Nikolaos G. Dervisis

Combined Gemcitabine and Carboplatin Therapy for Carcinomas in Dogs

BACKGROUND Response and adverse reactions to combined gemcitabine (GEM) and carboplatin (CARBO) therapy in dogs with carcinomas are not documented. HYPOTHESIS GEM and CARBO are safe for the treatment of dogs with carcinomas. ANIMALS Thirty-seven dogs with histologically or cytologically confirmed carcinomas. METHODS Prospective clinical trial. Dogs were treated with GEM (2 mg/kg, 20-30-minute infusion IV) on Days 1 and 8 and 4 hours later, CARBO (10 mg/kg IV) on Day 1. The cycle was repeated on Day 22. RESULTS Thirty-seven dogs (29 with measurable tumor) received a median of 2 cycles (range 0.5-6) for a total of 101 cycles administered. Twelve dogs (32%) developed neutropenia (3 Grade 3, and 5 Grade 4) and 9 (24%) thrombocytopenia (2 Grade 3, and 1 Grade 4). Dogs >20 kg were twice as likely to develop thrombocytopenia (P= .023). Twenty-seven dogs (73%) had evidence of gastrointestinal (GI) toxicosis, but most signs were of mild to moderate severity and self-limiting. One dog died of treatment-related complications. Overall tumor response rate was 13%. One dog with metastatic prostatic carcinoma achieved a complete remission and 1 dog with intestinal adenocarcinoma and 1 with tonsillar squamous cell carcinoma achieved partial remission. Twelve dogs achieved stable disease for a median of 72 days. CONCLUSION AND CLINICAL IMPORTANCE GEM and CARBO combination causes mild to moderate hematologic and GI toxicosis in dogs with carcinoma. Response rate in this study was modest, and optimization of dosing of this combination is required.

Treatment with DAV for Advanced-Stage Hemangiosarcoma in Dogs

Hemangiosarcoma (HSA) is an aggressive disease that is fairly common in the dog. The authors evaluated a doxorubicin, dacarbazine, and vincristine (DAV) combination protocol in dogs with nonresectable stage II and stage III HSA. Twenty-four dogs were enrolled in this prospective, phase 2 study. Doxorubicin and dacarbazine were administered on day 1 while vincristine was administered on days 8 and 15. The protocol was repeated every 21 days for a maximum of six cycles or until disease progression. Toxicity and efficacy were assessed by clinical and laboratory evaluation and by questionnaires completed by the owners. Of the 24 included dogs, 19 were evaluable for response. The response rate (including five complete responses and four partial responses) was 47.4%. Median time to tumor progression was 101 days and median overall survival was 125 days. Significant toxicities were noted, including 41 high-grade hematologic and 12 high-grade gastrointestinal toxic events. Five dogs discontinued treatment due to chemotherapy-related toxicities, but no treatment-related deaths occurred. Multivariate analysis identified patient age (relative risk [RR], 2.3, P=0.049) to be negatively associated with time to progression whereas dacarbazine dose reductions (RR, 0.06, P=0.031) were positively associated with time to progression. Dacarbazine dose reduction was the sole factor positively associated with overall survival (RR, 0.28, P=0.015). In conclusion, the DAV combination appears to offer clinical responses and may prolong survival in dogs with advanced-stage HSA.

Tolerability of gemcitabine and carboplatin doublet therapy in cats with carcinomas.

BACKGROUND This study was performed to determine the toxicity of gemcitabine-carboplatin doublet therapy in cats with carcinomas. HYPOTHESIS Gemcitabine and carboplatin are safe in tumor-bearing cats. ANIMALS Twenty cats with spontaneously occurring carcinomas. METHODS A cohort of 6 cats received gemcitabine (2 mg/kg IV) on days 1, 8, and 15 and carboplatin (10 mg/kg IV) immediately after gemcitabine on day 1 of a 21-day cycle. A 2nd cohort of 14 cats received carboplatin 4 hours after gemcitabine on day 1 and gemcitabine on day 8 but not day 15. The cycles were repeated every 21 days. RESULTS Cats in the 1st cohort received a median of 3.75 cycles per animal (range, 1-6). Two cats (33.3%) developed grade 3 or 4 neutropenia, 1 (16.7%) grade 4 thrombocytopenia, and 1 (16.7%) grade 3 gastrointestinal toxicity. Gemcitabine dose reductions and treatment delays occurred in 1 and 4 cats, respectively. Cats in the 2nd cohort received a median of 2 cycles per animal (range, 0.5-10). Two cats (14.3%) had grade 3 or 4 neutropenia and 1 (7.1%) had grade 3 and 4 gastrointestinal toxicity. One cat required gemcitabine dose reduction and 6 had treatment delays. In the 2nd cohort, of 11 cats with measurable tumors, there was 1 complete response (pancreatic carcinoma) and 1 partial response (squamous cell carcinoma, receiving concurrent nonsteroidal anti-inflammatory drugs). CONCLUSIONS AND CLINICAL IMPORTANCE Gemcitabine-carboplatin combination appears moderately well tolerated in tumor-bearing cats. Minimal patient benefit suggests that alternative schedules or combinations of gemcitabine with other agents should be explored.

