Association between genome-wide epigenetic and genetic alterations in breast cancer tissue and response to HER2-targeted therapies in HER2-positive breast cancer patients: new findings and a systematic review.

Recent evidence suggests that genetic and epigenetic mechanisms might be associated with acquired resistance to cancer therapies. The aim of this study was to assess the association of genome-wide genetic and epigenetic alterations with the response to anti-HER2 agents in HER2-positive breast cancer patients. PubMed was screened for articles published until March 2021 on observational studies investigating the association of genome-wide genetic and epigenetic alterations, measured in breast cancer tissues or blood, with the response to targeted treatment in HER2-positive breast cancer patients. Sixteen studies were included in the review along with ours, in which we compared the genome-wide DNA methylation pattern in breast tumor tissues of patients who acquired resistance to treatment (case group, n = 6) to that of patients who did not develop resistance (control group, n = 6). Among genes identified as differentially methylated between the breast cancer tissue of cases and controls, one of them, PRKACA, was also reported as differentially expressed in two studies included in the review. Although included studies were heterogeneous in terms of methodology and study population, our review suggests that genes of the PI3K pathway may play an important role in developing resistance to anti-HER2 agents in breast cancer patients. Genome-wide genetic and epigenetic alterations measured in breast cancer tissue or blood might be promising markers of resistance to anti-HER2 agents in HER2-positive breast cancer patients. Further studies are needed to confirm these data.

Health Impacts and Characteristics of Deprescribing Interventions in Older Adults: Protocol for a Systematic Review and Meta-analysis.

BACKGROUND: Deprescribing, a relatively recent concept, has been proposed as a promising solution to the growing issues of polypharmacy and use of medications of questionable benefit among older adults. However, little is known about the health outcomes of deprescribing interventions. OBJECTIVE: This paper presents the protocol of a study that aims to contribute to the knowledge on deprescribing by addressing two specific objectives: (1) describe the impact of deprescribing in adults ≥60 years on health outcomes or quality of life; and (2) determine the characteristics of effective interventions in deprescribing. METHODS: Primary studies targeting three concepts (older adults, deprescribing, and health or quality of life outcomes) will be included in the review. The search will be performed using key international databases (MEDLINE, EMBASE, CINAHL, Ageline, PsycInfo), and a special effort will be made to identify gray literature. Two reviewers will independently screen the articles, extract the information, and evaluate the quality of the selected studies. If methodologically feasible, meta-analyses will be performed for groups of intervention studies reporting on deprescribing interventions for similar medications, used for similar or identical indications, and reporting on similar outcomes (eg, benzodiazepines used against insomnia and studies reporting on quality of sleep or quality of life). Alternatively, the results will be presented in bottom-line statements (objective 1) and a matrix outlining effective interventions (objective 2). RESULTS: The knowledge synthesis may be limited by the availability of high-quality clinical trials on deprescribing and their outcomes in older adults. Additionally, analyses will likely be affected by studies on the deprescribing of different types of molecules within the same indication (eg, different pharmacological classes and medications to treat hypertension) and different measures of health and quality of life outcomes for the same indication. Nevertheless, we expect the review to identify which deprescribing interventions lead to improved health outcomes among seniors and which of their characteristics contribute to these outcomes. CONCLUSIONS: This systematic review will contribute to a better understanding of the health outcomes of deprescribing interventions among seniors. TRIAL REGISTRATION: PROSPERO International Prospective Register of Systematic Reviews CRD42015020866; https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42015020866. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): PRR1-10.2196/25200.

Association of Breast Tumour Expression of Cannabinoid Receptors CBR1 and CBR2 with Prognostic Factors and Survival in Breast Cancer Patients.

