Innogenics —Genomic Profiling for Pets: What information can it provide

Innogenics is a specialty genomics reference laboratory that helps veterinarians and owners of pets with cancer by providing state-of-the-art molecular diagnostic analysis of tumor tissue at costs affordable to the pet owner. We provide a molecular profile of a dog’s tumor that details the cancer type and subtype and provides biomarker information useful in guiding decisions about current and novel therapeutic options. 

At its core, cancer is a genetic disease. Unregulated growth, inappropriate growth signals, blunted or absent response to “death” signals—all of these characteristics are regulated by genes.  For this reason, the promise of genomics in oncology is huge. The expectation of improved diagnostics, prognostics and therapeutics is shared by patients and oncologists alike.  

Gene expression analysis is widely used in human oncology.

In the diagnostic phase of oncology, we certainly understand that an accurate diagnosis is necessary in order to prescribe the optimal therapy. A fantastic paper published in the Proceedings of the National Academy of Sciences (http://www.pnas.org/content/98/26/15149.short) reported that gene profiling was incredibly accurate in determining the type of cancer spanning 14 of the most common cancers. In addition, gene expression analysis showed that undifferentiated cancers based upon histology had gene expression patterns unlike their tissue of origin. The dramatically different gene expression accurately predicts the aggressive, abnormal biologic behavior of these tumors, similar to what most oncologists observe in the clinic.

Gene expression patterns can also aid in the prognostic phase of oncology. Accurate prognostication is important, as it allows the clinician to identify patients who will likely do well or those who will do poorly. In a highly cited paper in Nature (http://www.nature.com/nature/journal/v415/n6871/full/415530a.html) the authors evaluate the gene expression pattern of 117 patients and they were able to conclude that “This gene expression profile will outperform all currently used clinical parameters in predicting disease outcome. Our findings provide a strategy to select patients who would benefit from adjuvant therapy.” The number of genes that need to be evaluated does not have to be large. In one paper (http://www.sciencedirect.com/science/article/pii/S1535610804001412) the authors prove that the ratio of only TWO genes was prognostic in breast cancer patients. As a clinician, the question of how will MY pet do is one I hear every day. Wouldn’t it be wonderful to be able to accurately answer these pet owners?

When it comes to the cancer that is most treated in the veterinary world, lymphoma, can gene expression analysis help? The answer is a resounding yes. Diffuse large B-cell lymphoma (DLBCL) is the most common type of lymphoma in dogs and is very similar to DLBCL in humans. Gene expression analysis has proven useful in this disease for BOTH dogs and people (http://cancerres.aacrjournals.org/content/73/16/5029.abstract and http://www.nature.com/nature/journal/v403/n6769/full/403503a0.html). By routinely performing gene expression analysis on the lymphomas that we diagnose in dogs, it is possible, indeed expected, that therapy will be more effective. And at the end of the day, isn’t more effective therapy what we all want?

Gerald Post, DVM, MEM, DACVIM (Oncology)

The Veterinary Cancer Center

Canine Osteosarcoma - Jennifer McDaniel DVM (Practice Limited to Oncology)

Osteosarcoma (OSA) is the most common primary bone tumor of dogs. Unfortunately, it is a very common disease seen in veterinary medicine with greater than 10,000 dogs being affected every year in the United States alone. Although our canine patients are most commonly affected, osteosarcoma has been reported in horses, ferrets, rabbits, cats and human beings.  In Cats, OSA, as well as all other primary bone tumors, is less common.  Approximately 4.9 at risk cats per 100,000 develop primary bone tumors per year, with 80-90% of those tumors being osteosarcoma.  Although the disease is less common in humans than in dogs, osteosarcoma can be a devastating diagnosis in human beings affecting mainly children in their teenage years. Canine osteosarcoma has proven to be a very important comparative model in studying the disease and treatment options for children. There has been significant improvement in survival times of human beings with osteosarcoma. In 1980 there was a 20% 5 year-survival rate for human beings with OSA compared to a current long-term survival rate of 60%.  