Use of an axial pattern flap and nictitans to reconstruct medial eyelids and canthus in a dog.

A 10-year-old male neutered Boxer presented with recurrence of a mast cell tumor at the right medial canthal area. Following excision including 2 cm margins, the medial one-half of the upper and lower eyelids and the medial canthus were reconstructed using an axial pattern flap based on the cutaneous branch of the superficial temporal artery. The bulbar conjunctiva of the nictitans was preserved and sutured to the medial flap edge, thus creating a conjunctival lining to the deep aspect of the flap, protecting corneal epithelium. This is a valuable surgical technique for closing a large skin defect and reconstructing the medial eyelids, thus preserving the globe.

Measurement of serum carboxyterminal cross-linked telopeptide of type I collagen concentration in dogs with osteosarcoma

OBJECTIVE To evaluate the usefulness of carboxyterminal cross-linked telopeptide of type I collagen (ICTP) concentrations for screening dogs for the presence of osteosarcoma. SAMPLE POPULATION 32 client-owned dogs with osteosarcoma (27 dogs with osteosarcoma of the appendicular skeleton and 5 dogs with osteosarcoma of the axial skeleton) and 44 non-tumor-bearing control dogs. PROCEDURES Serum was obtained from blood samples collected from dogs with osteosarcoma and from clinically normal dogs. The serum ICTP concentration was determined by use of a commercially available radioimmunoassay for ICTP. RESULTS Mean +/- SD serum ICTP concentration in the tumor-bearing dogs was 7.32 +/- 2.88 ng/mL, and in clinically normal dogs, it was 6.77 +/- 2.31 ng/mL; values did not differ significantly. Mean serum ICTP concentration in dogs with appendicular osteosarcoma, compared with that of clinically normal dogs, was not significantly different. Mean serum ICTP concentration in dogs with axial skeletal tumor location was 10.82 +/- 2.31 ng/mL, compared with a value of 6.73 +/- 2.28 ng/mL in dogs with appendicular osteosarcoma. CONCLUSIONS AND CLINICAL RELEVANCE On the basis of the results of this study, serum ICTP concentrations are not a clinically useful screening tool for the detection of appendicular osteosarcoma in dogs. Despite the observation that serum ICTP concentration was higher in dogs with axial osteosarcoma than in clinically normal dogs, serum ICTP concentration determination is not a suitable screening test for osteosarcoma.

Treatment-related toxicities in tumor-bearing cats treated with temozolomide alone or in combination with doxorubicin: a pilot assessment

A retrospective study assessing treatment-related toxicities in tumor-bearing cats treated with temozolomide (TMZ) alone or in combination with doxorubicin was conducted. TMZ was administered orally once a day for 5 days every 3 weeks at a dose of 20 mg/cat. Tumor response was evaluated with standard World Health Organization criteria and toxicity was monitored using veterinary co-operative oncology group–common terminology criteria for adverse events (VCOG—CTCAE) criteria. Ten tumor-bearing cats with various types of malignancies were treated with TMZ-based chemotherapy. Eight cats were evaluable for response. Two cats achieved a complete response, one achieved stable disease and five achieved a partial response. Four grade III and one grade IV hematological toxicities, and one grade IV gastrointestinal toxicity were observed. Four cats were euthanased as a result of apparent toxicity. One cat was euthanased as a result of severe and prolonged myelosuppression with fever. Three were euthanased for grade III pleural and pericardial effusions. Effusion was seen in cats treated with higher cumulative dose of TMZ (P = 0.0046). Planned additional case accrual was discontinued because of unacceptable levels of toxicity despite evidence of efficacy in some of the cats. Additional investigation is needed to elucidate this unexpected apparent cumulative toxicity.