Cannabinoid receptors (CBR) are potential therapeutic targets for breast cancer. However, the role of CBR in breast cancer survival remains poorly understood. Data from a prospective cohort of 522 women diagnosed with invasive breast cancer between 2010 and 2012 were analysed. Clinical and pathological features were retrieved from electronic medical records. CBR expression was measured by immunohistochemistry. Adjusted partial Spearman correlations and multivariate Cox models were used to estimate associations with breast cancer prognostic factors and survival, respectively. The median follow-up was 92.0 months (range 7.0-114.0). CBR expression was heterogenous in tumours. Cytoplasmic expression of CBR1 was positively correlated with lymph node invasion (rs = 0.110; p = 0.0155) and positive status of the human epidermal growth factor receptor 2 (HER2) (rs = 0.168; p = 0.0002), while nuclear CBR2 was negatively correlated with grade (rs = -0.171; p = 0.0002) and positively correlated with oestrogen receptor and progesterone receptor-positive status (rs = 0.173; p = 0.0002 and rs = 0.121; p = 0.0084, respectively). High cytoplasmic expression of CBR2 was associated, with 13% higher locoregional and distant recurrences (HR = 1.13 [0.97-1.33]), though this association did not reach statistical significance. Although the few events occurring during follow-up may have limited the detection of significant associations, these results indicate that CBR expression in breast cancer deserves further investigation.

Cannabis against chronic musculoskeletal pain: a scoping review on users and their perceptions.

BACKGROUND: Chronic musculoskeletal pain (CMP) may lead to reduced physical function and is the most common cause of chronic non-cancer pain. Currently, the pharmacotherapeutic options against CMP are limited and frequently consist of pain management with non-steroidal anti-inflammatories, gabapentinoids, or opioids, which carry major adverse effects. Although the effectiveness of medical cannabis (MC) for CMP still lacks solid evidence, several patients suffering from it are exploring this therapeutic option with their physicians. OBJECTIVES: Little is known about patients' perceptions of their MC treatment for CMP. We aimed to increase this knowledge, useful for healthcare professionals and patients considering this treatment, by conducting a scoping literature review, following guidance by Arksey and O'Malley, to describe the views and perceptions of adult patients who had consumed MC to relieve chronic CMP. METHODS: Databases (PUBMED, EMBASE, Web of Science) and websites were searched using combinations of controlled and free vocabulary. All studies and study designs reporting on patients' perceptions regarding MC against CMP were considered. Studies had to include adult patients reporting qualitatively or quantitatively, i.e., through questionnaires, on MC use to treat CMP or other non-cancer pain, since studies reporting exclusively on perceptions regarding CMP were very rare. Study characteristics were extracted and limitations of the study quality were assessed. The review includes patients' demographic characteristics, patterns of MC use, perceived positive and negative effects, use of alcohol or other drugs, reported barriers to CM use, and funding sources of the studies. RESULTS: Participants of the 49 included studies reported that MC use helped them to reduce CMP and other chronic non-cancer pain, with only minor adverse effects, and some reported improved psychological well-being. In the included studies, men represent between 18 and 88% of the subjects. The mean age of participants in these studies (42/49) varied between 28.4 and 62.8 years old. The most common route of administration is inhalation. CONCLUSION: MC users suffering from CMP or other chronic non-cancer pain perceived more benefits than harms. However, the information from these studies has several methodological limitations and results are exploratory. These user-reported experiences must thus be examined by well-designed and methodologically sound clinical or observational studies, particularly regarding CMP, where reports are very scarce.

Discontinuation of bisphosphonates in seniors: a systematic review on health outcomes.

PURPOSE: Bisphosphonates are used to treat osteoporosis. Despite their benefits on bone mineral density (BMD) and fractures, they have shown adverse effects, sometimes severe, during chronic use. Taken for several years, they achieve long-term bone retention, making deprescribing feasible. This review aimed to synthesize evidence on the success and health outcomes of deprescribing of bisphosphonates in seniors, aged over 60 years. METHODS: The review followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, including articles in English, French, or German published before July 2020. Eligible studies included seniors having discontinued bisphosphonates and reported on health outcomes; some allowed meta-analyses on fracture risk. RESULTS: The review included 9 RCTs and 9 cohort studies of moderate quality. Bisphosphonates were discontinued after 2 to 7 years of use, and BMD or fractures were assessed during follow-up of 0.5 to 5 years. A significant reduction in BMD after discontinuation was observed in 9 of 10 studies. Results on fracture risk after discontinuation are mitigated: 6 RCT extensions showed no increase in the risk of any osteoporotic fractures after discontinuation. Meta-analyses including 4 RCTs showed an increased odds ratio of vertebral fractures of 2.04 (95% CI, 1.39-2.99) among discontinuers. Results from 2 large cohort studies showed no increased risks of any osteoporotic or vertebral fractures, while 2 studies found increased fracture risks. CONCLUSION: Bisphosphonates have successfully been discontinued low overall fracture risk after at least 3 years of use, but a risk for decreased BMD and increased vertebral fractures remained.