Osteosarcoma most commonly affects large and giant breed dogs. Breeds found to be at the highest risk of developing OSA include the Great Dane, Saint Bernard, Irish Setter, Greyhound, Rottweiler, German Shepherd and Golden Retriever. Most commonly patients are middle aged to geriatric (median of 7 years), although there is a peak in incidence at 18 to 24 months.

Most commonly the disease occurs in the appendicular skeleton (~75% of cases) although it can also be seen in the axial skeleton as well as soft tissue sites. The forelimbs are affected much more commonly than the hindlimbs with the metaphyseal region of long bones being the most common primary site. The distal radius and the proximal humerus are frequently affected, as are the distal femur and proximal tibia leading to the saying “towards the elbow, away from the knee”.  In the axial skeleton the tumor can present in the mandible, maxilla, spine, cranium, ribs, nasal cavity and pelvis. Extraskeletal sites are much less common but sites reported include the mammary tissue, subcutaneous tissue, spleen, bowel, liver, kidney, eye, gastric ligament, synovium, meninges and adrenal gland. OSA of extraskeletal sites tends to be very aggressive and is often associated with a poor prognosis.

Patients with appendicular lesions most commonly present with lameness and swelling of the tumor site. Owners often associate a recent, minor trauma with the limping and present the patient for evaluation. If the patient has a pathologic fracture (not uncommon with OSA) they may present with severe pain and non-weight bearing lameness.

Radiographs of the affected limb (lateral and craniocaudal views of the lesion) should be pursued following a thorough history and physical exam. Radiographic abnormalities most commonly noted include cortical lysis and osseous proliferation, often with soft tissue extension and/or swelling.  A presumptive diagnosis if often made based on the radiographic appearance of the lesion, signalment, history and physical exam findings. However, differential diagnosis should include other primary bone tumors (chondrosarcoma, fibrosarcoma, hemangiosarcoma, etc.), metastatic lesions, multiple myeloma, solitary plasma cell tumor, lymphoma, systemic mycosis, bacterial osteomyelitis and a bone cyst.

               

If there is a strong suspicion of osteosarcoma based on the radiographs and case findings, three-view thoracic radiographs should be obtained as the next step. Osteosarcoma is aggressive both locally and systemically and metastatic disease is common. At the time of diagnosis only about 15% of patients will have gross metastatic disease; however, approximately 90% of patients will have micrometastatic disease. OSA spreads hematogenously and the most common metastatic site is the lungs, although spread to other bones or soft tissue structures is possible. Along with thoracic radiographs a minimum metabolic database is recommended including a complete blood cell count, serum chemistry and urinalysis. Several studies have reported that dogs with an elevated alkaline phosphatase have a poorer prognosis and may develop metastatic lesions earlier than patients with a normal alkaline phosphatase.

In order to confirm diagnosis a sample (either cytology or biopsy) needs to be obtained. Cytology can be difficult as samples are often poorly cellular and non-diagnostic. Biopsy is the preferred method and can be obtained as an open incisional biopsy or a closed needle biopsy. If biopsy is performed the sample should be collected from the center of the lesion and in a location that will allow removal of the incision and biopsy tract at the time of definitive surgery. Bone fracture is a concern with this tumor, especially when a biopsy is performed. Owners should be warned of this risk and care taken to avoid fracture. If a primary bone tumor is highly suspected, many owners will forego a definitive diagnosis prior to pursuing definitive therapy.

Although OSA is an aggressive disease with survival times of 2-4 months without therapy; there are multiple treatment options available to these patients. These treatments are aimed at treating the local disease and managing the associated pain while decreasing the potential for systemic spread.  Local therapies include amputation, limb sparing surgery, palliative radiation therapy and bisphosphonates.  All of these options are palliative in nature and do not extend survival times.

