Genome-wide methylation patterns from canine nanopore assemblies.

Recent advances in long-read sequencing have enabled the creation of reference-quality genome assemblies for multiple individuals within a species. In particular, 8 long-read genome assemblies have recently been published for the canine model (dogs and wolves). These assemblies were created using a range of sequencing and computational approaches, with only limited comparisons described among subsets of the assemblies. Here we present 3 high-quality de novo reference assemblies based upon Oxford Nanopore long-read sequencing: 2 Bernese Mountain Dogs (BD & OD) and a Cairn terrier (CA611). These breeds are of particular interest due to the enrichment of unresolved genetic disorders. Leveraging advancement in software technologies, we utilized published data of Labrador Retriever (Yella) to generate a new assembly, resulting in a ∼280-fold increase in continuity (N50 size of 91 kbp vs 25.75 Mbp). In conjunction with these 4 new assemblies, we uniformly assessed 8 existing assemblies for generalized quality metrics, sequence divergence, and a detailed BUSCO assessment. We identified a set of ∼400 conserved genes during the BUSCO analysis missing in all assemblies. Genome-wide methylation profiles were generated from the nanopore sequencing, resulting in broad concordance with existing whole-genome and reduced-representation bisulfite sequencing, while highlighting superior overage of mobile elements. These analyses demonstrate the ability of Nanopore sequencing to resolve the sequence and epigenetic profile of canine genomes.

BET Inhibitor JQ1 Attenuates Feline Leukemia Virus DNA, Provirus, and Antigen Production in Domestic Cat Cell Lines.

Feline leukemia virus (FeLV) is a cosmopolitan gammaretrovirus that causes lifelong infections and fatal diseases, including leukemias, lymphomas, immunodeficiencies, and anemias, in domestic and wild felids. There is currently no definitive treatment for FeLV, and while existing vaccines reduce the prevalence of progressive infections, they neither provide sterilizing immunity nor prevent regressive infections that result in viral reservoirs with the potential for reactivation, transmission, and the development of associated clinical diseases. Previous studies of murine leukemia virus (MuLV) established that host cell epigenetic reader bromodomain and extra-terminal domain (BET) proteins facilitate MuLV replication by promoting proviral integration. Here, we provide evidence that this facilitatory effect of BET proteins extends to FeLV. Treatment with the archetypal BET protein bromodomain inhibitor (+)-JQ1 and FeLV challenge of two phenotypically disparate feline cell lines, 81C fibroblasts and 3201 lymphoma cells, significantly reduced FeLV proviral load, total FeLV DNA load, and p27 capsid protein expression at nonlethal concentrations. Moreover, significant decreases in FeLV proviral integration were documented in 81C and 3201 cells. These findings elucidate the importance of BET proteins for efficient FeLV replication, including proviral integration, and provide a potential target for treating FeLV infections.

Establishment and Characterization of Cell Lines from Canine Metastatic Osteosarcoma.

Despite the advancements in treatments for other cancers, the outcomes for osteosarcoma (OSA) patients have not improved in the past forty years, especially in metastatic patients. Moreover, the major cause of death in OSA patients is due to metastatic lesions. In the current study, we report on the establishment of three cell lines derived from metastatic canine OSA patients and their transcriptome as compared to normal canine osteoblasts. All the OSA cell lines displayed significant upregulation of genes in the epithelial mesenchymal transition (EMT) pathway, and upregulation of key cytokines such as CXCL8, CXCL10 and IL6. The two most upregulated genes are MX1 and ISG15. Interestingly, ISG15 has recently been identified as a potential therapeutic target for OSA. In addition, there is notable downregulation of cell cycle control genes, including CDKN2A, CDKN2B and THBS1. At the protein level, p16INK4A, coded by CDKN2A, was undetectable in all the canine OSA cell lines, while expression of the tumor suppressor PTEN was variable, with one cell line showing complete absence and others showing low levels of expression. In addition, the cells express a variety of actionable genes, including KIT, ERBB2, VEGF and immune checkpoint genes. These findings, similar to those reported in human OSA, point to some genes that can be used for prognosis, targeted therapies and novel drug development for both canine and human OSA patients.

Case report: MicroRNA-10b as a therapeutic target in feline metastatic mammary carcinoma and its implications for human clinical trials.

