J Vet Intern Med 2007;21:1340–1346 Adjuvant Immunother apy of Feline Fibrosarcoma with Recombinant Feline Interferon-v Verena Hampel, Bianca Schwarz, Christine Kempf, Roberto Ko¨stlin, Ulrike Schillinger, Helmut Ku¨chenhoff, Nora Fenske, Thomas Brill, and Johannes Hirschberger Background: Recombinant feline interferon-v (rFeIFN-v) was tested as a treatment option for cats with fibrosarcoma to assess safety and feasibility.
Hypothesis: Treatment with rFeIFN-v in cats with fibrosarcoma is safe and feasible.
Animals: Twenty domestic cats.
Methods: In an open-labeled uncontrolled clinical trial 12 injections of 1 3 106 U/kg rFeIFN-v were administered over a 5- week period: the 1st through 4th injections were given intratumorally, and the 5th through 12th injections were administeredsubcutaneously at the tumor excision site. Wide surgical excision of the tumors was carried out after the 4th injection andbefore the 5th injection of rFeIFN-v. A Common Terminology Criteria for Adverse Events (CTCAE) analysis wasconducted. Flow cytometry of fibrosarcoma cells after incubation with rFeIFN-v and recombinant feline interferon-c wasperformed to assess the biological effect of rFeIFN-v.
Results: Changes in blood cell count, increases in serum aspartate-amino-transferase activity, serum bilirubin concentration, serum creatinine and serum electrolyte concentrations, weight loss, anorexia, increased body temperature, and reducedgeneral condition were observed but were mostly minor (grade 1 and 2) and self limiting. Eosinophilia (P 5 .025), neutropenia(P 5 .021), and weight loss (P , .001) were statistically correlated with rFeIFN-v-treatment (analysis of parameters beforetreatment and after 3 injections of rFeIFN-v). Flow cytometry of 5 unrelated feline fibrosarcoma cell lines showed increasedexpression of major histocompatibility complex (MHC) class I molecules (P 5 .026) in response to in vitro incubation withrFeIFN-v, whereas expression of MHC class II molecules was not affected significantly.
Conclusions and Clinical Importance: RFeIFN-v for the treatment of feline fibrosarcoma is safe, well tolerated, and can be easily performed in practice. To assess the efficacy of the treatment, it should be tested in a placebo-controlled trial.
Key words: Antitumor immunity; Cat; Cytokines; Major histocompatibility complex; Soft-tissue sarcoma.
Fibrosarcomas are common in the cat and comprise free interval or reduce tumor recurrence or the rate of over 40% of all skin tumors in this species.1 Because of the invasive nature of fibrosarcomas, the recurrence Cancer immunotherapy refers to using the power and rate ranges from 30 to 70%,2,3 and metastasis occurs in the specificity of the immune system for the treatment of approximately 10 to 20% of affected cats.4–7 Currently, a malignancy. This therapy is based on the use of there is no successful treatment for fibrosarcoma in the biologically active proteins with the aim of altering the cat.7 Although surgery is the treatment of choice, specific and nonspecific immune responses of the complete excision often is difficult. Wide surgical patient. The immune system is capable, under certain excision extends the tumor-free interval and increases circumstances, of recognizing and eliminating tumor survival time.8,9 Pre- or postoperative radiation therapy extends the Interferons (IFN) are cytokines that have antiviral, median tumor-free time up to 422 days10 and may antiproliferative, and immunomodulatory effects. The reduce the recurrence rate to 42% in cats with clean family of IFNs consists of 2 major classes, type I and type surgical margins.11 In the study by Cohen et al,4 II. Type I IFNs include IFN-a, b, d, t, and v, whereas a recurrence rate of 41% after tumor excision, elec- IFN-c is the only type II IFN. The biological activities of tron-beam radiation, and, in some cases, chemotherapy type I IFNs include growth inhibition of tumor cells,16,17 was documented. Chemotherapy regimens that have induction of apoptosis,17,18 natural killer cell activation,19 been investigated include doxorubicin12,13 and carbopla- and an increase in the expression of major histocompat- tin.11 Martano et al14 showed that the combination of ibility complex (MHC) class I molecules.20,21 The amino surgery and doxorubicin does not increase the disease- acid sequence of recombinant feline IFN-v has approx-imately 60% homology to that of human IFN-v.22 The From the Department of Veterinary Internal Medicine, Veteri- antitumor efficacy of human IFN-v has been described in nary Faculty, LMU Munich, Munich, Germany (Hampel, Schwarz, vitro23,24 and in vivo.25 Recombinant feline IFN-v has Kempf, Hirschberger); Department of Veterinary Surgery, Veteri- approximately 60% homology to human IFN-a.22,26 nary Faculty, LMU Munich, Munich, Germany (Ko¨stlin); Institute Human IFN-a has been used in Germany since 1999 for Experimental Oncology, Klinikum Rechts der Isar, TU Munich, for the adjuvant therapy of malignant melanoma in Munich, Germany (Schwarz, Kempf, Schillinger, Brill); and the humans. Because of the close relationship of IFN-v and Statistical consulting unit, Department of Statistics, LMU Munich, IFN-a, adjuvant immunotherapy of feline fibrosarcoma Mu¨nchen, Germany (Ku¨chenhoff, Fenske).