·         Amputation is a readily available option and the majority of orthopedically sound dogs (even giant breeds and dogs with mild to moderate arthritis) handle this option very well.

·         Limb sparing surgeries can be very beneficial in orthopedically unsound dogs when amputation is not possible.  Limb sparing surgeries are most beneficial for tumors of the distal radius or ulna with absence of a pathologic fracture. However, these surgeries need to be performed by experienced surgeons at specialized surgical centers.

·         Radiation therapy and bisphosphonates are surgical alternatives for pain control. Survival time is not extended by either of these therapies but can be very beneficial in managing discomfort. Radiation therapy typically consists of one treatment per week for 3-4 weeks.  Approximately 70-90% of patients experience 2-3 months of pain relief from this treatment. Side effects are minimal and the patients do not experience systemic side effects as a result of radiation.

·         Bisphosphonates, such as pamidronate, are another option for pain relief. They are administered intravenously along with a fluid diuresis. These drugs reduce bone pain by inhibiting formation of osteoclasts and decreasing their ability to resorb bone. Bisphosphonates can be administered every 4 weeks and pain relief may last weeks to months.

As a result of the high metastatic rate chemotherapy is used in conjunction with local therapy in an effort to slow or prevent disease spread.  The most common chemotheraputic drugs used to treat OSA include carboplatin, cisplatin and adriamycin.  Cisplatin is used much less commonly as a result of its potential for nephrotoxicity necessitating a 6-hour diuresis at the time of drug administration. Carboplatin and adriamycin are used more commonly either as single agents or as part of a multiagent protocol (alternating carboplatin and adriamycin). Both carboplatin and adriamycin are administered intravenously every 3 weeks. Carboplatin is given for 4-6 treatments and adriamycin is given 5 times.  The majority of patients  (approximately 85%) handle these treatments very well with few to no side effects. When side effects are noted, we most commonly see vomiting, diarrhea, decreased appetite, decreased energy level and bone marrow suppression. These side effects tend to be short lived and self-limiting and most patients recover with no intervention. A smaller percentage of patients will require oral anti-emetic or anti-diarrheal drugs and less than 1% of patients have side effects that necessitate hospitalization.

Currently there are studies looking at the benefit of using the oral tyrosine kinase inhibitor, Palladia, or oral metronomic chemotherapy in addition to conventional chemotherapy.  With the addition of these therapies we may see survival times of greater than one year. In addition, osteosarcomas have been shown to overexpress Cox-2 and thus the use of non-steroidal anti-inflammatory drugs may also be beneficial in slowing disease progression and are commonly used in chemotherapy protocols.

With local therapy alone (surgery, radiation therapy, bisphosphonates) average survival times are 3-4 months. With surgery and chemotherapy survival times increase to approximately 12 months with 25% of patients living two years.

The goal with treatment is not only to improve our patient’s length of life but also to ensure an excellent quality of life. With the treatments discussed, the majority of patients will maintain an excellent quality of life and enjoy a significant extension of life.

Dr. Jennifer McDaniel

References

·         London C, Mathie T, Stingle N, et al: Preliminary evidence for biologi activity of toceranib phosphate (Palladia) in solid tumors, Vet Comp Oncol  2011.

·         Withrow SJ, Powers BE, Straw RC, et al: Comparative Aspects of osteosarcoma: dog versus man, Clin Orthop Relat Res Sept(270): 159-168, 1991.

·         Withrow S, Vail D, Page R. (2013).  Small Animal Clinical Oncology.  St. Louis, MO: Elselvier

Bailey’s Story - Our Journey with Hemangiosarcoma

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Bailey was an amazing dog.  A German Shepherd mix, she was strong, loyal and loving…and taking care of her family was her number one priority.  I would joke that she was my shadow – accompanying me to the bathroom, following me into the kid’s rooms in the middle of the night when they were sick, licking my face when she knew I needed a little extra love.  She was an incredibly special dog and healthy all her life.  And then one morning everything changed, a few weeks shy of her 11th birthday.