Ninety percent of deaths from cancer are caused by metastasis. miRNAs are critical players in biological processes such as proliferation, metastasis, apoptosis, and self-renewal. We and others have previously demonstrated that miRNA-10b promotes metastatic cell migration and invasion. Importantly, we also showed that miR-10b is a critical driver of metastatic cell viability and proliferation. To treat established metastases by inhibiting miR-10b, we utilized a therapeutic, termed MN-anti-miR10b, composed of anti-miR-10b antagomirs, conjugated to iron oxide nanoparticles, that serve as delivery vehicles to tumor cells in vivo and a magnetic resonance imaging (MRI) reporter. In our previous studies using murine models of metastatic breast cancer, we demonstrated the effectiveness of MN-anti-miR10b in preventing and eliminating existing metastases. With an outlook toward clinical translation of our therapeutic, here we report studies in large animals (companion cats) with spontaneous feline mammary carcinoma (FMC). We first investigated the expression and tissue localization of miR-10b in feline tumors and metastases and showed remarkable similarity to these features in humans. Next, in the first case study involving this therapeutic we intravenously dosed an FMC patient with MN-anti-miR10b and demonstrated its delivery to the metastatic lesions using MRI. We also showed the initial safety profile of the therapeutic and demonstrated significant change in miR-10b expression and its target HOXD10 after dosing. Our results provide support for using companion animals for further MN-anti-miR10b development as a therapy and serve as a guide for future clinical trials in human patients.

Identification of a Hypomorphic FANCG Variant in Bernese Mountain Dogs.

Bernese mountain dogs (BMDs), have an overall cancer incidence of 50%, half of which is comprised of an otherwise rare tumor, histiocytic sarcoma (HS). While recent studies have identified driver mutations in the MAPK pathway, identification of key predisposing genes has been elusive. Studies have identified several loci to be associated with predisposition to HS in BMDs, including near the MTAP/CDKN2A region, but no causative coding variant has been identified. Here we report the presence of a coding polymorphism in the gene encoding FANCG, near the MTAP/CDKN2A locus. This variant is in a conserved region of the protein and appears to be specific to BMDs. Canine fibroblasts derived from dogs homozygous for this variant are hypersensitive to cisplatin. We show this canine FANCG variant and a previously defined hypomorphic FANCG allele in humans impart similar defects in DNA repair. However, our data also indicate that this variant is neither necessary nor sufficient for the development of HS. Furthermore, BMDs homozygous for this FANCG allele display none of the characteristic phenotypes associated with Fanconi anemia (FA) such as anemia, short stature, infertility, or an earlier age of onset for HS. This is similar to findings in FA deficient mice, which do not develop overt FA without secondary genetic mutations that exacerbate the FA deficit. In sum, our data suggest that dogs with deficits in the FA pathway are, like mice, innately resistant to the development of FA.

Sorafenib and Doxorubicin Show Synergistic Effects in Human and Canine Osteosarcoma Cell Lines.

Osteosarcoma (OSA) is the most common bone tumor in both humans and dogs and has a nearly ten-fold higher incidence in dogs than humans. Despite advances in the treatment of other cancers, the overall survival rates for OSA have stagnated for the past four decades. Therefore, there is a great need to identify novel and effective treatments. We screened a series of tyrosine kinase inhibitors and selected sorafenib, a multi-kinase inhibitor, for further evaluation alone and in combination with cisplatin, carboplatin, and doxorubicin on canine and human OSA cell lines. Our data point to synergistic effects when sorafenib is combined with doxorubicin, but not when combined with cisplatin or carboplatin, in both human and canine OSA. Based on current findings, clinical trials using a combination of doxorubicin and sorafenib in proof-of-concept studies in dogs are warranted. These studies can be carried out relatively quickly in dogs where case load is high and, in turn, provide useful data for the initiation of clinical trials in humans.

Small Molecule 20S Proteasome Enhancer Regulates MYC Protein Stability and Exhibits Antitumor Activity in Multiple Myeloma.

Despite the addition of several new agents to the armamentarium for the treatment of multiple myeloma (MM) in the last decade and improvements in outcomes, the refractory and relapsing disease continues to take a great toll, limiting overall survival. Therefore, additional novel approaches are needed to improve outcomes for MM patients. The oncogenic transcription factor MYC drives cell growth, differentiation and tumor development in many cancers. MYC protein levels are tightly regulated by the proteasome and an increase in MYC protein expression is found in more than 70% of all human cancers, including MM. In addition to the ubiquitin-dependent degradation of MYC by the 26S proteasome, MYC levels are also regulated in a ubiquitin-independent manner through the REGγ activation of the 20S proteasome. Here, we demonstrate that a small molecule activator of the 20S proteasome, TCH-165, decreases MYC protein levels, in a manner that parallels REGγ protein-mediated MYC degradation. TCH-165 enhances MYC degradation and reduces cancer cell growth in vitro and in vivo models of multiple myeloma by enhancing apoptotic signaling, as assessed by targeted gene expression analysis of cancer pathways. Furthermore, 20S proteasome enhancement is well tolerated in mice and dogs. These data support the therapeutic potential of small molecule-driven 20S proteasome activation for the treatments of MYC-driven cancers, especially MM.