by using rFeIFN-v follows the therapy protocol for Reprint requests: Verena Hampel, Mangstrasse 7, D-80997 human IFN-a.27 Recombinant feline IFN-a is not Munich, Germany; e-mail: [email protected].
commercially available, whereas rFeIFN-v is the only Submitted August 6, 2006; Revised December 12, 2006, March 3, IFN licensed for use in cats.
2007, April 3, 2007; Accepted June 19, 2007.
IFN-c increases antigen presentation by upregulation Copyright E 2007 by the American College of Veterinary Internal of MHC expression on antigen presenting cells and by activation of natural killer cells.21,28,29 Tumor cells often Fibrosarcoma Therapy with Interferon-v have a decreased expression of MHC class I mole- through 4th injection) and then subcutaneously (SC) (5th through cules.15,30–33 This so-called tumor escape mechanism 12th injection) at the tumor excision site. In the first week of prevents recognition of tumor antigens by cytotoxic T treatment, the cats received 1 intratumoral injection daily for 3 cells, because tumor antigens are presented by MHC consecutive days. In the second week, the cats again received 1 class I molecules on the surface of malignant cells.
injection daily for 3 consecutive days, but the 4th injection wasadministered intratumorally and the 5th and 6th injections were given Because IFN-v and IFN-c may be able to increase SC at the excision site after surgical excision of the tumor. In the next expression of MHC class I molecules and, therefore, 3 weeks, the cats received 2 injections of rFeIFN-v SC per week.
antigen presentation, these cytokines may render tumorcells susceptible to attack by natural killer cells,cytotoxic T cells, activated macrophages, or antibo- dies.32,34 To assess the biological effect of rFeIFN-v on Wide surgical excision (ie, tumor excision en bloc with 3-cm MHC class I and II expression on feline fibrosarcoma margins to palpable evidence of disease) was carried out during the cells, flow cytometry from 5 unrelated fibrosarcoma cell second week of treatment, after the 4th and before the 5th injection lines was carried out. The effect of rFeIFN-v was of rFeIFN-v. All of the operations were performed by the same compared with that of rFeIFN-c. RFeIFN-v was surgeon (RK). Cross-sectional imaging was not performed in any administered to domestic cats before and after tumor excision. To evaluate the safety and the feasibility of thisadjuvant treatment, an open-label prospective clinical Pain Relief and Antibiotic Treatment trial was conducted in 20 cats.
For perioperative analgesia, buprenorphineb was given at a dosage of 0.01 mg/kg on the day of surgery. For postoperative Materials and Methods analgesia, all cats received meloxicamc for 5 days (1st day aftersurgery: 0.3 mg/kg; 2nd through 5th days after surgery: 0.1 mg/kg).
Furthermore, the cats were treated with amoxicillin-clavulanicacidd at a dosage of 25 mg/kg per day for 5 days.
