ~3-5 days DISEASE CD MOLECULES ROUTINELY ASSESSED AND CONSIDERED MOST USEFUL FOR DIAGNOSIS AND PROGNOSIS LYMPHOMA* CD3, CD3, CD4, CD5, CD8, CD8, CD11d, CD21, CD34, CD45, CD79a, TCR, TCR LEUKEMIA* All of above plus CD1a, CD1c, CD11b, CD11c, CD14, CD56. CD34 is very useful for differentiating primary leukemia (+ve) from marked leukemic phase of lymphoma (-ve) and occasionally for separating acute leukemia (+ve) versus chronic leukemia (-ve). As expected, a very high proportion of primary acute Leukemias in the dog (myeloid and lymphoid) express CD34. HISTIOCYTIC CD1a, CD1c, CD4, CD11b, CD11c, CD11d, CD18 (paraffin especially), CD90 * Adjunctive tests: Clonality assessment by PCR analysis of antigen receptor gene rearrangement in both T cells and B cells is performed in those cases where a diagnostic dilemma still exists ie. small cell indolent lymphoma, “mixed” lymphomas, lymphocytic CSF, endoscopic gut biopsies with mature lymphocyte infiltration, phenotypically primitive or ambiguous leukemias (can assist with lineage assigment, although many caveats in this situation). From W. Vernau, P. Moore, UCD Pathology • High grade/Intermediate grade • Most common • Rapid onset • Low grade • Indolent form • Long hx of enlarged lymph nodes • May not need therapy Canine Lymphoma - Prognosis • Clinical Staging • Stage I: Involvement of 1 LN or organ Stage II: Involvement of 1 or > regional LNs Stage III: Generalized LN involvement Stage IV: +/- Liver/Spleen involvement Stage V: Bone marrow involvement or other organ systems • • • • • A - Healthy (absence of significant clinical signs) • B - Sick (clinical signs attributable to systemic disease) Similar Prognosis Poorer Prognoses Lymphoma: Prognostic factors • Stage of dz V vs other • Advanced clinical stage • no significant difference (if healthy) • Substage a > b • Most consistent prognostic indicator • Immunophenotype • B>T • Anatomic form: • Mediastinal/GI/unusual site < multicent • Prior prednisone • Inc MDR if > 3 weeks • Histologic grade: • Low grade = better long term prognosis • Hypercalcemia • Mediastinal form/T cell Hitting the Wall Remission Rate: 60-90% Remission Duration: 228-330d Survival Time: 240-360d COP COPLA PACO AMC UW MOPP DMAC COAP Lymphoma Therapy • Definitions: • Induction Phase: • Weekly chemotherapy: • Designed to decrease tumor volume • Chemo kills a constant fraction of cells • Independent of the # of cells • Maintenance chemotherapy • Increased dose interval • Uses modestly toxic but effective drugs • • Designed to maintain low tumor volume Impt in duration of remission Long vs. Short Protocol Protocol • Long: • Induction: • Set # of cycles (weekly) • Maintenance: • • • • Increase interval (EOW) Vincristine Cytoxan/Leukeran Methotrexate • Short • Induction • Set # of cycles (weekly) • Discontinuation of chemo • • If in remission Monthly PE/Bloodwork until relapse Long vs. Short Protocol Is Maintenance Chemo Needed? Yes! • Cont chemo will dec tumor volume • Designed to maintain low cell # • Maintains remission until resistance develops • Should improve duration of remission Long vs. Short Protocol • NO! • Residual LSA volume is low post completion of chemo • Average dog will not relapse for several months • Dogs benefit from time off chemo • Approx 10-15% of dogs are cured of LSA • Additional chemo given to a cured patient? • Chemo is palliative tx, short protocol should inc QOL Long vs. Short Protocol Cost Differences? • Higher for Long Protocol due to continued chemotherapy • Short: • m2 dog = $3500.00 • Long: • m2 dog = $4,500.00 Long vs. Short Protocol Ability to Reinduce? • Long Protocol: • 50-60% RR • Short Protocol: • 80-90% RR WHY THE DIFFERENCE? Long vs. Short Protocol Efficacy of each protocol? Long Protocol • Remission rate: • Remission duration: • Survival time: 100 90 80 70 