Dogs, Cancer and Mathematics An SME Perspective on University Collaboration.

1. Dogs, Cancer and MathematicsAn SME Perspective on University Collaboration. Kevin Slater Ilias Alexandrakis, Renu Tuli Alexander Gorban, EvgenyMirkes

2. Why Dogs? Why Lymphoma? USA dog population = 78 Million UK dog population = 8-10 Million Canine Lymphoma - Incidence • 20% of all canine tumours are lymphoma cases • 0.1% of older dogs will develop lymphoma • Very high incidence in some breeds, e.g. Golden Retrievers 25% in USA Canine Lymphoma - Symptoms • Lymphadenopathy • Lethargy • Weakness • Fever • Anorexia • Pu/Pd

3. Canine Lymphoma – Treatment • Predominantly treated with chemotherapy • Diverse range of treatment protocols • Initially responds well to treatment • Canine Lymphoma – Prognosis • B-cell lymphoma favorable to T-cell lymphoma • Clinical stage (Stage V has poorer prognosis against Stage I) • Dogs treated with chemotherapy experience a greater survival time • Recurrence almost inevitable

4. Types of Lymphoma • Multicentric lymphoma - usually shows up first as painless but enlarged peripheral lymph nodes. Owners may see or feel these in areas under the jaw, in the armpits, in the groin area or behind the knees. Enlargement of the liver and/or spleen can also occur, causing the abdomen to distend. This is the most common form of lymphoid cancer in dogs. • Gastrointestinal (alimentary) lymphoma - is a malignant form of cancer that can show up anywhere along the gastrointestinal tract (stomach, small intestine, large intestine, rectum). Clinical signs of gastrointestinal lymphoma include vomiting, diarrhoea, weight loss, lethargy, depression, diarrhoea and melena. Low serum albumin levels and elevated blood calcium levels commonly accompany alimentary lymphoma, although these can only be detected by veterinary evaluation of blood samples. This is the second most frequent form of lymphoma in dogs. • Mediastinal lymphoma - where the cancer is localized to tissues in the chest cavity - can cause fluid to build up around the lungs. This can lead to coughing and laboured breathing (dyspnoea), mimicking the signs of congestive heart failure. • Lymphoma of the skin (cutaneous lymphoma) - is uncommon in dogs. When it does occur, it usually shows up with hair loss (alopecia) and visible bumps on the skin. It can also be itchy (pruritic) and vary widely in appearance, ranging from a single lump to large areas of bruised, ulcerated and/or hairless skin. • Lymphoma of the central nervous system (CNS) - is very uncommon in dogs. When lymphoma is localized in the CNS, dogs typically present with neurological signs such as circling, seizures, behaviour changes and incoordination

5. Canine Lymphoma Diagnosis • Cytology • Histology • Immunophenotyping (T or B cell) • Generally invasive procedures • FNA prone to non diagnostic samples • Not suitable to treatment monitoring

6. Serum Biomarkers • Serum easily accessible • Potential for picking up circulating biomarkers • Diverse array of cancers whereby potential biomarkers identified • Prostate cancer • Breast cancer • Melanoma • Developed a serum biomarker approach to assist with detection of canine lymphoma

7. Summary of Biomarker Identification Studies • Protein sequence analysis identified 3 different biomarkers • Limited information in the literature about the function of 2 biomarkers and their involvement in lymphoma • No antibodies available to the unique biomarkers, therefore had to work with human antibodies with poor cross reactivity to the canine proteins. • Third biomarker identified as Haptoglobin, known to be upregulatedin canine lymphoma.

8. Acute Phase Protein Response in Dogs Infection Inflammation White Blood Cells IL-1 TNF-α IL-6 C-RP Haptoglobin SAA AGP

9. APP in Malignant Lymphoma Sig Diff from control P <0.0001 P <0.0001;<0.0001; <0.001; <0.02 Haptoglobin C-reactive protein lymphoma (n=16), acute lymphoblastic leukaemia (ALL) (n=11), chronic lymphocytic leukaemia (CLL) (n=7) and multiple myeloma (n=9) Control (n=25) Mischkeet al Vet J 2006 174:188-92

10. Multi Vs Single Biomarker tests in Human Testing

11. From MS to ELISA Development of a multi marker test using Acute Phase Proteins (Haptoglobin, CRP) Use of Biomarker Pattern Software to create unique algorithms More than 19 protein peaks identified as significantly different on MS Further investigation in order to characterise and identify the proteins Identification of Haptoglobin A unique new method of quickly and accurately diagnosing canine lymphoma . The combination of two Acute Phase Protein Assays, Haptoglobin and a specific canine CRP, combined with a unique Diagnostic Algorithm provide a diagnostic system

