Prospective evaluation of toceranib phosphate in metastatic canine osteosarcoma.

Efficacious therapies for measurable metastatic canine osteosarcoma (OSA) are generally lacking. Preliminary retrospective studies suggested that approximately 50% of dogs with measurable metastatic OSA experienced clinical benefit (objective response or clinically meaningful disease stabilisation) following toceranib (TOC) treatment. The purpose of this clinical trial was to prospectively evaluate the clinical outcome following TOC treatment in dogs with measurable pulmonary metastatic OSA. A secondary goal was to identify potential biomarkers of clinical benefit by measuring changes in plasma vascular endothelial growth factor (VEGF) and circulating regulatory T-cell (Treg) percentage. Twenty-two dogs with pulmonary metastasis from appendicular OSA having undergone previous amputation were treated prospectively with TOC. Adverse events (AEs) were common but predominantly low grade. Nine patients were withdrawn from the study prior to the week 8 assessment of response either due to progressive disease (PD), decreased quality of life or owner perceived unacceptable AEs. Of the patients evaluable for disease progression at week 8 (or earlier), 3/17 (17.6 %) had stable disease with the remainder having PD. The median progression-free survival time for all patients was 57 days (range 7-176 days) with a median overall survival time of 89 days (range 7-574 days). Plasma VEGF concentrations were significantly elevated in patients after 4 weeks of TOC treatment, but no changes were observed in percentage of Treg in peripheral blood. Overall, the results of this clinical trial do not support the use of TOC as single agent therapy for canine metastatic OSA.

Phase I lead-in and subsequent randomized trial assessing safety and modulation of regulatory T cell numbers following a maximally tolerated dose doxorubicin and metronomic dose cyclophosphamide combination chemotherapy protocol in tumour-bearing dogs.

Maximally tolerated dose (MTD) and metronomic dose chemotherapeutic approaches alter the immune system and the angiogenic process in different yet potentially complementary ways. A combination of MTD doxorubicin (MTD-DOX) and metronomic cyclophosphamide (mCTX) protocol was evaluated for safety and effect on circulating regulatory T (Treg) cells. We found that mCTX can be safely administered with MTD-DOX in tumour-bearing dogs. Both combination DOX/mCTX and single-agent DOX resulted in significant depletions of circulating lymphocytes throughout the chemotherapy cycle without apparent selectivity for Tregs. The indiscriminant lymphocyte depletions were similar between dogs randomized to receive DOX and dogs randomized to receive DOX/mCTX, suggesting this effect is because of DOX alone. These findings may have implications as to the therapeutic benefit (or lack thereof) of concurrent combination MTD and metronomic protocols. Future investigations are required to determine the effects and indeed the efficacy of concurrent versus sequential applications of MTD and metronomic chemotherapy protocols.

Safety evaluation of combination doxorubicin and toceranib phosphate (Palladia®) in tumour bearing dogs: a phase I dose-finding study.

Combination chemotherapy holds promise for improving outcomes in malignancy when compared with single-agent approaches. Care must be taken to avoid overlapping toxicity and to utilize agents with differing mechanisms of action. A phase I dose-finding trial was performed to determine the maximally tolerated dose (MTD) of a concurrent toceranib and doxorubicin (DOX) combination protocol where toceranib dose was maintained at or near 2.75 mg kg-1 by mouth every other day (PO EOD) while escalating DOX dosage. The dose-limiting toxicity was found to be neutropenia and the MTD of the combination was determined to be 25 mg m-2 of DOX q 21 days given concurrently with toceranib 2.75 mg kg-1 PO EOD. This combination was well tolerated with no excessive gastrointestinal toxicity nor novel adverse events (AEs) noted. Anti-tumour activity was observed in the majority of cases. This combination warrants further investigation in the context of phase II/III clinical trials to characterize efficacy and long-term AE profiles.

Clinical outcome in 20 cases of lingual hemangiosarcoma in dogs: 1996-2011.