ATP7A, ATP7B, and RETN genotypes in Labrador Retrievers with and without copper-associated hepatopathy.

OBJECTIVE: To determine the frequency of previously reported coding variants in the ATP7A, ATP7B, and RETN genes in a US population of Labrador Retrievers and to explore potential associations of these genotypes with pathologic hepatic copper accumulation. SAMPLE: Archived hepatic specimens from 90 Labrador Retrievers collected between 2013 and 2021. PROCEDURES: The Michigan State University Veterinary Diagnostic Laboratory database was searched to identify archived tissues from Labrador Retrievers that had undergone hepatic histopathologic assessment. Cases were classified into control, copper-associated hepatopathy (CAH), and intermediate populations on the basis of histopathologic features and hepatic copper accumulation. The DNA was extracted from archived tissues and genotyped for reported variants in the 3 genes. Allele frequencies were determined, and associations of genotypes with CAH status were evaluated. RESULTS: 29 control dogs, 45 CAH dogs, and 16 intermediate dogs were included in the study. The overall ATP7A and RETN variant allele frequencies were 30% and 13%, respectively, and were not significantly different among control, CAH, and intermediate populations. The ATP7B variant allele frequency was significantly higher in the CAH population (30%) as compared to the control population (13%). However, 21 of 45 (47%) CAH dogs did not have an ATP7B variant allele whereas 7 of 28 (25%) control dogs did have an ATP7B variant allele. CLINICAL RELEVANCE: Study results supported a contributory role for the ATP7B variant in CAH pathogenesis in Labrador Retrievers. However, the application of genetic testing in a clinical setting is complicated by genotypic variability within healthy and diseased dogs.

Design and Synthesis of Bovine Leukemia Virus-Associated Peptide-Based Qß Conjugate Eliciting Long-Lasting Neutralizing Antibodies in Mice.

Bovine leukemia virus (BLV) is a C-type retrovirus of cattle that causes huge economic losses with high infection rates in the majority of countries worldwide. To develop an anti-BLV vaccine, we constructed a peptide conjugate using the envelope glycoprotein gp51-peptide epitope, a putative receptor-binding site. This highly antigenic peptide was covalently linked to a mutant bacteriophage carrier (mQß) using two different linker strategies, isothiocyanate (NCS) and dinitrophenyl adipate. Both constructs elicited higher anti-BLV peptide IgG titers than the corresponding conjugate with keyhole limpet hemocyanin protein carrier (gold standard) in mice with the NCS linker strategy requiring less sample processing. The mQß-gp51-peptide construct is the first BLV peptide-based vaccine candidate to generate durable immunity (>539 days), which recognized both native gp51 protein and BLV particles and significantly decreased fusion of a susceptible cell line exposed to infectious BLV. These results support the high translational and animal health potential of the vaccine construct.

Proposed Diagnostic Criteria and Classification of Canine Mast Cell Neoplasms: A Consensus Proposal.

Mast cell neoplasms are one of the most frequently diagnosed malignancies in dogs. The clinical picture, course, and prognosis vary substantially among patients, depending on the anatomic site, grade and stage of the disease. The most frequently involved organ is the skin, followed by hematopoietic organs (lymph nodes, spleen, liver, and bone marrow) and mucosal sites of the oral cavity and the gastrointestinal tract. In cutaneous mast cell tumors, several grading and staging systems have been introduced. However, no comprehensive classification and no widely accepted diagnostic criteria have been proposed to date. To address these open issues and points we organized a Working Conference on canine mast cell neoplasms in Vienna in 2019. The outcomes of this meeting are summarized in this article. The proposed classification includes cutaneous mast cell tumors and their sub-variants defined by grading- and staging results, mucosal mast cell tumors, extracutaneous/extramucosal mast cell tumors without skin involvement, and mast cell leukemia (MCL). For each of these entities, diagnostic criteria are proposed. Moreover, we have refined grading and staging criteria for mast cell neoplasms in dogs based on consensus discussion. The criteria and classification proposed in this article should greatly facilitate diagnostic evaluation and prognostication in dogs with mast cell neoplasms and should thereby support management of these patients in daily practice and the conduct of clinical trials.