60 50 40 30 Short Protocol 70-90% 240-330d 240-360d 100 90 80 70 60 50 40 No Maintenance Median = 315 d Historical with Maintenance Median = 220 d 20 10 0 250 500 Days 750 1000 70-90% 300-315d 318-397d No Maintenance Median = 397 d Historical with Maintenance Median = 303 d 30 20 10 0 P = 0.79 0 • Remission rate: • Remission duration: • Survival time: P = 0.30 0 250 500 Days 750 1000 Discontinuous Protocols n Protocol ORR DFI MST Moore et al 82 VELCAP-S 68% 20 wks 36% @ 1 yr Chun et al 49 UW-25 --- 300d 270d Garrett et al 53 UW-25 94% 282 d 397 d MacDonald et al 115 UW-19 89% 206 d 310 d Simon et al 77 CHOP-12 76% 243 d 28% @ 1 yr Head to Head Study Hosoya et al. JVIM 2007 • Long Protocol COP/COAP • N=71 • 1st Remission • 94 days • MST • 309 days • UW-19 week CHOP • N=30 • 1st Remission • 174 days • MST • 270 days **Long Protocol contained no doxorubicin Post relapse: protocols varied Canine indolent lymphomas • Veterinary literature is scarce • Effect on prognosis? • Optimal therapeutic approach? • May be underdiagnosed? • Lymphoid “hyperplasia” • • Requires histopath & IHC Based on WHO classification, most common variants include: • B-cell: follicular LSA, marginal zone LSA, mantle cell LSA • T-cell: T-zone LSA Clinical presentation • Most are middle-age to older dogs • Typically no clinical signs (Substage “a”) • Incidental finding by owner or veterinarian • No true breed or sex predilection • May have only local disease, or multicentric • No hypercalcemia reported, even with TZL • Diagnosis: • Indolent”: clinical history provides information • Present with chronically enlarged lymph nodes Definitive Diagnosis • • Cytology is NOT enough Tissue biopsies and histopathology • Large tru-cut, or lymphadenectomy • Immunohistochemistry necessary • Splenectomy (MCL or MZL) • Clonality assays • Recommendations • Regular clinical staging MCL Therapy • If solitary (node, spleen) • Surgical resection • Splenectomy? • Obrien et al. 2010 VCS (n=34) • MST 347d • No adjuvant chemo? Splenic MZL • Multicentric • Depends on variant • MZL – may progress late, similar to high-grade LSA • TZL – very slow progression Therapy and prognosis (cont’d) • Multicentric MZL or FL • If rapid clinical progression • Multiagent chemotherapy • Most respond well at that stage • Multicentric TZL • Often slow progression • May live years with lymphadenopathy • Could chemotherapy be worse than no Tx? Lymphoma: What’s New? Diagnostics • Proteomics • Pet Screen • Thymidine kinase LSA Therapy • Lurie et al JVIM 2009 • ½ body radiation + chemotherapy • 1st remission 410d • 1yr=54%, 2yr=42%, 3yr=31% • MST: 684d • 1yr=66%, 2yr=47%, 3yr=44% TK Inhibitors • Stopping abnormal signal may lead to cancer cell death TK Inhibitor Enters Cancer Cell and Stops Signal Cancer Cell Death Xenogenic Vaccine Concept Dog Differentiation Antigen (cd21) No Immunity Human Differentiation Antigen (cd21) Immunity Questions? Cliffdoc2000@yahoo.com Evaluation of a discontinuous protocol (UW19 week) for canine lymphoma: 118 cases (2003-2005) RE Risbon1, K Drobatz2, CE Goldkamp3, KE Burgess4, and CA Clifford1 Veterinary Cancer Society 2006 Study Design • Primary Objective • Evaluation of UW-19 • Response rate • Remission duration (DFI) • Overall survival (MST) • Multidrug resistance (MDR) • Methods: • Retrospective (2003-2005) • Inclusion criteria • Lymphoma • Intent to treat • Follow up Study Design • Methods: Statistical Analysis • Immunophenotype • Ca++ • Mediastinal mass • MDR • Stage/Substage • Prior steroid tx • Age • Wt • Univariate Analysis (K-M) • p ≤ 0.05 Materials/Methods: UW-19 Protocol Week L-Asparaginase 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 ● 400IU/kg SQ Vincristine 0.5mg/m2 IV 0.7mg/m2 IV ● ● ● ● Cytoxan ● ● ● ● ● ● ● ● 250mg/m2 IV/PO ● Adriamycin ● ● 30mg/m2IV;1mg/kg if < 15kg