Biomarkers for Lymphoma Diagnostics

Dogs develop spontaneous hematopoietic tumors with clinical, histologic, and molecular similarities to humans. Beyond the veterinary interest, the understanding of canine neoplasia has also comparative interest for human oncology, increased by the fact that dogs are exposed to the same environment as are humans and develop these tumors spontaneously, which is a significant advantage over experimental models.1,² The completion of the human genome sequencing in 2001 and subsequent publishing of the first canine genome has made it possible to apply high-throughput protocols used for the study of molecular features of human cancer in dogs.³ In particular, molecular studies of canine lymphoma and leukemia have revealed evolutionary conserved cytogenetic abnormalities comparable with the corresponding human conditions. This opened the possibility that some evolutionary conserved expressed or regulatory genes have a role in the pathogenesis of canine lymphoma and leukemia similar to those in humans. In the past few years, initial studies have focused on two particular entities: (1) expressed genes—by measuring and quantifying messenger RNA (mRNA), which is a transcribed copy of involved genes; and (2) the quantification of regulatory components of expressed genes, the so-called micro-RNA (miRNA).

Lymphoma Profiling Using Expressed Gene Analysis

Canine lymphoma is a heterogeneous group of diseases that share malignant transformation of lymphocytes as a common property. The most updated World Health Organization (WHO) classification system for B- and T-cell lymphomas includes approximately 30 subtypes. The classification is based on morphology, topography, immunophenotype, and clinical progression. Six subtypes are most commonly observed, including diffuse large B-cell lymphoma (DLBCL), marginal-zone lymphoma (MZL), Burkitt and Burkitt-like lymphoma (BL), lymphoblastic T-cell lymphoma (LBT), T-zone lymphoma (TZL), and peripheral T-cell lymphoma (PTCL). Although this classification has been confirmed in several clinical studies, a molecular basis for this classification has been elusive until recently. On the human counterpart, transcriptional profiling provided further insight by allowing subclassification of the larger lymphoma classes. For example, the human DLBCL is subdivided further into three subtypes, each of which has a distinct transcriptional signature, biologic behavior, and response to therapy, indicating that molecular profiling also provides prognostic opportunities.

In 2011, an international group reached consensus to subclassify canine lymphomas into six subtypes based on the modified WHO criteria, including DLBCL, BL, MZL, LBT, PTCL, and TZL.⁴ Subsequently, a multi-institutional group of veterinary and human geneticists, pathologists, and diagnosticians, found molecular correlates classifying these six subtypes into three general subgroups consisting of (1) high-grade T-cell lymphomas (LBT, PTCL), (2) low-grade T-cell lymphomas (TZL), and (3) B-cell lymphomas (BLBCL, BL, and MZL).⁵ The molecular workup included array hybridization, confirmation by real-time polymerase chain reaction (PCR), immunophenotyping, and PCR for antigen receptor rearrangement (PARR). Candidate genes were selected to form pairs of genes with the following characteristics: To give high p-value between groups, to give large and opposing fold changes between groups, and to have low within group variance. The group was able to define a simplified test based on four genes used in two pairs to (1) define the separation of B-cell and T-cell tumors (genes CD28 and ABCA5), and (2) for T-cell tumors, to separate high- and low-grade lymphomas (genes CCDC3 and SMOC2). Kaplan-Meier analysis confirmed that the classification based on the molecular four-gene signature allowed correct assignment of (1) low-grade T-cell lymphomas (TZL) with longest survival times, (2) high-grade T-lymphomas (TZL) with shortest survival times, and (3) B-cell lymphomas with intermediate survival times. Statistical analysis showed that the B- and T-cell classification was highly accurate with a risk for an incorrect classification in less than 1 in 1 billion. Similarly, the risk to misclassify a high-grade T-cell or a low-grade T-cell lymphoma was less than 1 in 10,000. Although this test, compared with the PARR test, offers prognostic value, it is not yet commercially available to veterinarians.

Lymphoma Classification Based on Micro–Ribonucleic Acid Quantification

In the last decade, a flurry of studies has elucidated the role of micro-RNA (miRNA) in human cancer. miRNAs are a new class of short, approximately 21 to 22 nucleotides long, noncoding RNAs that are involved in negative regulation of gene expression through sequence-specific base pairing with target mRNAs, usually in their 3’-UTR part. Each miRNAs can control hundreds of gene targets, with potential influence on almost every pathway. A large body of evidence that already exists suggests that miRNAs play important roles in cellular growth and differentiation, including apoptosis (programmed cell death), and oncogenesis.⁶ So-called Oncomirs comprises an miRNA subclass that includes genes associated with several types of cancer. Although some miRNAs promote cell proliferation and survival and, thus, act as oncogenes, others diminish cell proliferation and survival and, thus, act as tumor suppressors. Although miRNAs may not be the causal event of tumorigenesis, they still may be useful for classifying tumors and predicting outcomes. Most miRNA species that are necessary for the differentiation of immune functions are found to be abnormally expressed in hematopoietic tumors. This derives from the fact that miRNAs are not only differentially expressed but also seem to modulate immunologic processes at the pathophysiologic level toward normal or aberrant. For this and other reasons, miRNA profiling holds great promise for biomarker and novel target discovery in cancer research. In this regard, it is noteworthy that miRNA represents a class of RNA molecules with great stability at room temperature, compared with mRNA from transcribed genes, which remain intact for minutes before recycled by physiologic mechanisms. From a diagnostic perspective, increased stability is a significant advantage, as it leads to easy collection recommendations and more robust results with fewer possibilities for artifacts. Moreover, as is true for mRNA, miRNA may be readily extracted from formalin-fixed, paraffin-embedded (FFPE) samples, and it has already been confirmed that miRNA profiles in fresh tissues correlate well with FFPE tissues.⁷

Progress has been made in the canine genome by the computational analysis for 357 miRNA candidates, among which 300 were 100% identical to already characterized human miRNAs.⁸ However, the use of this information in canine oncology is still in its early stages.

In a study published in 2010, a panel of 11 human miRNA real-time PCR tests with a minimal identity of 95% to canine miRNA species was used to screen a set of 22 canine lymphoma samples.⁹ In this study, it was found that miR-181a was significantly overexpressed and miR-29b was significantly downregulated in T-cell lymphoma cases compared with B-cell lymphomas, and a significant overexpression of miR-17-5p in B-cell lymphomas compared with T-cell lymphomas. By using differentially expressed miRNA such as miR-181a and miR-17-5p in a ratio, the authors were able to show that the test was able to reliably separate T-cell lymphomas from B-cell lymphomas. However, with the set of 11 miRNAs used for this study, no differentiation between high-grade and low-grade lymphomas was found.