Actionable mutations in canine hemangiosarcoma

<div><p>Background</p><p>Angiosarcomas (AS) are rare in humans, but they are a deadly subtype of soft tissue sarcoma. Discovery sequencing in AS, especially the visceral form, is hampered by the rarity of cases. Most diagnostic material exists as archival formalin fixed, paraffin embedded tissue which serves as a poor source of high quality DNA for genome-wide sequencing. We approached this problem through comparative genomics. We hypothesized that exome sequencing a histologically similar tumor, hemangiosarcoma (HSA), that occurs in approximately 50,000 dogs per year, may lead to the identification of potential oncogenic drivers and druggable targets that could also occur in angiosarcoma.</p><p>Methods</p><p>Splenic hemangiosarcomas are common in dogs, which allowed us to collect a cohort of archived matched tumor and normal tissue samples suitable for whole exome sequencing. Mapping of the reads to the latest canine reference genome (Canfam3) demonstrated that >99% of the targeted exomal regions were covered, with >80% at 20X coverage and >90% at 10X coverage.</p><p>Results and conclusions</p><p>Sequence analysis of 20 samples identified somatic mutations in <i>PIK3CA</i>, <i>TP53</i>, <i>PTEN</i>, and <i>PLCG1</i>, all of which correspond to well-known tumor drivers in human cancer, in more than half of the cases. In one case, we identified a mutation in <i>PLCG1</i> identical to a mutation observed previously in this gene in human visceral AS. Activating <i>PIK3CA</i> mutations present novel therapeutic targets, and clinical trials of targeted inhibitors are underway in human cancers. Our results lay a foundation for similar clinical trials in canine HSA, enabling a precision medicine approach to this disease.</p></div

Decision tree for filtering and curation of variants.

<p>Single nucleotide variants (SNVs) and small insertion-deletion variants (indels) were filtered as shown, on the left and right sides of the Fig, respectively. The final filtered SNVs (167) and indels (32) were subjected to various annotation steps, such as pathway analysis, comparative genomics analysis and candidate gene comparison, as schematized at the bottom of the Figure.</p

Distribution of alterations in the <i>PIK3CA</i> gene.

<p>(A) The table shows the distribution of <i>PIK3CA</i> mutations in our cohort of canine HSA cases, the corresponding human amino acid change, and the functional impacts of the mutants based on previous human studies. "GOF" denotes gain-of-function. (B) Schematic diagram of homologous mutations in human PIK3CA. The x axis represents amino acid positions and domain structure of human PIK3CA, and the y axis represents the number of times each mutation observed in our cohort.</p

Distribution of alterations in the <i>TP53</i> gene.

<p>(A) Table shows the distribution of <i>TP53</i> mutations in our cohort, the mutation types, the corresponding human amino acid change, and the predicted functional impact of each mutation based on previous studies. (B) Schematic diagram of homologue mutations in human TP53. The x axis represents amino acid positions and domain structure of human TP53, and the y axis represents the number of times each mutation was identified in our cohort.</p

Summary of somatic mutations in HSA cases lacking strong driver mutation candidates.

<p>Green blocks denote missense mutations. Shown in black are predicted loss-of-function mutations (essential splice-site variants, nonsense mutations, and frameshift indels).</p

Number of mutations in each tumor.

<p>Nonsynonymous somatic mutation loads in canine HSA exomes: the numbers of somatic nonsynonymous mutations is displayed for each case of 20 HSA cases, ranked from left to right by the total number of mutations. Sequence coverage is listed under each case. Note that the number of mutations per case is not correlated with sequence coverage.</p

Candidate driver mutations and recurrent mutations.

<p>Each row represents a mutated gene and each column represents an individual tumor. Shown in green blocks are non-synonymous, non truncating variants which could represent gain-of-function mutations. Shown in black are predicted inactivating mutations, including truncating mutations, essential splice-site variants, and nonsense mutations. A: Candidate driver mutations, B: additional recurrent mutations.</p

Supplemental Material, DS1_VET_10.1177_0300985818776054 - Characteristics of the Epithelial-Mesenchymal Transition in Primary and Paired Metastatic Canine Mammary Carcinomas

<p>Supplemental Material, DS1_VET_10.1177_0300985818776054 for Characteristics of the Epithelial-Mesenchymal Transition in Primary and Paired Metastatic Canine Mammary Carcinomas by Talita M. M. Raposo-Ferreira, Becky K. Brisson, Amy C. Durham, Renee Laufer-Amorim, Veronica Kristiansen, Ellen Puré, Susan W. Volk, and Karin Sorenmo in Veterinary Pathology</p

Identification of prognostic collagen signatures and potential therapeutic stromal targets in canine mammary gland carcinoma

<div><p>Increasing evidence indicates that the tumor microenvironment plays a critical role in regulating the biologic behavior of breast cancer. In veterinary oncology, there is a need for improved prognostic markers to accurately identify dogs at risk for local and distant (metastatic) recurrence of mammary gland carcinoma and therefore would benefit from adjuvant therapy. Collagen density and fiber organization have been shown to regulate tumor progression in both mouse and human mammary tumors, with certain collagen signatures predicting poor outcomes in women with breast cancer. We hypothesized that collagen signatures in canine mammary tumor biopsies can serve as prognostic biomarkers and potential targets for treatment. We used second harmonic generation imaging to evaluate fibrillar collagen density, the presence of a tumor-stromal boundary, tumor associated collagen signatures (TACS) and individual collagen fiber characteristics (width, length and straightness) in grade I/II and grade III canine mammary tumors. Collagen density, as well as fiber width, length and straightness, were inversely correlated with patient overall survival time. Notably, grade III cases were less likely to have a tumor-stromal boundary and the lack of a boundary predicted poor outcome. Importantly, a lack of a defined tumor-stromal boundary and an increased collagen fiber width were associated with decreased survival even when tumor grade, patient stage, ovariohysterectomy status at the time of mammary tumor excision, and histologic evidence of lymphovascular invasion were considered in a multivariable model, indicating that these parameters could augment current methods to identify patients at high risk for local or metastatic progression/recurrence. Furthermore, these data, which identify for the first time, prognostic collagen biomarkers in naturally occurring mammary gland neoplasia in the dog, support the use of the dog as a translational model for tumor-stromal interactions in breast cancer.</p></div

Collagen density predicts poor outcome in canine mammary tumors.

<p>Integrated density of collagen signal from SHG images was quantified using Fiji (Image J) software. Graph represents averages from 5 images per tumor from 11 grade I/II and 9 grade III mammary tumors. *p<0.05 via an unpaired Mann-Whitney test (A). Kaplan-Meier survival curve for 18 dogs with collagen integrated density higher or lower than the mean integrated density value (B). The log-rank test was used to evaluate whether the collagen density significantly impacted survival (p = 0.013, HR 4.099, 95%CI 0.860–19.520).</p