Localized and disseminated histiocytic sarcoma complex: A canine model

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Canine histiocytic sarcoma is an aggressive and uniformly-fatal round-cell neoplasm. The localized, peri-articular form of the disease (PAHS) appears to have statistically significant prolonged survival, when compared to the non - peri-articular form of the disease (non-PAHS). Knowledge of the molecular basis for the aggressiveness of the canine histiocytic sarcoma is foundational for developing effective treatments. The aim of this study was to characterize the molecular expression profiles of the different sub-types of the disease. Canine patients, with spontaneously-arising histiocytic sarcoma, were recruited for the study. Tumor tissue samples and peripheral blood were obtained and used for primary cell line development and gene expression analysis. Eight PAHS and 8 non-PAHS tumor samples were selected. RNA extraction, amplification and labeling were performed using standard techniques. Raw gene expression values were obtained using the Affymetrix GeneChip Canine Genome 2.0 Array. Background correction, normalization, PM correction, summarization, and expression analysis were performed using R/affy/limma. Gene set analysis was performed using BRB-ArrayTools, and the Molecular Signatures Database (MSigDB), to investigate gene ontology groups of genes whose expression was differentially regulated. Primary cell lines from tumor biopsies and peripheral blood samples were developed and characterized based on cellular morphology, growth characteristics, and immunophenotyping. In our gene expression array experiments, we identified specific gene transcripts that are more likely to represent true and biologically meaningful differences in expression levels between PAHS and non-PAHS. More specifically, metallopeptidase genes, and homeobox domain gene expression levels were enriched in the PAHS group, while ATP-binding cassette genes, and interleukin 1 receptor signaling gene expression levels were enriched in the non-PAHS group. Our results suggest presence of gene expression signatures that can provide insight in the diverse biologic behavior of the disease and highlight canine spontaneous tumors in understanding tumor biology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5092. doi:1538-7445.AM2012-5092

Abstract 1354: Whole transcriptome analysis of primary and metastatic osteosarcoma in the canine model

Canine osteosarcoma is an excellent model of the human disease. Understanding the molecular events leading to metastatic disease is foundational for developing effective treatments. The objective of this study was to characterize single nucleotide variations (SNVs) acquired during metastasis. Primary and metastatic tumor samples were obtained from a dog with spontaneously occurring osteosarcoma, treated with surgery and adjuvant chemotherapy. Tumor total RNA was isolated and processed using standard techniques. Created libraries were used for paired-end sequencing (2 lanes of 2x80 per sample), in the Illumina Platform. Alignments to canFam2 reference sequence were performed using the TopHat splice aligner and quantified expression levels for all canFam2 ENSEMBL gene models using CuffLinks with GC-correction and upper-quartile normalization. After SNV discovery via custom pipeline, quantification of their abundance was performed by Benjamini-Hochberg corrected Fisher exact tests, on SNVs read counts. Thesequence data were visualized using Integrated Genome Viewer, while Polyphen2 was used to assess the potential impact of SNVs in protein function. Tumor samples from a 6-year-old neutered male Golden Retriever, diagnosed with spontaneously-occurringosteosarcoma, treated with surgery and adjuvant cheemotherapy were used. Lung metastases were confirmed to be metastatic osteosarcoma. Tumor RNA of adequate quantity and quality was isolated from primary and metastatic lesions. Seventy-four and 89 million pair-end reads were generated from the primary and the metastatic site, respectively. There were 13,292 heterologous and 19,780 homologous SNVs identified in the primary tumor, while 48,453 heterologous and 72,737 homologous SNVs identified in the metastatic tumor. At 20% false discovery rate, we identified 190 SNVs with increased abundance in the metastatic site compared to the primary site. The majority of these SNVs (>95%) were nearly or fully homozygous in the metastatic sample and either heterozygous or low abundance in the primary tumor sample. These results suggest frequent and potentially convergent loss-of-heterozygosity in the metastasis compared to the primary. More specifically, a missense SNV with high probability of affecting protein function was identified in p53, only in the metastatic sample. A putativealternative splicing event was identified in BRCA1, skipping exons 4, 5, and 6. A missense SNV was identified in BRCA2, with 1/9 reads having a T>C change in the primary tumor, but 25/25 reads having the same variant in the metastatic site (adjusted p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1354. doi:1538-7445.AM2012-1354

Abstract 4310: Gene expression analysis of axial and appendicular osteosarcoma in the canine model

