The difficulty of detecting HTLV-1 proviral sequences in patients with mycosis fungoides

Although most patients with cutaneous T cell lymphomas, including mycosis fungoides (MF) and its leukemic variant, the Sezary syndrome, are seronegative for antibodies to the human T cell lymphotropic viruses (HTLV-I/II), it has recently been shown that >95% of such patients harbor proviral DNA sequences related to the region of the HTLV genome that encodes the transregulatory/transforming gene, tax, However, the demonstration of HTLV sequences, even after amplification by polymerase chain reaction (PCR), has not been universally successful, and some investigators continue to question this observation. In an effort to resolve this controversy, we have compared published methodologies that have been less successful with techniques currently used in this laboratory. Major differences were found in (a) the nature of the cells used [freshly isolated versus cultured peripheral blood mononuclear cells (PBMC)] and (b) the methods used to prepare samples for PCR (whole cell lysates versus DNA extracts). PBMC from 10 different MF patients and the healthy daughter of 1 of the patients were subjected to comparative analyses. While all of the PBMC lysates were positive, the DNA extract from only one of these individuals revealed HTLV tax sequences. Studies were also conducted comparing cell lysates and DNA extracts of cultured cells derived from tax sequence-positive PBMC from seven different MF patients. The cells from four of the seven were shown to have retained tax sequences after varying times in culture, when whole-cell lysates were used as targets for PCR amplification and Southern analysis, whereas none of the DNA extracts wore positive. It appears that the use of whole-cell lysates instead of DNA extracts and the use of fresh instead of cultured cells greatly enhance the ability to detect HTLV-1 tax sequences in specimens from MF patients.