Chemoenzymatic Synthesis of 9NHAc-GD2 Antigen to Overcome the Hydrolytic Instability of O-Acetylated-GD2 for Anticancer Conjugate Vaccine Development.

Ganglioside GD2 is an attractive tumor-associated carbohydrate antigen for anti-cancer vaccine development. However, its low immunogenicity and the significant side effects observed with anti-GD2 antibodies present significant obstacles for vaccines. To overcome these, a new GD2 derivative bearing an N-acetamide (NHAc) at its non-reducing end neuraminic acid (9NHAc-GD2) has been designed to mimic the 9-O-acetylated-GD2 (9OAc-GD2), a GD2 based antigen with a restricted expression on tumor cells. 9NHAc-GD2 was synthesized efficiently via a chemoenzymatic method and subsequently conjugated with a powerful carrier bacteriophage Qß. Mouse immunization with the Qß-9NHAc-GD2 conjugate elicited strong and long-lasting IgG antibodies, which were highly selective toward 9NHAc-GD2 with little cross-recognition of GD2. Immunization of canines with Qß-9NHAc-GD2 showed the construct was immunogenic in canines with little adverse effects, paving the way for future clinical translation to humans.

Activating Mutations in PTPN11 and KRAS in Canine Histiocytic Sarcomas.

While the genetic contributions to the predisposition of Bernese mountain dogs (BMDs) to histiocytic sarcoma (HS) remains unclear, some insights into key genetic drivers have been gained. Our group recently reported a mutation in the PTPN11 gene (E76K). We have now identified a second missense mutation in PTPN11 (G503V), and a mutation in KRAS (Q61H) present in HS cell lines. These mutations are associated with malignancies in humans, and known to be gain-of-function mutations that result in activation of the mitogen-activated protein kinase (MAPK) pathway. The goal of the present study was to evaluate the prevalence of these mutations in a large sample of HS cases from BMDs and golden retrievers, and in lymphoma cases, from a cohort of BMDs. Mutations in PTPN11 were present in HS in 41/96 (43%) BMDs, and in 3/13 (23%) golden retrievers. PTPN11 mutations E76K and G503V did not coexist in the same neoplasm. The KRAS mutation was much less frequent, with a prevalence of 3.1% (3/96). We did not identify either PTPN11 nor KRAS mutations in any of the lymphoma samples. These results point out the potential relevance of PTPN11 and KRAS mutations as activators of the oncogenic MAPK pathway for canine HS, particularly in BMDs.

Development of an Orthotopic Intrasplenic Xenograft Mouse Model of Canine Histiocytic Sarcoma and Its Use in Evaluating the Efficacy of Treatment with Dasatinib.

Canine histiocytic sarcoma is a highly aggressive and metastatic hematopoietic neoplasm that responds poorly to currently available treatment regimens. Our goal was to establish a clinically relevant xenograft mouse model to assess the preclinical efficacy of novel cancer treatment protocols for histiocytic sarcoma. We developed an intrasplenic xenograft mouse model characterized by consistent tumor growth and development of metastasis to the liver and other abdominal organs. This model represents the metastatic or disseminated form of canine histiocytic sarcoma, which is considered the most clinically challenging form of the disease. Transfection of tumor cells with a luciferase vector supported the use of in vivo bioluminescence imaging to track tumor progression over time and to assess the response of this murine model to novel chemotherapeutic agents. Dasatinib treatment of the mice with intrasplenic xenografts decreased tumor growth and increased survival times, compared with mice treated with vehicle only. Our findings indicate the potential of dasatinib for the treatment of histiocytic sarcoma in dogs and for similar diseases in humans. These results warrant additional studies to clinically test the efficacy of dasatinib in dogs with histiocytic sarcoma.

Investigation of novel chemotherapeutics for feline oral squamous cell carcinoma.

