Melting curve analysis in canine lymphoma by calculating maximum fluorescence decrease

Abstract

PARR is widely used in the diagnostics of canine lymphoma. In human and veterinary medicine, melting curve analysis (MCA) has successfully been introduced to facilitate the process. Since visual interpretation of melting curves can be rather subjective, the purpose of this study was to develop an objective interpretation of melting curves by calculating the maximum fluorescence decrease (dF ) within a defined rise of temperature. Lymph node aspirates and blood of 34 dogs with lymphoma and 28 control dogs were tested. 27/34 lymphoma cases were correctly detected to be monoclonal (sensitivity 79%). 2/28 control dogs showed a monoclonal rearrangement (specificity 93%). B- and T-cell neoplasia were still detectable using DNA amount as low as 10 ng. In serial dilutions of tumor DNA with DNA of normal tonsils, the detection limit was 25% for B-cell lymphomas and 100% for T-cell lymphoma, suggesting that PCR conditions could still be optimized.

Dogs with Acute Myeloid Leukemia Have Clonal Rearrangements in T and B Cell Receptors

“…The positive result was confirmed with repeat testing at CSU. The sensitivity of our assay falls within that reported for similar primer sets in other studies (5–7, 10, 20). …”

“…Clonal rearrangements in both B and T cell receptors have been reported in dogs with lymphoid neoplasms; however, the proportion of biclonality in dogs with AML is higher than that reported for dogs with lymphoid neoplasia, which ranges from 1 to 10% (3, 5, 6, 10). These data indicate that if clonality testing is done in a dog with acute leukemia of unclear phenotype, a B and T cell biclonal result should raise the index of suspicion for an underlying AML and prompt further testing for AML, such as expanded testing for myeloid antigen expression on flow cytometric analysis or cytochemical staining.…”

“…We found a similar result in this study, in which tumor cells in dogs with AML expressed B or T cell surface antigens, yet displayed non-clonal, biclonal, or B or T cell receptor clonal rearrangements. Although non-clonal or polyclonal results on clonality testing have been used to support a diagnosis of AML in previous reports (6, 10, 14, 15), false-negative reactions for clonality do occur in lymphoid neoplasms, with reported sensitivities ranging from 67 to 98% (5–7, 10, 20), using similar primer sets to that used herein.…”

“…A lower sensitivity of clonality detection in this study could mean that an even higher proportion of dogs with AML had clonal rearrangements. In addition, the assessment of clonality with high-resolution melt curve analysis is subjective, and there are more quantitative techniques that can be done with this method to reduce subjectivity (10). However, we always performed confirmatory polyacrylamide gel electrophoresis with equivocal or inconclusive results for the high-resolution melt curve analysis and defaulted to the results of the gel if there were discrepancies in interpretation.…”

“…However, we always performed confirmatory polyacrylamide gel electrophoresis with equivocal or inconclusive results for the high-resolution melt curve analysis and defaulted to the results of the gel if there were discrepancies in interpretation. Because a direct comparison between quantitative and subjective assessments was not made in the aforementioned study (10), it remains to be determined if quantitation is truly superior. It is intriguing that clonality in the CDR3 region of the B cell receptor was detected more frequently with primer set 2 than primer set 1.…”