With the exception of solar-induced dermal hemangiosarcoma (HSA), the biologic behaviour of canine HSA is characterised by rapid tumour growth, a high metastatic rate and short survival times. Outcome of dogs with HSA of the tongue has not been previously reported. The purpose of this study was to assess outcome and prognostic factors in dogs with lingual HSA. Clinical data was collected retrospectively and histopathology was reviewed for 20 dogs. Median progression free survival was 524 days and the median overall survival time was 553 days. All dogs had low or intermediate grade tumours; most tumours were small and located on the ventral surface of the tongue. Prognostic factors significantly associated with increased survival included small tumour size and absence of clinical signs of an oral mass at the time of diagnosis. Dogs with HSA confined to the tongue may have a better prognosis compared with HSA in other organs.

Intensity-modulated and image-guided radiation therapy for treatment of genitourinary carcinomas in dogs.

BACKGROUND: External beam radiation therapy can be used to treat pelvic tumors in dogs, but its utility is limited by lack of efficacy data and associated late complications. HYPOTHESIS/OBJECTIVES: The objective of this study was to assess local tumor control, overall survival, and toxicosis after intensity-modulated and image-guided radiation therapy (IM/IGRT) for treatment of genitourinary carcinomas (CGUC) in dogs. ANIMALS: 21 client-owned dogs. METHODS: A retrospective study was performed. Medical records of dogs for which there was intent to treat with a course of definitive-intent IM/IGRT for CGUC between 2008 and 2011 were reviewed. Descriptive and actuarial statistics comprised the data analysis. RESULTS: Primary tumors were located in the prostate (10), urinary bladder (9), or urethra (2). The total radiation dose ranged from 54-58 Gy, delivered in 20 daily fractions. Grade 1 and 2 acute gastrointestinal toxicoses developed in 33 and 5% of dogs, respectively. Grade 1 and 2 acute genitourinary and grade 1 acute integumentary toxicoses were documented in 5, 5, and 20% of dogs, respectively. Four dogs experienced late grade 3 gastrointestinal or genitourinary toxicosis. The subjective response rate was 60%. The median event-free survival was 317 days; the overall median survival time was 654 days. Neither local tumor control nor overall survival was statistically dependent upon location of the primary tumor. CONCLUSIONS AND CLINICAL IMPORTANCE: IM/IGRT is generally well-tolerated and provides an effective option for locoregional control of CGUC. As compared with previous reports in the veterinary literature, inclusion of IM/IGRT in multimodal treatment protocols for CGUC can result in superior survival times; controlled prospective evaluation is warranted.

Induction of remission results in spontaneous enhancement of anti-tumor cytotoxic T-lymphocyte activity in dogs with B cell lymphoma.

Characterization of the tumor microenvironment, particularly the immune cells that infiltrate tumors, provides important predictive and prognostic information in humans with lymphoma and other types of cancer. Tumor associated T lymphocytes have not been previously described in dogs with lymphoma. Therefore, we investigated the phenotype and function of T cells in the lymph nodes of dogs with B cell Non-Hodgkin's lymphoma (NHL), as well as the function of T cells in circulation of these dogs. We found that CD4+ and CD8+ T lymphocytes were few in number and minimally responsive to mitogenic stimuli compared to T cells in lymph nodes of normal dogs. Additionally, regulatory T cells (Treg) were significantly increased in tumor tissues compared to lymph nodes of healthy dogs. To better understand cell mediated antitumor immune responses we developed a non-radioactive assay to measure cytotoxic T lymphocyte (CTL) mediated killing of autologous tumor cells. Using this assay, we found that spontaneous CTL activity in the blood of dogs with lymphoma improved significantly following induction of tumor remission using doxorubicin. Coincident with the improvement in CTL activity, circulating Treg numbers were significantly decreased compared to pretreatment levels. We conclude from these studies that CTL activity in dogs with lymphoma can be significantly improved following induction of tumor remission using chemotherapy, as assessed using a new non-radioactive CTL assay.