Trastuzumab effects depend on HER2 phosphorylation in HER2-negative breast cancer cell lines.

The breast cancer (BC) biomarker HER2 (Human Epidermal Receptor 2) is overexpressed in 25% of BC. Only patients with HER2-positive tumors receive HER2-targeting therapies, like trastuzumab (Herceptin). However, some women with a HER2-negative BC could benefit from trastuzumab. This could be explained by the activation/phosphorylation of HER2 that can be recognized by trastuzumab. The aim of this study is to examine trastuzumab effects on HER2 phosphorylation at tyrosine Y877 (pHER2Y877). HER2 and pHER2Y877 status were evaluated in a cohort of BC patients representative of molecular subtypes distribution (n = 497) and in a series of BC cell lines (n = 7). Immunohistochemistry against pHER2Y877 was performed on tissue micro arrays. Cellular proliferation assays were performed on BC cell lines presenting different combinations of HER2 and pHER2Y877 status and treated with increasing doses of trastuzumab (0-150 µg/ml). The prevalence of pHER2Y877 in this cohort was 6%. Nearly 5% of patients with HER2-negative tumors (n = 406, 82%) overexpressed pHER2Y877. Among triple negative BC patients (n = 39, 8%), 7.7% expressed pHER2Y877. Trastuzumab treatment decreased cell proliferation in HER2-/pHER2Y877+ BC cell lines, to an extent comparable to what occurs in HER2+ cell lines, but did not affect HER2-/pHER2Y877- cell lines. Trastuzumab sensitivity in HER2-/pHER2Y877+ cell line is specific to HER2 tyrosine 877 phosphorylation. Hence, with further confirmation in a bigger cohort, trastuzumab treatment could be envisaged as a treatment option to women presenting with HER2-/pHER2+ tumors, representing more than 1000 BC women in Canada in 2019.

Performance of preoperative plasma tumor markers HE4 and CA125 in predicting ovarian cancer mortality in women with epithelial ovarian cancer.

Clinical utility of new biomarkers often requires the identification of their optimal threshold. This external validation study was conducted to assess the performance of the preoperative plasma tumor markers HE4 and CA125 optimal cut-offs to predict cancer mortality in women with epithelial ovarian cancer (EOC). Participating women had upfront debulking surgery in the University Hospital of Quebec City (Canada) between 1998 and 2013. A total of 136 women participated in the training cohort (cohort 1) and 177 in the validation cohort (cohort 2). Preoperative plasma HE4 and CA125 levels were measured by Elecsys. Optimal thresholds were identified in the cohort 1 using time-dependent receiver operating characteristic (ROC) curves. Multivariate Cox models were used to validate the biomarkers using their optimal cut-offs in the cohort 2. The likelihood ratio (LR) test was done to test whether the biomarkers added prognostic information beyond that provided by standard prognostic factors. The Areas Under the Curves (AUC) for HE4 and CA125 were respectively 64.2 (95% CI: 54.7-73.6) and 63.1 (95%CI: 53.6-72.6). The optimal thresholds were 277 pmol/L for HE4 and 282 U/ml for CA125. Preoperative plasma HE4 (≥277 pmol/L) was significantly associated with EOC mortality (adjusted hazard ratio (aHR): 1.90; 95% CI:1.09-3.29). The prognostic effect of HE4 was strongest in the subgroup of women with serous ovarian cancer (aHR: 2.42; 95% CI: 1.25-4.68). Using a multivariate model including all standard prognostic factors, this association was maintained (aHR: 2.21; 95% CI: 1.15-4.23). In addition, preoperative plasma HE4 added prediction for death over the standard prognostic markers in women with serous tumors (p-value for LR-test: 0.01). Preoperative CA125 was not associated with cancer mortality, both in women with EOC and in those with serous tumors. Preoperative HE4 is a promising prognostic biomarker in EOC, especially in serous tumor.

Association of Tobacco Use, Alcohol Consumption and HER2 Polymorphisms With Response to Trastuzumab in HER2-Positive Breast Cancer Patients.