The study was an open-label, single-arm, monocentric, pro- spective, safety and feasibility study. From January 2004 untilAugust 2004, cats with fibrosarcoma were evaluated for inclusion in the study. Cats were enrolled in the study if they met definedinclusion criteria: resectable tumor (excision possible without The cats were reevaluated for tumor recurrence on days 28, 42, opening a visceral cavity, amputation or partial resection of parts 90, 180, 270, and 360 after surgery or on an ad hoc basis if the of the scapula or the spine), absence of previous immunomodu- owner reported regrowth of the tumor. At each reevaluation, the latory treatment within 6 weeks of the start of IFN-v treatment, cats underwent a clinical examination, which included weighing, as and life expectancy .1 year. Twenty cats with fibrosarcoma met well as evaluation of wound healing, potential adverse effects, the inclusion criteria: 18 Domestic Shorthair, 1 Domestic Longhair potential recurrence, and metastasis of the tumor. On days 28, 42, and 1 Persian cat. They ranged in age from 4 to 16 years (mean, 180, and 360, CBC count and serum biochemistry were performed.
10 years), and there were 11 castrated male and 9 spayed female The final evaluation on day 360 also included thoracic (left and cats. Eleven cats had primary tumors, and 9 cats had tumors that right lateral and ventrodorsal views) and abdominal (lateral view) had recurred after surgery.
radiographs and abdominal ultrasonography.
The tumors were mainly located in the interscapular region (11 cats). Other sites were the thoracic wall (5 cats), the lumbar area (3cats), and the shoulder region (1 cat). The tumors all were located Common Terminology Criteria for Adverse Events at sites that are commonly used for administration of vaccines.
All data were recorded in detail in case report forms and were However, it was not possible to determine whether vaccines had analyzed to determine the toxicity of the treatment. To provide indeed been given at the tumor site. Nine cats had stage I tumors a basis for the evaluation of toxicity, the ‘‘Veterinary Co-operative (,2 cm), 8 cats had stage II tumors (2–3 cm), and 3 cats had stage Oncology Group—Common terminology criteria for adverse III tumors (.3 cm). Two cats had multiple tumors at 1 site. In both events following chemotherapy or biological antineoplastics cats, 3 tumors were present at 1 site. In these cats, the diameters of therapy in dogs and cats'' V 1.0 was used.37 the tumors were added (tumor staging according to Hirschbergerand Kessler35). Nine cats could not be included in the study becauseof metastasis in the liver (1 cat), nonresectable tumor (4 cats), no clinical evidence of fibrosarcoma (3 cats), and 2 tumors at 2different sites (1 cat).
Five unrelated feline fibrosarcoma cell lines (fibrosarcomas from control animals of another study) were cultured at theInstitute for Experimental Oncology, TU Munich, Munich, Reagents and Products Germany, by standard methods: cells were mechanically andenzymatically dissociated and cultured in Dulbecco's Modified Recombinant FeIFN-va was provided by Virbac, S.A., France.
Eagle Mediume supplemented with 10% fetal calf serum,f 1% The 5 3 106 U powder fraction was diluted in 1 ml of isotonic glutamine,g 100 U/mL penicillin,h and 100 mg/mL streptomycini at saline solution immediately before use.
37uC in a 5% CO2 atmosphere.
Immunotherapy with rFeIFN-v Incubation with rFeIFN-v and rFeIFN-c The treatment protocol consisted of 12 injections of 1 3 106 U/ kg rFeIFN-va over 5 weeks. The dosage of 1 3 106/kg rFeIFN-v Fibrosarcoma cells lines (1.75 3 106 cells each) were incubated has been established in cats and is recommended by the in vitro for 48 hours with 50 mL isotonic saline solution (negative manufacturer.36 RFeIFN-v was administered intratumorally (1st control), 1,000 ng rFeIFN-c,j or 25,000 U rFeIFN-v.a Staining of the Cells Modulation of MHC Molecules on Fibrosarcoma Cells by rFeIFN-v and rFeIFN-c The flow cytometry protocol described by Schwarz28 was used.