Clinical and immunomodulatory effects of toceranib combined with low-dose cyclophosphamide in dogs with cancer.

BACKGROUND: Tyrosine kinase inhibitors (TKIs) and metronomic dosing of cyclophosphamide (CYC) can improve tumor control by suppression of regulatory T cells (Treg) and restoration of T cell-mediated immune responses in mice and humans. The immunomodulatory effects of the TKI toceranib, as a single agent or in combination with metronomic CYC, have not been previously investigated in dogs. HYPOTHESIS: The primary objectives of this study were to determine the effects of toceranib and metronomic CYC treatment on lymphocyte subsets including Treg and on interferon-gamma (IFN-γ) secretion in dogs with cancer. We hypothesized that toceranib would selectively decrease Treg numbers and increase IFN-γ production and that addition of CYC would further enhance these effects. ANIMALS: Fifteen client-owned dogs with advanced tumors were entered into a prospective clinical trial. METHODS: Dogs received toceranib at 2.75 mg/kg once every other day. After 2 weeks, oral CYC was added at 15 mg/m(2) daily. Numbers of Treg and lymphocyte subsets were measured in blood by flow cytometry during the 8-week study period. Serum concentrations of IFN-γ were measured by ELISA. RESULTS: Administration of toceranib significantly decreased the number and percentage of Treg in the peripheral blood of dogs with cancer. Dogs receiving toceranib and CYC demonstrated a significant increase in serum concentrations of IFN-γ, which was inversely correlated with Treg numbers after 6 weeks of combination treatment. CONCLUSIONS: In addition to antitumor effects, these data support further investigations into the immunomodulatory effects of toceranib, administered alone or in combination with CYC in dogs with cancer.

Low-dose cyclophosphamide selectively decreases regulatory T cells and inhibits angiogenesis in dogs with soft tissue sarcoma.

BACKGROUND: Low-dose, continuous (metronomic) chemotherapy improves tumor control by inhibiting tumor angiogenesis and suppressing regulatory T cells (Treg) in mice and humans. The effects of metronomic chemotherapy on Treg and tumor angiogenesis in dogs has not been investigated previously. OBJECTIVE: To determine whether metronomic cyclophosphamide (CYC) therapy decreases Treg or exhibits antiangiogenic activity or both in dogs with soft tissue sarcoma (STS). We hypothesized that Treg numbers would be increased in dogs with STS and that continuous dosing of CYC would decrease Treg in a dose-dependent manner, as well as exhibit antiangiogenic activity. ANIMALS: Eleven client-owned dogs with grade I or II STS. Twenty-one healthy dogs were used as controls. METHODS: Prospective, open, clinical trial. Dogs with STS were enrolled in 2 dose cohorts and administered CYC at 12.5 or 15 mg/m(2) p.o. once daily for 28 days. Whole blood and tumor biopsy specimens were obtained on days 0, 14, and 28 to assess changes in T lymphocyte subsets by flow cytometry and tumor microvessel density (MVD), respectively. RESULTS: Administration of CYC at 12.5 mg/m(2)/d significantly decreased the number of Treg from days 0 to 28, but there was no change in the percentage of Treg or tumor MVD. In dogs that received CYC at 15.0 mg/m(2)/d, both the number and percent of Treg as well as tumor MVD were significantly decreased over 28 days. CONCLUSIONS: CYC administered at 15 mg/m(2)/d should be used in further studies examining the antitumor properties of low-dose CYC in dogs.

Decreased ratio of CD8+ T cells to regulatory T cells associated with decreased survival in dogs with osteosarcoma.