PURPOSE: Although the administration of trastuzumab has improved the survival of human epidermal growth factor receptor 2 (HER2)-positive breast cancer patients, resistance remains a major clinical obstacle. We retrospectively evaluated the association of HER2 polymorphisms, tobacco use and alcohol consumption with disease-free survival (DFS) in HER2-positive breast cancer patients. PATIENTS AND METHODS: Clinicopathologic and survival data (median follow-up, 7.4 years) were collected from medical records for 236 nonmetastatic trastuzumab-treated HER2-positive breast cancer patients. Tobacco and alcohol consumption were assessed using validated questionnaires, and HER2 polymorphisms (Ile655Val and Ala1170Pro) were determined by TaqMan assay. Multivariate Cox proportional hazard models were used to analyze DFS. RESULTS: Compared to nonsmokers, patients who smoked before breast cancer diagnosis showed a worse DFS (hazard ratio [HR], 2.63, P = .001), and this association was stronger among patients who smoked > 20 cigarettes per day or who spent more than 2 decades smoking before their diagnosis (HR, 3.65, P = .01, and HR, 3.19, P = .002, respectively). Smoking during trastuzumab treatment was associated with DFS, but only among patients with estrogen receptor-negative tumors (HR, 4.49, P = .02). Compared to nondrinkers, patients who consumed alcohol before breast cancer diagnosis had a significantly better DFS (HR, 0.56, P = .03). No association was observed between alcohol consumption during trastuzumab treatment and DFS. Concerning HER2 polymorphisms, patients with Ile/Val or Val/Val genotype had a significantly worse DFS than those with the Ile/Ile genotype (HR, 4.96, P = .01). CONCLUSION: Tobacco and alcohol consumption as well as HER2 Ile655Val polymorphism could influence trastuzumab response. These results need to be confirmed in a larger cohort study.

Concordance of HER2 Immunohistochemistry and Fluorescence In Situ Hybridization Using Tissue Microarray in Breast Cancer.

AIM: Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are common methods for assessment of human epidermal growth factor receptor 2 (HER2) in breast cancer. MATERIALS AND METHODS: In a cohort of 498 consecutive patients with breast cancer, we examined concordance between IHC and FISH for HER2 on tissue microarray (TMA) sections. In a subset of 116 specimens, we examined HER2 concordance from the block used for diagnostics and a randomly-chosen additional block (a proxy of the core biopsy). RESULTS: Overall concordance between both methods on TMA sections was 93.8% and between HER2, determined on diagnostic and additional blocks, was 93.6% for IHC and 98.0% for FISH. CONCLUSION: Since some cases were discordant, we suggest that both methods be used for HER2 assessment. The lower concordance rate between diagnostic and additional blocks using IHC compared to FISH suggests a greater variability of IHC staining across tumor regions than for FISH results.

MCM2: An alternative to Ki-67 for measuring breast cancer cell proliferation.

Breast cancer is a heterogeneous disease comprising a diversity of tumor subtypes that manifest themselves in a wide variety of clinical, pathological, and molecular features. One important subset, luminal breast cancers, comprises two clinically distinct subtypes luminal A and B each of them endowed with its own genetic program of differentiation and proliferation. Luminal breast cancers were operationally defined as follows: Luminal A: ER+, PR+, HER2-, Ki-67<14% and Luminal B: ER+ and/or PR+, HER2-,Ki-67≥14% or, alternatively ER+ and/or PR+, HER2+, any Ki-67. There is currently a need for a clinically robust and validated immunohistochemical assay that can help distinguish between luminal A and B breast cancer. MCM2 is a family member of the minichromosome maintenance protein complex whose role in DNA replication and cell proliferation is firmly established. As MCM2 appears to be an attractive alternative to Ki-67, we sought to study the expression of MCM2 and Ki-67 in different histological grades and molecular subtypes of breast cancer focusing primarily on ER-positive tumors. MCM2 and Ki-67 mRNA expression were studied using in silico analysis of available DNA microarray and RNA-sequencing data of human breast cancer. We next used immunohistochemistry to evaluate protein expression of MCM2 and Ki-67 on tissue microarrays of invasive breast carcinoma. We found that MCM2 and Ki-67 are highly expressed in breast tumors of high histological grades, comprising clinically aggressive tumors such as triple-negative, HER2-positive and luminal B subtypes. MCM2 expression was detected at higher levels than that of Ki-67 in normal breast tissues and in breast cancers. The bimodal distribution of MCM2 scores in ER+/HER2- breast tumors led to the identification of two distinct subgroups with different relapse-free survival rates. In conclusion, MCM2 expression can help sorting out two clinically important subsets of luminal breast cancer whose treatment and clinical outcomes are likely to diverge.