12. cLBT Assay Development • Serum samples collected from dogs with lymphoma, healthy dogs and dogs with other diseases (many with similar presentation to lymphoma). Positive samples were confirmed by either FNA or excisional biopsy. Non lymphoma dogs were confirmed to be free of the disease at a minimum of six months after providing the serum sample • Samples were tested in batches for HAPT & CRP levels • Ciphergen Biomarker Pattern Software was used to generate a series of algorithms using the Classification and Regression Tree (CART) procedure. Through an iterative process, the software uses the training set of data to build trees to a point when optimal differentiation between the populations is achieved. • Blinded sample test performed.

13. Classification and Regression Tree Breiman L, Friedman JH, Olshen RA, Stone CJ. Classification and Regression Trees. Chapman & Hall (Wadsworth, Inc.): New York, 1984.

14. Database Two cohorts Developments with The University of Leicester Healthy – 71 Lymphoma – 97, Other disease – 135 Healthy Clinically suspected Problems Differential diagnosis Screening Challenge: The Estimation of Lymphoma Risk

15. Methodologies Risk maps • K nearest neighbours • Classic kNN with k from 1 to 30 • kNN with Fisher’s distance transformations • kNN with adaptive distance transformations • Decision tree • Information gain (C4.5) • Gini gain (CART) • DKM • Probability density function estimation • Radial-basis function (statistics kernel) • Three random values (Lymphoma, Other diseases, Healthy) x-axis CRP, y-axis Hapt

16. Software tools • Database maintenance • Add new data • Delete old data Microsoft Excel Selection of the best methods for each problem and input data set. Best solutions are exported to the applet Canine lymphoma software Providing access for practitioner vets to the diagnosis applet

17. http://arxiv.org/abs/1305.4942

18. Clinical Utility of Avacta Canine Lymphoma Blood Test • Initial test for differentiation between benign and malignant lymphadenopathy • Add-on test for the routine diagnosis of lymphoma • Remission monitoring • Screening population • Reflex testing as a follow on test for hypercalcaemia – Antech perform 10,000 biochemistry tests per day, of which 570 are hypercalcaemic.

19. Add new diagnostic data

20. Case Studies

21. Retrospective Study of 196 canine serum samples blind samples from Netherlands and United Kingdom • Serum samples were stored at -20oC Monitoring Canine Lymphoma

22. Materials-Methods • Canine population: 57 (33 Female, 24 Male) • Period of sample collection: 2008-2012 • Age Range: 2.5 – 14 years, Median: 8 years

23. Computational Diagnosis in Monitoring Canine Lymphoma

26. 0-19 20-39 40-59 60-79 80-100

30. Vet Diagnosis

31. CLBT

32. N=24

33. Life Expectancy predicted by CLBT • Patients that gained remission based on Physical Examination • Survival Rate Median: 34.5weeks (avg 44.6) • Patients that never obtained remission based on Physical Examination • Survival Rate Median: 4 weeks (avg 13.8) • ttest 0.011048 • Patients that gained remission based on CLBT • Survival Rate Median: 59 weeks (avg 56.3) • Patients that never obtained remission based on CLBT • Survival Rate Median: 10 weeks (avg 17.8) • ttest 0.001485 CLBT is 7.4 times more accurate in predicting remission status based on survival rate

34. Prognostic Value Group <80% Mean survival 55.1 weeks Group >80% Mean survival 28.53 weeks

35. Conclusions • Both Physical Examination (vet Diagnosis) and CLBT correlate extremely well • CLBT is more accurate compared to Vet Diagnosis • CLBT can detect a lymphoma relapse up to 5 weeks in advance before any clinical signs are shown • In Remission patients that gave a low risk of lymphoma lived longer • CLBT may be useful as a prognostic tool before treatment • A tool to support vets with less experience in chemotherapy treatment and referral practices

36. Next Steps/ Applications • Should a vet continue treatment up to the point that the CLBT returns a very low risk? • Does this study support a framework for monitoring other animals’ treatment with other diseases?

37. Comparison of CRP and Hapt levels in confirmed lymphoma patients when either treated with steroids or free of the drug n = 17 dogs n = 21 dogs

38. Advantages • A simple blood test • Can be used as part of blood work up at initial appointment • Complete test takes approximately one hour • Results available same day • Similar cost to FNA but less invasive • Early diagnostic • No anaesthetic required