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Spontaneously occurring canine osteosarcoma (OSA) is considered an excellent model of the human disease. Canine axial and appendicular osteosarcoma appear histopathologically identical. The clinical course of the two forms, however, seems to diverge. Following surgery, the axial form may be associated with a lower metastatic rate and longer survival than the appendicular form. Identifying gene expression profiles associated with decreased metastatic potential may be crucial for the development of targeted therapies for the human disease. We hypothesized that there are distinct gene expression patterns for canine axial and appendicular OSA. Six axial osteosarcoma tumor samples and six age, breed, and weight matched appendicular osteosarcoma clinical tumor samples were selected from the Center for Comparative Oncology, Michigan State University tissue bank. RNA extraction, amplification and labeling were performed using standard techniques. Raw gene expression values were obtained using the Affymetrix GeneChip Canine Genome 2.0 Array. Background correction, normalization, PM correction, summarization, and expression analysis were performed using the R software with the affy/limma/sva packages. Gene set enrichment analysis was performed using GSEA software. All RNA samples passed quality control and were included in the data set. The expression analysis took into account RNA degradation, which was similar in all tumor samples. A total of 184 genes were differentially expressed by >16-fold, between the two groups. Surrogate variable analysis did not result in any statistically significant differences in gene expression. Gene set enrichment analysis resulted in 9 gene sets enriched in axial, and 4 gene sets enriched in appendicular osteosarcoma samples at FDR < 25%. Gene ranking was similar between the two methods of analysis, with MMP1, ATP6AP1, SLC6A8, and dysadherin expression increased in axial tumor samples, while ICAM2 expression increased in appendicular samples. Expression analysis indicates no statistically significant differences in gene expression between the axial and appendicular OSA. Gene set enrichment analysis indicates potential differences in gene set enrichment between the two types of osteosarcoma. Further quantitative RT-PCR analysis is required to confirm our results. These data will guide hypothesis-driven translational studies, which may have direct clinical implications on the treatment of osteosarcoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4310. doi:10.1158/1538-7445.AM2011-4310

Abstract 2661: Evaluation of genetic variability of cytochrome P-450 members in the canine preclinical model

Dogs are used as pharmacokinetic and preclinical models, but no study has yet evaluated polymorphisms in a large dog population. We hypothesize that genetic variation exists in the coding region of the canine cytochrome P-450 homologue genes CYP1A1, CYP1A2, CYP2E1, and CYP2B6. The canine CYP1A1, CYP1A2, CYP2E1, and CYP2B11 (homologue to CYP2B6) genomic sequences were identified using the UCSC Genome Browser. Intronic primers, flanking individual exons were designed using Primer3. Amplification of all exons was carried out by PCR, products were electrophoresed in agarose gel, and visualized using ethidium bromide. Genomic DNA from 20 dogs (10 breeds, 2 dogs/breed) from our lab9s genomic DNA bank was used as template. Direct sequencing of the PCR amplicons was performed and the results were analyzed using the DNASTAR Lasergene 7. PCR restriction fragment length polymorphism (RFLP) assay was designed for each polymorphism identified by sequencing. PCR-RFLP assays were used to evaluate identified polymorphic sites in dogs of 10 different breeds (10 dogs/breed). The potential effect of each polymorphism to the protein function was estimated using PolyPhen and SIFT software. In CYP1A1 a total of 6 SNPs were identified: three SNPs in exon 1, and three SNPs in exon 6. One SNP was a synonymous change (c.1362T>A), while five were nonsynonymous (c.53C>G → p.A18G; c.146G>T → p.W49L; c.345C>G → p.F115L; c.C1305C>A → p.F435L; and c.1561T>C → p.S521P). In CYP1A2, A total of 6 SNPs were identified. Four of these SNPs have been already described (c.1117C>T; c.1173C>G; c.1299C>T; and c.1303G>A), while 2 novel SNPs were identified (c.1165A>G; and c.1451A>G). Of the novel SNPs, one is a synonymous change (1451A>G), and one is nonsynonymous (c.1165A>G → p.N389D). A potential InDel was identified at the 6th exon in 7/13 Siberian huskies initially assayed. Sequencing of the cloned potential InDel - containing exon revealed a 10 base long duplication (1464_1473dupCCCCATCTAT). All 7 dogs harboring the duplication, appeared to be heterozygotes for it, hence we expanded our screening of the duplication to a set of a total of 274 Siberian husky genomic DNA. We identified 75 homozygotes for the wild type, and 199 dogs that appeared heterozygotes for the duplication. When we assayed 28 Alaskan malamutes for the duplication identified in the Siberian huskies, we failed to identify any dog positive for this duplication. In CYP2E1, a total of 2 SNPs in the coding region and 2 intronic SNPs were identified. The 2 exonic SNPs are nonsynonymous (c.85C>T → p.R29C; c.1453T>C → p.Y485H). The two intronic SNPs are located at the 5th (IVS5+5G>A), and the 6th intron, (IVS6+27C>T). In CYP2B11 a total of 3 SNPs were identified. Two SNPs were synonymous (c123G>A; c966G>A) and 1 was nonsynonymous (c220C>T → p.R74C). Genetic variation in the canine CYP1A1, CYP1A2, CYP2E1, and CYP2B11 genes exists, and may have significant implications in the use of dogs as preclinical models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2661.