 It was Saturday morning, September 24th, 2011 and the family was up early getting ready to hit the road for a weekend away.  I noticed only our other dog, Misty, was down in the kitchen with me so when my husband came down, I asked him where Bailey was.  He said she was laying at the top of the stairs and I immediately knew something was not right.  When I got to her, her breathing was labored and she couldn’t stand up.  Her gums were white.  We got her into the car and raced her to the emergency hospital where they quickly diagnosed a ruptured spleen.  They performed emergency surgery to stop the bleeding but we knew the odds were now against us.  A few days later, the biopsy returned and our hearts broke – Bailey  had hemagiosarcoma, a deadly, aggressive form or canine cancer and her outlooks was bleak – 2-3 months without any treatments, 6-9, maybe a year, if we did chemo.

But we refused to give up and a few days later, we had our first visit at the Veterinary Cancer Center. She had been through so much, but after meeting with the doctors we were confident they would do everything they could to give her as much quality time as possible – and that was important.  We didn’t just want her to have time – we wanted her to be full of life, chasing squirrels, stealing pizza crusts, playing with the kids and tearing up the yard with her sister, Misty.  And with their amazing care, she defied the odds.  She lived for three more amazing years – and died just shy of her 14thbirthday.  And those years were just as incredible as her first 11 before cancer struck.    Bailey’s passing was still devastating to our family, but we knew she lived an amazing life and we knew we did everything we could to ensure she had as much time as possible.

Thanks to the amazing doctors at Veterinary Cancer Center, they gave us three more years with our beloved dog! We hope her story gives hope to other families who are faced with this terrible prognosis.

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Hemangiosarcoma

Leiomyosarcoma Case Report

Clinical Presentation:

A 9 year old male castrated Labrador Retriever presented to the emergency service for vomiting, lethargy and discomfort.  The day prior to presentation, he started vomiting and had been evaluated by his primary veterinarian.  He was treated on an out-patient basis with supportive therapy (Maropitant, Metronidazole and a bland diet).

He continued to vomit overnight and was reevaluated by his primary veterinarian. He was depressed, had pale mucus membranes and bloodwork revealed hyperglycemia (129mg/dl, N:75-125), decreased sodium (139 mEq/l; N:141-152), mild anemia(Hct: 36.2%; :N:37-55), and thrombocytopenia (151 10^3/uL; N:200-500).  Abdominal radiographs revealed a soft tissue mass effect cranial ventral to the stomach on the lateral view and decreased contrast.  Thoracic radiographs had no significant findings.  

He was referred to HopeVS’s emergency service for further treatment. A cursory emergency ultrasound documented mild peritoneal effusion and a mixed echogenic mass in the cranial abdomen. An abdominocentesis revealed nonclotting hemorrhagic effusion with a PCV of 49% and Total Solids of 5.8 g/dl.   Extended data base noted increased lactate of 2.7 (0.6-2.5) and PCV: 37%/TS: 6.4g/dl.  Coagulation profiles (PT/PTT) were within normal limits.

A complete abdominal ultrasound revealed a very large mass along the lesser curvature of the stomach measuring 6-8 cm in thickness and 12-16 cm in length.  The mass was partially compartmentalized and occasionally cavitated but primarily consisted of a semi-solid disorganized clump of coalescing hyper and hypoechoic material that was closely associated with the gastric wall causing the lesser curvature to be concave and occasionally effacing the layers of the gastric wall. The was no direct evidence of a transmural lesion or of perforation.  Portions of the mass close to the gastric wall were variably vascular though the majority of the mass was avascular.  Several large pockets of faintly echogenic effusion throughout the ventral abdomen and the fat, omentum and mesentery were hyperechoic throughout the cranial abdomen surrounding the gastric mass. (Figure 1) After consultation with the owner, an abdominal exploratory with resection of the mass was recommended.