For each tumor cell line, 13 FACS tubesk were prepared (3–4 3 105 Expression of MHC class I and II antigens on feline cells each): 3 for negative control, 5 for each rFeIFN-v and fibrosarcoma cell lines was evaluated by using flow rFeIFN-c. The monoclonal antibodies used were mouse anti-MHC cytometry (see Fig 2). A 1-fold increase of expression of class I v6/32l and mouse anti-feMHC class II 42.3.m The antibodies MHC class I molecules was observed in fibrosarcoma were added as follows: (a) no antibody (negative control), (b) 10 mL cell lines in response to in vitro incubation with immunogloublin (Ig) G1,n and (c) 10 mL IgG2ao (isotype controls),(d) 60 mL MHC I antibody (v6/32), and (e) 10 mL MHC II 25,000 U rFeIFN-v (P 5 .026). The expression of antibody (42.3). The cells were washed twice and incubated with MHC class II molecules also was influenced by rFeIFN- 10 mL fluorescein isothiocyanate-conjugated rabbit anti-mouse v, but this effect was not statistically significant (P 5 IgG antibodyp for 20 minutes in the dark and resuspended .22). The incubation of feline tumor cell lines with 1,000 ng rFeIFN-c caused a 1.5-fold increase ofexpression of MCH class I (P 5 .010) and a 3-foldincrease of expression of MHC class II molecules (P 5 Flow Cytometric Analysis .020). This observation means that rFeIFN-v is able to Expression of MHC class I and II on the cell surface was increase the expression of MHC class I molecules on evaluated by performing flow cytometric analysis with FACS- feline fibrosarcoma cells, whereas rFeIFN-c can mod- Vantage.q Negative and isotype controls were used as previously ulate the expression of MHC class I and II molecules.
described.28 The Cell Quest Programr was used for data analysis.
Statistical Analysis Immunotherapy has been investigated in several Kaplan-Meier analysis of recurrence was carried out by using studies in cats with fibrosarcoma. King et al38 reported SPSS v.13.Os. The number of events (3 in the group of cats without that the immunostimulant acemannan may be an previous recurrence, 6 in the group of cats with at least 1 previous effective adjunct to surgery and radiation therapy in recurrence) was too small for additional statistical analyses. A the treatment of fibrosarcoma. Acemannan enhances paired Student's t-test was used to evaluate differences between the macrophage release of interleukin-1, interleukin-6, serum results and blood counts of days –7 (before 1st treatment) tumor necrosis factor-a, and IFN-c. Jourdier et al39 and 0 (day before surgery) and to test for change of expression ofMCH class I and II. In all statistical analyses, a P value of ,.05 studied local immunotherapy of spontaneous feline was considered significant.
fibrosarcomas by using recombinant poxviruses expres-sing interleukin-2. This study demonstrated a decrease intumor recurrence rates from 61% (11 of 18 cats) in control animals (treated with surgery and iridium-basedradiotherapy) to 39% (7 of 18) and 28% (5 of 18) in cats Common Terminology Criteria for Adverse Events who received human or feline interleukin-2 in addition The Common Terminology Criteria for Adverse to surgery and iridium-based radiotherapy.
Events (CTCAE) analysis produced the results shown Recombinant feline IFN-v has antiviral40,41 and in Table 1. The statistical analysis of CBC count, serum antiproliferative effects,42,43 and has been used in the biochemistry results, and body weight on days –7 treatment of feline calicivirus infection44; feline herpetic (before treatment) and 0 (after 1 week of treatment keratitis45; feline leukemia virus (FeLV) and feline but before surgery) produced the following results: the immunodeficiency virus (FIV) infection46; feline infec- parameters neutropenia (P 5 .021), eosinophilia (P 5 tious peritonitis47; gingivitis, stomatitis, and orophar- .025), and weight loss (P , .001) showed significance yngitis48; and canine parvovirus infection.49 De Mari et and were positively correlated with the rFeIFN-v al46 tested rFeIFN-v in 81 cats with FeLV and with FeLV and FIV-coinfection. In this placebo-controlledtrial, the cats were treated with 1 3 106 U/kg rFeIFN-vfor 5 consecutive days in 3 series on day 0, day 14, and Clinical Findings day 60. The investigators observed systemic adverse The tumor recurred locally in 9 of 20 cats (45%); 1 events in 5 of 81 of the cats, including lethargy, transient other cat developed pulmonary metastases and died on vomiting, and diarrhea, but with no statistically day 258. The tumor had not recurred at the original site.
significant differences between the IFN-treated group The disease-free interval of this cat was censored at day and the placebo group.