Feline oral squamous cell carcinomas (FOSCC) are highly aggressive neoplasms with short survival times despite multimodal treatment. FOSCC are similar to squamous cell carcinomas of the head and neck (SCCHN) in humans, which also present therapeutic challenges. The current study was undertaken to identify novel chemotherapeutics using FOSCC cell lines. A high throughput drug screen using 1,952 drugs was performed to identify chemotherapeutics for further investigation. Two of the drugs identified in the drug screen, actinomycin D and methotrexate, and two drugs with similar molecular targets to drugs found to be efficacious in the screening, dinaciclib and flavopiridol, were selected for further investigation. Drug inhibition profiles were generated for each drug and cell line using an MTS assay. In addition, the effects of the drugs of interest on cell cycle progression were analyzed via a propidium iodide DNA labeling assay. Changes in caspase-3/7 activity after treatment with each drug were also determined. The findings demonstrated effectiveness of the drugs at nanomolar concentrations with sensitivity varying across cell lines. With all of the drugs except for actinomycin D, evidence for G1 arrest was found. Dinaciclib and flavopiridol were demonstrated to induce apoptosis. The results of the study suggest that the selected drugs are potential candidates for developing novel chemotherapeutic approaches to FOSCC. Through these studies, novel therapeutic strategies for the treatment of FOSCC can be developed to provide better care for affected cats which can also serve as proof of concept studies to inform translational studies in SCCHN in humans.

Targeting MEK in a Translational Model of Histiocytic Sarcoma.

Histiocytic sarcoma in humans is an aggressive orphan disease with a poor prognosis as treatment options are limited. Dogs are the only species that spontaneously develops histiocytic sarcoma with an appreciable frequency, and may have value as a translational model system. In the current study, high-throughput drug screening utilizing histiocytic sarcoma cells isolated from canine neoplasms identified these cells as particularly sensitive to a MEK inhibitor, trametinib. One of the canine cell lines carries a mutation in PTPN11 (E76K), and another one in KRAS (Q61H), which are associated with the activation of oncogenic MAPK signaling. Both mutations were previously reported in human histiocytic sarcoma. Trametinib inhibited sensitive cell lines by promoting cell apoptosis, indicated by a significant increase in caspase 3/7. Furthermore, in vitro findings were successfully recapitulated in an intrasplenic orthotopic xenograft mouse model, which represents a disseminated aggressive form of histiocytic sarcoma. Mice with histiocytic sarcoma xenograft neoplasms that were treated with trametinib had significantly longer survival times. Target engagement was validated as activity of ERK, downstream of MEK, was significantly downregulated in neoplasms of treated mice. Additionally, trametinib was found in plasma and neoplastic tissues within projected therapeutic levels. These findings demonstrate that in dogs, histiocytic sarcoma may be associated with a dysfunctional MAPK pathway, at least in some cases, and may be effectively targeted through MEK inhibition. Clinical trials to test safety and efficacy of trametinib in dogs with histiocytic sarcoma are warranted, and may provide valuable translational information to similar diseases in humans. Mol Cancer Ther; 17(11); 2439-50. ©2018 AACR.

A novel canine histiocytic sarcoma cell line: initial characterization and utilization for drug screening studies.

BACKGROUND: Histiocytic sarcoma is a rare disorder in humans, however it is seen with appreciable frequency in certain breeds of dogs, such as Bernese mountain dog. The purpose of this study was to fully characterize a novel canine histiocytic sarcoma cell line, and utilize it as a tool to screen for potential therapeutic drugs. METHODS: The histiocytic sarcoma cell line was characterized by expression of cellular markers as determined by immunohistochemistry and flow cytometry techniques. The neoplastic cells were also evaluated for their capability of phagocytizing beads particles, and their potential to grow as xenograft in an immunodeficient mouse. We investigated the in vitro cytotoxic activity of a panel of thirteen compounds using the MTS proliferation assay. Inhibitory effects of different drugs were compared using one-way ANOVA, and multiple means were compared using Tukey's test. RESULTS: Neoplastic cells expressed CD11c, CD14, CD18, CD45, CD172a, CD204, MHC I, and vimentin. Expression of MHC II was upregulated after exposure to LPS. Furthermore, the established cell line clearly demonstrated phagocytic activity similar to positive controls of macrophage cell line. The xenograft mouse developed a palpable subcutaneous soft tissue mass after 29 days of inoculation, which histologically resembled the primary neoplasm. Dasatinib, a tyrosine kinase pan-inhibitor, significantly inhibited the growth of the cells in vitro within a clinically achievable and tolerable plasma concentration. The inhibitory response to dasatinib was augmented when combined with doxorubicin. CONCLUSIONS: In the present study we demonstrated that a novel canine histiocytic sarcoma cell line presents a valuable tool to evaluate novel treatment approaches. The neoplastic cell line favorably responded to dasatinib, which represents a promising anticancer strategy for the treatment of this malignancy in dogs and similar disorders in humans.