Evaluation of human epidermal growth factor receptor 2 (HER2) single nucleotide polymorphisms (SNPs) in normal and breast tumor tissues and their link with breast cancer prognostic factors.

Amplification of the human epidermal growth factor receptor 2 (HER2) gene is associated with worse prognosis and decreased overall survival in breast cancer patients. The HER2 gene contains several polymorphisms; two of the best-characterized HER2 polymorphisms are Ile655Val and Ala1170Pro. The aim of this study was to evaluate the association between these two HER2 polymorphisms in normal breast and breast cancer tissues and known breast cancer prognostic factors in a retrospective cohort study of 73 women with non-metastatic HER2-positive breast cancer. HER2 polymorphisms were assessed in breast cancer tissue and normal breast tissue using TaqMan assay. Ala1170Pro polymorphism in normal breast tissue was associated with age at diagnosis (p = 0.007), tumor size (p = 0.004) and lymphovascular invasion (p = 0.06). Similar significant associations in cancer tissues were observed. No association between the Ile655Val polymorphism and prognostic factors were observed. However, we found significant differences in the distribution of Ile655Val (p = 0.03) and Ala1170Pro (p = 0.01) genotypes between normal breast and breast tumor tissues. This study demonstrates that only the Ala1170Pro polymorphism is associated with prognostic factors in HER2-positive breast cancer patients. Moreover, our results suggest that both HER2 polymorphisms could play a significant role in carcinogenesis in non-metastatic HER2-positive breast cancer women.

Tissue Microarray Is a Reliable Tool for the Evaluation of HER2 Amplification in Breast Cancer.

AIM: We examined an economical method for evaluating the amplification of the human epidermal growth factor receptor 2 (HER2) gene in breast cancer specimens. MATERIALS AND METHODS: We compared HER2 amplification determined by fluorescence in situ hybridization (FISH) on whole-tissue (WT) blocks used for diagnostic and on tissue microarray (TMA) sections for a cohort of 521 consecutive patients with breast cancer. In a subset of 116 patients, we examined HER2 concordance from the WT section and a TMA section from a randomly chosen additional block (a proxy of the core biopsy). RESULTS: Overall concordance for HER2 amplification between WT and TMA sections was 98.2%, and between sections from WT and from the additional block was 99.0%. CONCLUSION: The high concordance rates support the use of TMA for the evaluation of HER2 amplification in breast cancer and suggest that FISH can be used to assess HER2 using core biopsies.

Validation of EP1 Antibody Clone for Estrogen Receptor Immunohistochemistry for Breast Cancer.

BACKGROUND: SP1 Rabbit monoclonal antibody to estrogen receptor (ER) has long been the standard for determination of ER status in breast cancer but has been replaced by the rabbit EP1 clone. AIM: To validate the EP1 antibody clone for use in determination of breast cancer ER status in a large clinical population against the previous standard SP1. MATERIALS AND METHODS: ER immunohistochemistry was assessed in 523 consecutive cases from a clinical setting using tissue microarrays. RESULTS: The kappa statistic showed that the agreement of ER status between SP1 and EP1 was considered to be almost perfect (kappa=0.97, 95% confidence interval=0.94-1.00). Sensitivity was 99.3%, specificity was 98.6% and overall agreement was 99.2%. CONCLUSION: The EP1 antibody was herein validated regarding its use in breast cancer with almost perfect agreement with the previously used standard SP1 antibody.

Advantages and disadvantages of technologies for HER2 testing in breast cancer specimens.

OBJECTIVES: Human epidermal growth factor receptor 2 (HER2) plays a central role as a prognostic and predictive marker in breast cancer specimens. Reliable HER2 evaluation is central to determine the eligibility of patients with breast cancer to targeted anti-HER2 therapies such as trastuzumab and lapatinib. Presently, several methods exist for the determination of HER2 status at different levels (protein, RNA, and DNA level). METHODS: In this review, we discuss the main advantages and disadvantages of the techniques developed so far for the evaluation of HER2 status in breast cancer specimens. RESULTS: Each technique has its own advantages and disadvantages. It is therefore not surprising that no consensus has been reached so far on which technique is the best for the determination of HER2 status. CONCLUSIONS: Currently, emphasis must be put on standardization of procedures, internal and external quality control assessment, and competency evaluation of already existing methods to ensure accurate, reliable, and clinically meaningful test results. Development of new robust and accurate diagnostic assays should also be encouraged. In addition, large clinical trials are warranted to identify the technique that most reliably predicts a positive response to anti-HER2 drugs.