Surgery:

The patient was placed in dorsal recumbency and the ventral abdomen was clipped and prepped using standard aseptic technique.  A ventral abdominal incision was made and approximately 500 mls of hemorrhagic effusion  was removed from the abdomen.  (Figure 2) Exploration of the abdomen revealed bleeding from a small break in the serosal surface on the ventral aspect of the stomach.  Approximately 1/2 of the stomach palpated irregular (mildly thickened) and was bruised.  The bruising extended around the majority of the pylorus and into the beginning of the duodenum on the dorsal aspect of the stomach.  The remainder of the abdomen appeared normal.  A pylorectomy was performed obtaining 1 cm margins around the bruised, thickened area followed by a gastroduodenostomy (Billroth I).  Care was taken to assure that the biliary system was not disrupted.  The major duodenal papilla was identified and was ~ 1 cm from the site of resection of the proximal duodenum.  The gallbladder was easily expressed.  A liver biopsy was obtained from the left lateral liver lobe using a guillotine method and the abdomen was closed routinely.  All resected tissue, including the draining lymph node, was submitted for histopathology.   

Histopathology:

No significant lesions were noted in the liver or lymph node biopsies. Within the gastrointestinal tract an acute mural hematoma was noted with, mild lymphoplasmacytic gastritis and the presence of helicobacter. Further gross evaluation revealed a small white, firm nodule (1cm in diameter) at the periphery of the hematoma but within the gastric wall.  Within this section, the wall was expanded by an unencapsulated neoplastic moderately to highly cellular proliferation of spindle cells arranged in interwoven bundles.  The cells were described as elongated to cigar shaped nuclei with stippled chromatin and small to moderate amounts of eosinophilic cytoplasm with indistinct cell borders.  Anisokaryosis was mild to moderate and mitotic activity was 44  per 10 high power fields.  There was multifocal necrosis within the lesion containing accumulations of eosinophilic and karyorrhectic debris.  There are dense infiltrates of small well differentiated lymphocytes and fibroplasia along the periphery of the tumor.  The mass was greater than 5mms from a surgical margin. A diagnosis of a high grade sarcoma was made with the differentials of a leiomyosarcoma and gastrointestinal stromal tumor.  Immunohistochemistry staining was negative for C-Kit, confirming a diagnosis of a high grade leiomyosarcoma. 

Treatment:

The patient recovered uneventfully post-surgery and was discharged at 4 days post- surgery.  A follow oncology consultation was performed and systemic therapy with Doxorubicin IV q 3 weeks for 4-5 cycles was recommended since the tumor was deemed high grade. A baseline abdominal ultrasound was performed prior to starting chemotherapy at 28 days postop and no overt evidence of recurrence was noted. The patient has received 2 cycles thus far with little issues noted.

Overview:

Gastric tumors make up approximately 1-3% of malignant neoplasia in dogs and cats. In dogs, the most common gastric tumor is gastric adenocarcinoma, which accounts for 42-72% of malignant gastric neoplasia.  Leiomyosarcoma, a tumor of smooth muscle,  is the second most common type of gastrointestinal tumor in dogs but it occurs much less frequently than adenocarcinoma.  It can occur anywhere in the gastrointestinal tract but occurs most commonly in the stomach and small intestine. Clinical signs are influenced by tumor location and can include vomiting, weight loss, inappetence, melena, lethargy, hematochezia and hematemesis. Perforation of the intestinal wall secondary to tumor infiltration has been reported but is more common with gastrointestinal stromal tumors than leiomyosarcomas.  A hemoabdomen, as noted in this patient’s history, is not a common finding.