BACKGROUND: Increased numbers of regulatory T cells (Treg) and decreased ratios of CD8+ T cells to Treg have been shown to correlate with decreased survival times (ST) in humans with certain malignancies. A possible connection between Treg and ST in dogs with cancer has not been investigated previously. HYPOTHESIS: The purpose of this study was to compare numbers of Treg and T lymphocyte subsets in dogs with osteosarcoma (OSA) to those of healthy dogs and to determine whether pretreatment values were associated with disease-free interval or with ST. We hypothesized that Treg numbers would be increased in dogs with cancer and that dogs with a high percentage of Treg would have a poorer prognosis. ANIMALS: Twelve client-owned dogs with appendicular OSA were entered into a prospective clinical trial. Twenty-two healthy dogs were used as controls. METHODS: The percentages and numbers of Treg and CD4+ and CD8+ T cells in blood, lymph nodes, and tumors were determined with flow cytometry and compared between dogs with OSA and control dogs. RESULTS: Dogs with OSA had significantly fewer circulating CD8+ T cells and significantly more Treg compared with healthy dogs. The CD8/Treg ratio also was significantly lower in dogs with OSA compared with control dogs. In dogs with OSA, a decreased CD8/Treg ratio was associated with significantly shorter STs. CONCLUSIONS: These data support a role for Treg in the immune control of canine OSA and suggest that determination of the CD8/Treg ratio may be useful for assessing outcomes.

Use of FoxP3 expression to identify regulatory T cells in healthy dogs and dogs with cancer.

Regulatory T cells (Treg) are a distinct group of T lymphocytes with immunosuppressive properties that serve normally to prevent harmful autoimmune responses. However, Tregs can also interfere with beneficial immune responses such as anti-tumor and anti-viral immunity in humans and rodents. Given the overall importance of Tregs, it is likely that they play an important role in diseases of dogs as well. However, at present reagents required for identification of Tregs in dogs are not available. Therefore, we investigated whether expression of FoxP3, a transcription factor that is highly expressed in Tregs in humans and rodents could also be used to identify Tregs in dogs. We found that a cross-reactive FoxP3 antibody identified a subset of CD4(+) T cells in blood and lymph nodes of dogs. By flow cytometry the mean percentage of FoxP3(+)CD4(+) T cells in normal dogs was 4.3% in blood and 9.8% in the lymph nodes. In dogs with cancer, there was a significant increase in numbers of Treg in blood (7.5%) and tumor-draining lymph nodes (17.1%) compared to age-matched healthy control dogs. We also found that FoxP3(+)CD4(+) T cells in dogs could be significantly expanded in vitro by TCR activation together with addition of TGF-beta and IL-2. Treated cells also significantly increased expression of TGF-beta and IL-10mRNA. We conclude from these studies that a cross-reactive FoxP3 antibody can be used to identify Tregs in dogs and that this reagent may serve as a useful tool for investigating the role of Treg in a variety of diseases of dogs.

Telomerase activity as a marker for malignancy in feline tissues.

OBJECTIVE: To establish the diagnostic significance of the telomeric repeat amplification protocol (TRAP) assay in detecting feline malignancies. SAMPLE POPULATION: Solid tissue specimens collected from 33 client-owned cats undergoing diagnostic or therapeutic procedures at the University of Illinois Veterinary Medical Teaching Hospital between July 1997 and September 1999 and an additional 20 tissue samples were collected from 3 clinically normal control cats euthanatized at the conclusion of an unrelated study. PROCEDURE: The TRAP assay was used for detection of telomerase activity. Each result was compared to its respective histopathologic diagnosis. RESULTS: Twenty-nine of 31 malignant and 1 of 22 benign or normal tissue samples had telomerase activity, indicating 94% sensitivity and 95% specificity of the TRAP assay in our laboratory. CONCLUSIONS AND CLINICAL RELEVANCE: The diagnostic significance of telomerase activity has been demonstrated in humans and recently in dogs by our laboratory. We tested feline samples to determine whether similar patterns of telomerase activity exist. On the basis of our results, the TRAP assay may be clinically useful in providing a rapid diagnosis of malignancy in cats. The telomerase enzyme may also serve as a therapeutic target in feline tumors.