Prednisone* ● ● ● ● *2mg/kg po sid x7d, 1.5mg/kg po sid x7d, 1mg/kg po sid x7d, 1mg/kg po eod x7d then d/c ● Materials/Methods: UW-10 + CCNU 1 Week L-Asparaginase 2 3 4 5 6 7 8 9 10 12 15 ● ● 18 21 24 27 ● ● ● ● ● 400IU/kg SQ Vincristine ● ● ● ● 0.5mg/m2 IV 0.7mg/m2 IV ● Cytoxan ● 250mg/m2 IV/PO ● Adriamycin ● 30mg/m2 IV; 1mg/kg if < 15kg CCNU 60-80mg/m2 PO Prednisone* ● ● ● ● *2mg/kg po sid x7d, 1.5mg/kg po sid x7d, 1mg/kg po sid x7d, 1mg/kg po eod x7d then d/c Results: Response to UW-19 • Response Criteria: • CR: 100% reduction in measurable tumor volume • PR: 50-99% reduction in measurable tumor volume • SD: <49% reduction or <10% increase in measurable tumor volume • PD: >10% increase in measurable tumor volume Results: Signalment • n=118 • Breed • Mixed: n=15 • Purebred: n=103 • • • Golden: n=23 Labrador: n=8 Rottweiler: n=7 • Sex • M=14; MC=49 • F=5; FS=50 • M:F = 1.1:1 • Age (yrs) • Median 7.7 (1.9-13.7) • Weight (kgs) • Median 32.8 (4.1-68.2) Results: WHO Stage • Stage • • • • • • I: n=4 II: n=1 III: n=65 IV: n=41 V: n=7 Substage • a: n=95 • b: n=23 Results: Diagnostics • Method of diagnosis: • • Cytology:: n=94 Immunophenotyping: n=79 • IHC:: n=2 • ICC: n=40 • PCR:: n=37 • Histopath:: n=24 • CD79a+: n=64 • CD3+: n=13 • CD79a-/CD3-:: n=2 • MDR: n=31 • Positive: n=11 • Negative: n=20 Results: Response to UW-19 • Overall Response Rate = 99% (CR + PR) • • • • • CR: n=108 (92%) PR: n=8 (7%) SD: n=1 (<1%) PD: n=1 (<1%) Median Duration of Tx • 20 wks (9-29 wks) Overall (MST) • 417 days (33-1302d) Median: 267d Range: 33-1170d 0.00 • Proportion Remaining In Remission 0.25 0.50 0.75 1.00 Duration of 1st Remission (DFI) 0 200 400 600 800 Duration of Remission (Days) 1000 1200 Results: Prognostic Factors (DFI) Prognostic Factor n DFI (days) p value Immunophenotype B cell T cell 77 64 13 289 120 <0.001 Age <7.7yrs >7.7yrs 118 59 59 281 226 0.03 1.00 Overall Median Survival Time (MST) 0.75 0.25 0.50 Range: 33-1302d 0.00 Proportion Surviving Median: 400d 0 200 400 600 800 Duration of Survival (Days) 1000 1200 1400 Results: Prognostic Factors (MST) Prognostic Factor n MST (days) p value Immunophenotype B cell T cell 77 64 13 522 257 <0.001 Weight >18kg <18kg 118 96 22 418 270 0.02 Results: Relapse • Overall: n=101 • 38: During UW-19 • 63: Completed UW-19 • Tx free remission: 143d • MDR: n=24 • 8/24 positive • 1/8 + at dx • 2/8 – at dx • 5/8 not tested at dx Relapse during UW-19: n = 38 n Relapse relevance 10 T cell 25 Age > Median 9 Substage b 4 Stage V 4 MDR + 5 Mediastinal mass 7 PR 3 Hypercalcemia 6 None 20 > 1 Factor DFI 79 days MST 158 days Factors associated with shortened DFI Results: Reinduction n Overall 93 CR + PR 56 UW-10 + CCNU 40 CR 33 PR 4 CCNU CR PR Other CR PR 40 9 5 13 1 4 Response Rate (CR+PR) 60% 2nd Remission (med) 126 days 93% 170 days 35% 59 days 38% 126 days Results: Status • Relapsed, in remission • n = 16 • Med Follow-up = 834d • 2nd remission ≥ 1st remission • n = 13 • Med Follow-up = 662d • Still in 1st Remission • n = 11 • Median DFI = 757d Conclusions • • • ORR, DFI, MST Prognostic Factors • Immunophenotype • Weight? • Drug Resistance Subpopulations • Early relapse • No relapse DFI and MST MST only Not prognostic Study Limitations • • • • Retrospective No control group Staging Histopathologic diagnosis Summary RBVH LSA Study Historical Short LSA • Remission Rate: 99% • Remission rate: 70-90% • 1st Remission 267d • 1st Remission: 300-315d • Survival time: 318-397d • 11 no relapse @ • Survival time: • 28 still alive @ 757d 400d 699d • Reinduction Rate: 93% • Reinduction Rate: 80-90% Summary • Short protocol is beneficial for a subset of dogs • % dogs still in 1st remission • % dogs successfully re-induced • Cost effective • What subset? • Immunophenotyping • If T cell LSA then maintenance chemo? • MDR • If MDR positive then maintenance chemo?