Imaging modalities most commonly used to evaluate a gastric mass include radiography (+/-contrast), ultrasonography and endoscopy.  In addition, when working up a gastric mass, three view thoracic radiographs, abdominal ultrasound and full blood work are recommended to look for any evidence of spread, and evaluate for any concurrent disease.  Paraneoplastic  hypoglycemia has been reported in dogs with leiomyosarcoma.  The exact mechanism is unclear ,but it is normally reversible following surgical treatment.

Surgical excision is the initial treatment of choice for gastric leimyosarcoma.  Also, at the time of surgical exploration, biopsy of the draining lymph node is recommended.  Histopathology with margin evaluate should be performed in all cases as adjuvant treatment recommendations will be made based on the margins and aggressiveness of the lesion.   

On histopathologic evaluation, gastrointestinal stromal tumors and leiomyosarcoma have a similar appearance.  Gastrointestinal stromal tumors arise from the interstitial cells of Cajal.  They express the CD117 (c-KIT) transmembrane receptor and therefore stain positive on c-KIT immunohistochemistry staining.  Leiomyosarcoma is negative for CD117.  Therefore, c-KIT immunohistochemistry staining is often used to differentiate between the two types of neoplasia. 

Currently there is not a published prognostically relevant grading scheme for gastric leiomyosarcoma specifically, but in cutaneous and subcutaneous soft tissue sarcomas,  grade is based on differentiation, necrosis and mitotic activity.    If considered low or moderate grade with clean margins, there is low chance for spread in general with sarcomas and surgery alone is recommended with no further adjuvant treatment postop.  If sarcomas, including leiomyosarcoma, are considered high grade, even with complete excision, chemotherapy is recommended, however data regarding efficacy is lacking.

In one study, 10 dogs with gastrointestinal leiomyosarcoma, a median survival time of 7.8 months was noted with surgery alone was noted, however, only 3 of the 10 dogs had gastric leiomyosarcoma.  Currently, no large scale studies exist comparing survival times in dogs treated with surgery alone vs. surgery and adjuvant chemotherapy.

Submitted by Dr. Lauren May VMD, DACVS

http://hopevs.com/our-doctors/surgery/dr-lauren-may/

References:

Bacon N. Soft Tissue Sarcomas. In: Dobson JM, Lascelles BDX, eds. BSAVA Manual of Canine and Feline Oncology. 3rded. Gloucester: BSAVA, 2011;178-190.

Culp WTN, Cavanaugh RP, et al. Alimentary tract. In: Kudnig ST, Sequin B, eds. Veterinary Surgical Oncology. UK: Wiley-Blackwell, 2012;179-271.

Dennis MM, McSporran KD, et al. Prognostic factors for cutaneous and subcutaneous soft tissue sarcomas in dogs. Vet Pathol 2011;48(1):73-84.

http://vet.sagepub.com/content/48/1/73.short

Kuntz CA, Dernell WS, et al. Prognostic factors for surgical treatment of soft-tissue sarcomas in dogs: 75 cases (1986–1996). J Am Vet Med Assoc 1997;211:1147–1151.

http://europepmc.org/abstract/med/9364229

Liptak JM, Forrest LJ. Soft Tissue Sarcomas. In: Withrow ST, Vail DM, eds. Withrow & MacEwen’s Small Animal Clinical Oncology. 4th ed. St. Louis: Saunders Elsevier, 2007; 425-449.

Russell KN, Mehler SJ, et al. Clinical and immunohistochemical differentiation of gastrointestinal stromal tumors from leiomyosarcomas in dogs 42 cases (1990-2003) J Am Vet Med Assoc 2007;230:1329-33.

http://avmajournals.avma.org/doi/abs/10.2460/javma.230.9.1329

Figures:

Figure 1. Ultrasound image showing in cross section the proximity of the mass to the pylorus. The mass is indicated by the red arrow and the pylorus is labeled by the white arrow.

Figure 2

Intraoperative photo showing stomach wall with severe bruising.  Region of stomach wall that had been bleeding is indicated by black arrow. 

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