Evaluation of an assay for detecting telomerase activity in neoplastic tissues of dogs.

OBJECTIVE: To determine feasibility of using the telomere repeat amplification protocol (TRAP) assay to detect telomerase activity in tumors of dogs. SAMPLE POPULATION: Samples of tumor or normal tissue were obtained from client-owned dogs that underwent surgical biopsy during the period of January 1996 through December 1997. PROCEDURE: The TRAP assay was used to detect telomerase activity in malignant or benign tumors of dogs. Telomerase status (positive or negative) was compared with results of histologic examination for each sample to estimate specificity and sensitivity of this assay for the diagnosis of malignancy. RESULTS: Of 26 malignant tumors, 24 were telomerase positive on TRAP assay, whereas 3 of 4 benign tumors and 3 of 3 normal tissues were telomerase negative. Analysis of these results indicated an estimated sensitivity of 92% and specificity of 86% for tumor analysis, using the TRAP assay. CONCLUSION: The TRAP assay can be used to measure telomerase activity in malignant tumors of dogs. CLINICAL RELEVANCE: Because telomerase activation may be required for indefinite longevity of cells, it may also serve as a tumor marker and therapeutic target. The TRAP assay can be used to detect telomerase in samples of fluid as well as tissues obtained from solid tumors. Therefore, it may have considerable clinical value in rapid and noninvasive diagnosis of neoplasia in dogs. Additional studies must be completed to more accurately determine sensitivity and specificity of the assay.

Long-term follow-up of transsphenoidal selective adenomectomy for prolactinoma.

Although transsphenoidal selective resection of prolactinoma has high cure rates and low morbidity, the frequency of late recurrence of adenoma is controversial. Long-term follow-up data were available on 29 of our patients having initial return to normal prolactin levels after microadenoma resection; in 24, prolactin levels remained normal at 50 +/- 3 months' (range, 11 to 81 months) follow-up. Five patients manifested hyperprolactinemia again after intervals of six to 16 months. In five patients with normal prolactin levels immediately after macroadenoma resection, who were available for long-term follow-up, prolactin levels remained normal in four at 41 +/- 3 months; one patient had a relapse 78 months after surgery. Testing of prolactin secretory dynamics six weeks postoperatively was not predictive of who would suffer relapse. It is not known whether these relapses are due to regrowth of tumor remnants or represent new tumor formation.

Surgical cure of prolactinoma reverses abnormal prolactin repsonse to carbidopa/L-dopa.

To determine whether the abnormalities in dopaminergic regulation of PRL secretion in patients with prolactinomas persist after resection of the adenoma, we evaluated PRL inhibitory responses to L-dopa alone and L-dopa given after pretreatment with the dopa decarboxylase inhibitor carbidopa before and after transsphenoidal selective resection of prolactinomas in 23 women. Eighteen women were cured by surgery (normal PRL, menses, no galactorrhea), while 5 women were not cured. Preoperatively, the PRL inhibitory responses to L-dopa cured, 4 .3 +/- 3.8%; uncured, 50.1 +/- 5.5% of baseline) was blunted by pretreatment with the decarboxylase inhibitor carbidopa (cured, 79.1 +/- 4.1%; uncured, 76.8 +/- 9.2%). Postoperatively, this blunting disappeared in the cured patients (L-dopa, 49.1 +/- 3.5%; carbidopa/L-dopa, 56.3 +/- 5.1%), but the blunting persisted in the uncured patients (L-dopa, 49.3 +/- 7.9%; carbidopa/L-dopa, 69.3 +/- 4.2%). The return to normal of the carbidopa/L-dopa test in cured prolactinoma patients after surgery is evidence that in these individuals, preoperative abnormalities of secretion are due to either intrinsic abnormalities of the tumor or alteration of hypothalamic function secondary to tumor secretion. In those patients not cured by surgery, dynamic tests of function remain abnormal, findings attributable to either incomplete tumor resection or the presence, in some patients, of underlying hypothalamic dysregulation.