Investigation of blood lead concentrations in dogs living in Flint, Michigan.

OBJECTIVE To measure blood lead concentrations (BLCs) in dogs living in Flint, Mich, following a declared water crisis and to assess potential associations of BLCs with demographic data, water sources, and clinical signs in these dogs. DESIGN Cross-sectional study. ANIMALS 284 dogs residing in Flint, Mich (test population), and 47 dogs residing in East Lansing, Mich (control population), and immediately adjacent areas. PROCEDURES Blood samples were collected at free screening clinics in Flint (test population) and at the Michigan State University College of Veterinary Medicine Veterinary Medical Center (control population). Owners of test population dogs completed questionnaires providing demographic and clinical information. Hematologic evaluations were performed; BLCs were measured by inductively coupled plasma-mass spectrometry. RESULTS 4 of 284 test population dogs had BLCs > 50 ppb; an additional 20 had BLCs > 20 ppb. Overall, BLCs of test population dogs were higher than those of control dogs. Within the test population, young dogs (≤ 2 years of age) had higher BLCs than old dogs (≥ 6 years of age). Only 7.2% of test population dogs were drinking unfiltered tap water at the time of screening; however, dogs that had been receiving filtered or bottled water for ≤ 3 months before screening had higher BLCs than did those that received such water for > 3 months. CONCLUSIONS AND CLINICAL RELEVANCE Taken together, findings suggested that the impact of the Flint water crisis extended to companion animals. Results highlighted the importance of maintaining awareness of lead exposure and considering both human and animal well-being in cases of environmental toxicant exposures.

Evaluation of Immunomodulatory Properties of Feline Mesenchymal Stem Cells.

Mesenchymal stem cells (MSC) offer a novel approach to treatment of inflammatory disorders in humans and companion animals. Cats spontaneously develop a wide variety of inflammatory disorders and may potentially benefit from MSC-based therapies. Multiple genes are involved in immunomodulation by MSC and interspecies differences between expressions of these genes exist. The goals of the study were to characterize the expression of genes known to be involved in MSC-based immunomodulation and determine the effect of MSC on proliferation of T cells in coculture experiments with peripheral blood mononuclear cells (PBMC). Unstimulated MSC expressed all immunomodulatory genes studied except for IL-10. Levels of iNOS and FASL were low or undetectable at the RNA level. INFγ stimulation resulted in significant dose-dependent upregulation of IDO1, PD-L1, IL-6, COX2, and HGF. Levels of kynurenine were increased after 3-day incubation with INFγ. TNFα stimulation increased expression of IL-6 at both RNA and protein level as well as upregulated COX2 gene expression and PTGES1.Stimulation with both INFγ and TNFα resulted in significant increase in PGE2 levels in cell culture medium. MSC significantly decreased proliferation of ConA-stimulated PBMC in coculture experiments at 1:5 ratio. Our results suggest that feline MSC have similar immunomodulatory gene expression and react to inflammatory cytokines in a manner similar to human MSC. Thus, MSC may play an important role in treatment of feline disease as well as serve as an important translational species to evaluate MSC-based therapies of diseases common to both humans and cats.

Comparison of FTIR transmission and transfection substrates for canine liver cancer detection.

FTIR spectroscopy is a widely used technique that provides insights into disease processes at the molecular level. Due to its numerous advantages it is becoming an increasingly powerful tool for the study of biological materials and has the potential to become an excellent diagnostic method, especially considering the low cost of transflection substrates. However, questions about the usefulness of the transflection measurement mode due to the complicated nature of physical processes occurring during the measurement and in particular the Electric Field Standing Wave (EFSW) effect have been raised. In this paper we present a comparison of the two most common FT-IR measurement modes: transmission and transfection using healthy and pathologically altered tissue (histiocytic sarcoma). We found that the major differences between normal and cancerous tissue were associated with changes DNA and carbohydrate content. In particular we identified a band at 964 cm(-1) assigned to a nucleic acid phosphodiester backbone mode, which appeared more pronounced in cancerous tissue irrespective of the substrate. We applied Principal Component Analysis, Unsupervised Hierarchical Cluster Analysis and k-means clustering to transmission and transflection substrates and found that both measurement modes were equally capable of discrimination normal form cancerous tissue. Moreover, the differences between spectra from cancerous and normal tissue were significantly more important than the ones arising from the measurement modes.