Validation of a new classifier for the automated analysis of the human epidermal growth factor receptor 2 (HER2) gene amplification in breast cancer specimens.

Amplification of the human epidermal growth factor receptor 2 (HER2) is a prognostic marker for poor clinical outcome and a predictive marker for therapeutic response to targeted therapies in breast cancer patients. With the introduction of anti-HER2 therapies, accurate assessment of HER2 status has become essential. Fluorescence in situ hybridization (FISH) is a widely used technique for the determination of HER2 status in breast cancer. However, the manual signal enumeration is time-consuming. Therefore, several companies like MetaSystem have developed automated image analysis software. Some of these signal enumeration software employ the so called "tile-sampling classifier", a programming algorithm through which the software quantifies fluorescent signals in images on the basis of square tiles of fixed dimensions. Considering that the size of tile does not always correspond to the size of a single tumor cell nucleus, some users argue that this analysis method might not completely reflect the biology of cells. For that reason, MetaSystems has developed a new classifier which is able to recognize nuclei within tissue sections in order to determine the HER2 amplification status on nuclei basis. We call this new programming algorithm "nuclei-sampling classifier". In this study, we evaluated the accuracy of the "nuclei-sampling classifier" in determining HER2 gene amplification by FISH in nuclei of breast cancer cells. To this aim, we randomly selected from our cohort 64 breast cancer specimens (32 nonamplified and 32 amplified) and we compared results obtained through manual scoring and through this new classifier. The new classifier automatically recognized individual nuclei. The automated analysis was followed by an optional human correction, during which the user interacted with the software in order to improve the selection of cell nuclei automatically selected. Overall concordance between manual scoring and automated nuclei-sampling analysis was 98.4% (100% for nonamplified cases and 96.9% for amplified cases). However, after human correction, concordance between the two methods was 100%. We conclude that the nuclei-based classifier is a new available tool for automated quantitative HER2 FISH signals analysis in nuclei in breast cancer specimen and it can be used for clinical purposes.

Amylin reduces plasma glucagon concentration in cats.

Pancreatic amylin plays an important role in the control of nutrient fluxes and is an established therapy in human diabetes as it reduces post-prandial glucagon secretion and slows gastric emptying. Given the similar pathophysiology of human type-2 and feline diabetes mellitus, we investigated whether amylin reduces plasma glucagon levels in cats. Healthy cats were tested using an intravenous arginine stimulation test (IVAST), a meal response test with the test meal comprising 50% of average daily food intake, and an IV glucose tolerance test (IVGTT). Non-amyloidogenic rat amylin injected 5 min before the respective stimulus significantly reduced plasma glucagon levels under all test situations. In the IVAST and IVGTT, cats treated with amylin also had lower plasma insulin concentrations. It was concluded that amylin does reduce plasma glucagon levels in cats, a feature that has treatment potential in diabetic animals as co-administration of amylin would reduce the insulin requirement to control glycaemia.

The dipeptidyl peptidase IV inhibitor NVP-DPP728 reduces plasma glucagon concentration in cats.

Glucagon-like peptide-1 (GLP-1) analogues and inhibitors of its degrading enzyme, dipeptidyl peptidase IV (DPPIV), are interesting therapy options in human diabetics because they increase insulin secretion and reduce postprandial glucagon secretion. Given the similar pathophysiology of human type 2 and feline diabetes mellitus, this study investigated whether the DPPIV inhibitor NVP-DPP728 reduces plasma glucagon levels in cats. Intravenous glucose tolerance tests (ivGTT; 0.5 g/kg glucose after 12 h fasting) and a meal response test (test meal of 50% of average daily food intake, offered after 24 h fasting) were performed in healthy experimental cats. NVP-DPP728 (0.5-2.5 mg/kg i.v. or s.c.) significantly reduced glucagon output in all tests and increased insulin output in the ivGTT. Follow-up studies will investigate the potential usefulness as therapy in diabetic cats.