Bromocriptine therapy in acromegaly: use in patients resistant to conventional therapy and effect on serum levels of somatomedin C.

Seven patients with clinically active acromegaly who had not responded completely to previous surgical or radiation therapy were treated with bromocriptine. Bromocriptine was well tolerated; only one of the seven patients discontinued treatment secondary to side effects. Six of the seven patients improved during bromocriptine therapy, although GH levels were normalized in only two patients. All patients had elevated levels of somatomedin C (Sm-C) before therapy even when basal levels of GH were less than 10 ng/ml. One patient normalized both GH and Sm-C during bromocriptine therapy and had an excellent clinical response. Five patients had moderately good clinical responses; four of these patients had substantial falls in GH levels, but Sm-C levels fell minimally if at all in four and actually increased in one patient. In one patient, there was no change in clinical status, GH levels, or Sm-C levels. Thus, the clinical response did not correlate well with changes in Sm-C in most patients. The patterns of response to provocative stimuli of GH secretion in acromegaly were maintained during bromocriptine therapy, as has been previously been reported. Based on our experience, bromocriptine appears to be a useful adjunct in the therapy of acromegaly, even in patients who have had prior ablative therapy.

Galactorrhea, oligo/amenorrhea, and hyperprolactinemia in patients with craniopharyngiomas.

We report three patients with craniopharyngiomas who had galactorrhea, oligo/amenorrhea, and abnormal sellar tomograms, clinically suggesting the presence of a prolactinoma. One patient had an intrasellar craniopharyngioma (Rathke's cleft cyst) diagosed during surgical exploration of the pituitary fossa for removal of a suspected prolactinoma, and two had suprasellar caraniopharyngiomas whose presence was suspected on the basis of computed tomography. This finding emphasizes the importance of computed tomography in the evaluation of patients with the clinical presentation of a prolactinoma. In two patients, PRL levels were elevated before surgery and remained elevated after removal of the craniopharyngioma. In the third case, an initially normal serum PRL level became elevated after removal of the tumor.

Selective transsphenoidal adenomectomy in women with galactorrhea-amenorrhea.

Thirty women with prolactin (PRL)-secreting adenomas underwent selective adenomectomy via a transsphenoidal route. All had abnormal sella polytomes or visual fields, amenorrhea with low basal serum gonadotropin levels despite decreased serum estradiol concentrations, and elevated basal serum PRL levels with blunted PRL response to neuroendocrine stimulation tests )thyrotropin-releasing hormone, levodopa, chlorpromazine, and hypoglycemia). Of 17 patients with microadenomas, 14 (82.4%) were cured and three (17.6%) improved. None were unchanged or worse. Three (60%) of five patients with larger, but still intrasellar tumors, had normalization of PRL levels, return of menses, and resolution of galactorrhea. The patients with tumors extending out of the sella did not fare as well. Overall, 21 (70%) were cured, six (20%) improved, two (6.7%) were unchanged, and the condition of one (3.3%) became worse. All preoperative neurologic deficits resolved. Postoperative complications were minimal with no neurologic morbidity. When tumors are small, surgical results are excellent with minimal risk.

The value of computed tomography in evaluating patients with prolactinomas.

The use of computed tomography (CT) in 28 patients with surgically proved prolactinomas is described. The authors' experience suggests that in evaluating patients with galactorrhea-amenorrhea syndrome, CT in combination with pluridirectional tomography usually provides sufficient information to make further invasive studies unnecessary. In selected cases with low-density areas within the sella as seen on CT, pneumoencephalography is still advisable to distinguish between the empty sella and the necrotic tumor. Angiography is still necessary to exclude vascular causes for suprasellar and parasellar masses such as aneurysms and meningiomas.