258, but it was included in the statistical analysis. At the In another clinical trial,48 20 cats with chronic final evaluation, on day 360, 10 cats were disease free.
gingivitis, stomatitis, or oropharyngitis were treated by To investigate the influence of previous recurrences on using subgingival and SC injections of 1 3 106 U/kg or recurrence-free time, the study population was divided 2.5 3 106 U/kg rFeIFN-v. Mihaljevic48 found transient into 2 groups: a group without previous recurrence (10 lethargy, increased body temperature, and anorexia in cats) and a second group with at least 1 previous some cats. These adverse events were mainly observed in recurrence (10 cats). The Kaplan-Meier curves for both cats treated at a dosage of 2.5 3 106 U/kg. In the present groups are shown in Fig 1.
study, cats were treated at a dosage of 1 3 106 U/kg, as Fibrosarcoma Therapy with Interferon-v Number of adverse events during the study according to the Veterinary Co-operative Oncology Group Adjuvant Treatment (28 adverse events) (53 adverse events) (101 adverse events) (68 adverse events) 28 (last treatment Segmented neutropenia 3 Wound healing disorder q, increased parameter; Q, decreased parameter.
a From Ref. 37.
recommended by the manufacturer36 to avoid the above- weight loss, which indicated a correlation to the mentioned adverse events of the higher dosage.
treatment with rFeIFN-v. The manufacturer36 describes The results of the CTCAE analysis of the study neutropenia during treatment with rFeIFN-v. Mild correspond with the adverse effects specified by the eosinophilia may be induced by rFeIFN-v. Weight loss manufacturer.36 These included hyperthermia; vomiting; showed a clear statistical significance (P , .001), which diarrhea; mild decrease in the total number of leuko- could be correlated to the therapy or stress caused by cytes, thrombocytes and erythrocytes; increased activity frequent visits in the clinic. Weight loss was not observed of serum aspartate-amino-transferase; and transient in the above-mentioned studies.46,48 lethargy. However, these changes generally were minor In the present study, there was a recurrence rate of grade and self limiting. The statistical analysis of blood 45% (9 of 20 cats); metastases were found in 1 cat. The test results and body weight on days –7 (before current study design was not able to a demonstrate treatment) and 0 (after 1 week of treatment) showed decreased recurrence rate, because of a lack of a control significant differences in neutropenia, eosinophilia, and group and a lack of surgical margin assessment after

study, cats were enrolled in the trial as they werepresented in the clinic, therefore, 17 cats randomly hadtumor sizes of ,3 cm. This observation can beattributed to the awareness of the referring veterinariansabout fibrosarcomas.
The cats had to meet defined inclusion criteria: 4 cats were not included in the study, because they hadnonresectable tumors. In these cats, tumor masses wereso large that tumor excision was not possible withoutopening a visceral cavity or performing amputation orpartial resection of parts of the scapula or the spine.
Cats with 2 tumors at 2 different sites (eg, thoracic wall and lumbar area) could not be included in thestudy. It cannot be assumed that 2 tumors at 2 differentsites originated from the same tumor cells. Two differentfibrosarcomas might not have the same biologicalbehavior and potentially may have different antigenicproperties. The cats received recombinant feline IFN-vintratumorally or SC at the excision site, and for thisreason only 1 tumor could be treated. Consequently, 1cat with tumors at different sites was excluded from the Analysis of recurrence (Kaplan-Meier) of cats without study. Two cats had multiple tumors at 1 site. In both previous recurrences (n 5 10) and cats with at least 1 previous cats, 3 tumors were present at 1 site, for 1 cat, it was the recurrence (n 5 10).
first recurrence and, for the other cat, it was the secondrecurrence. This finding was thought to represent surgery. Histologic grading of the surgical specimens as a recurrence of 1 fibrosarcoma as multiple small nodules described previously was not performed.
at the surgical site.
Tumors in the study described here all were located at Tumors were resected en bloc with 3-cm margins to vaccination sites. However, it was not possible to palpable evidence of disease, and tumor excisions always determine whether vaccines had indeed been given at were carried out by the same experienced surgeon. Cohen the tumor site. Hendrick et al50 reported that 39% of cats et al4 stated that the location and the size of the primary with vaccination site sarcomas had tumor sizes of 2.0– tumor and the ability to remove it are important 3.9 cm and 33% of the cats had tumor sizes of 4.0– prognostic factors. The recurrence rate in the present 5.0 cm. However, 33% of cats with nonvaccination site study could have been influenced by the fact that most of sarcomas had tumor sizes of 2.0–3.9 cm. In the present the cats had small tumors and that the surgical procedurewas performed by an experienced surgeon.
Cohen et al4 also reported that the prognosis of recurrent fibrosarcoma treated by surgical excision isworse than that of excision of primary fibrosarcoma. Inthe present study, the number of tumor recurrencesbefore rFeIFN-v treatment also may have had animpact on the outcome. Seven cats that had tumorrelapses (6) or metastases (1) had a minimum of 1fibrosarcoma removed surgically before inclusion in thestudy. Of these 7 cats, 4 had 1 previous fibrosarcomaand 3 had 2 previous tumors. In contrast, only 3 catswith a primary tumor had tumor recurrence in thisstudy. Of the 10 cats that were tumor free at the end ofthe study, 7 had a primary tumor and 3 had had 1previous fibrosarcoma at the time of admission. Visual Results of flow cytometry of feline fibrosarcoma cell lines inspection of the Kaplan-Meier curve (Fig 1) indicated incubated in vitro with 25,000 U (rFeIFN-v) and 1,000 ng an influence of previous recurrences on recurrence-free rFeIFN-c for 48 hours. The figure shows 1 of 5 experiments time, but, presumably because of small sample sizes, this representative of all 5 fibrosarcoma cell lines tested. The y-axis effect did not achieve statistical significance.
shows the counts, and the x-axis shows the intensity of the All of the cats received meloxicam for postoperative fluorescence of fluorescein isothiocyanate-conjugated rabbit anti- pain once daily for 5 days, as described in other mouse immunogloublin (Ig) G antibody (FITC). 2.1.: solid line: studies.51,52 Nonsteroidal anti-inflammatory drugs also major histocompatibility complex (MHC) class I after stimulation have been beneficial in the treatment of transitional-cell with rFeIFN-c, dotted line: negative control; 2.2.: solid line: MHC carcinoma in dogs.53 These substances not only decrease class II after stimulation with rFeIFN-c; 2.3.: solid line: MHC classI after stimulation with rFeIFN-v; 2.4.: solid line: MHC class II the inflammatory reaction in the tumor tissue but also after stimulation with rFeIFN-v.
inhibit tumor growth. It seems unlikely that meloxicam Fibrosarcoma Therapy with Interferon-v retarded tumor growth in this study, because it was Faculty and Prof Dr Walter Hermanns, Department of administered for only 5 days.
Veterinary Pathology of the Veterinary Faculty, LMU The expression of MHC class I and II antigens on Munich, Munich, Germany, and Edelburga Ham- feline fibrosarcoma cell lines was evaluated by using merschmid and Gerlinde Stejskal from the Institute for flow cytometry. An increase in expression of MHC class Experimental Oncology, Klinikum Rechts der Isar, TU I molecules was observed in all tested fibrosarcoma cell Munich, Munich, Germany. The trial was funded by lines after in vitro incubation with recombinant feline Virbac, Carros, France.
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Carbamate and Pyrethroid Resistance in the Leafminer J. A. ROSENHEIM,' AND B. E. T ABASHNIK Department of Entomology, University of Hawaii at Manoa, Honolulu, Hawaii 96822 J. Econ.Entomol.83(6): 2153-2158 (1990) ABSTRACT Populations of D1glyphus begini (Ashmead), a parasitoid of Lirlomyza leafminers, showed resistance to oxamyl, methomyl, fenvalerate, and permethrin in labo-ratory bioassays. Relative to a susceptible strain from California, maximum resistance ratiosfor these pesticides were 20, 21, 17, and 13, respectively. Three populations that had beentreated frequently with insecticides were significantly more resistant to all four insecticidescompared with an untreated Hawaii population and a California population with an unknownspray history. Parasitoids from a heavily sprayed tomato greenhouse on the island of Hawaiihad LC",'s for permethrin and fenvalerate that were 10 and 29 times higher than the fieldrate, respectively. Populations resistant to oxamyl and methomyl had LC",'s two- and sixfoldbelow the field rate, respectively. D. begini is one of the few parasitoids resistant to pyre-throids, with LC",'s exceeding field application rates. Resistant D. begini may be useful forcontrolling leafminers in management programs that integrate biological and chemical con-trols.


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