Graft-Versus-Lymphoma Effect After Non-Myeloablative Allogeneic Transplant Induces Molecular Remission Assessed by High-Throughput Sequencing of T Cell Receptor in Patients with Advanced Stage Mycosis Fungoides and Sezary Syndrome

Abstract 3114

Mycosis fungoides (MF) is a mature T-cell lymphoma arising primarily from the skin. Patients with advanced stage disease either with skin tumor lesions or its leukemic form, Sezary syndrome (SS), consistently require therapy for disease control and symptom relief. Currently, there is no curative therapy and none of the available therapies provides a long-term remission. We have performed non-myeloablative allogeneic transplant in 11 MF/SS patients using total skin electron beam therapy (TSEBT), total lymphoid irradiation (TLI) and anti-thymocyte globulin (ATG) preparative regimen in an attempt to provide prolonged disease control. The median age was 63 years (range 20–73). All but one patient had stage IV disease at the time of preparative regimen. The median number of prior systemic therapies was 5 (range 2–8). Eight patients achieved complete response after transplant and 3 patients had partial response (ORR 100%). However, 4 patients experienced disease progression post transplant, requiring additional therapy. Patients tolerated the transplant extremely well. Only one patient developed grade 2 acute GVHD (skin), and another patient developed extensive chronic GVHD (skin, oral, GI). All patients were alive at the last evaluation (median follow-up, 10.6 months).

The current method for monitoring residual disease in MF/SS uses flow cytometry to detect circulating Sezary cells and pathological evaluation of skin biopsy samples. However, there are no MF/SS specific tumor markers that can easily and consistently differentiate malignant clones from normal T cells and these standard diagnostic tools lack sensitivity in detecting minimal residual disease (MRD). Here, we tested whether high-throughput sequencing of T cell receptor (TCR) provides a new tool for monitoring MRD after allogeneic transplant. Genomic DNA was extracted from either peripheral blood mononuclear cells or selected skin biopsy samples. The rearranged VDJ of TCR ß was amplified using V ß-specific forward and J ß-specific reverse primers. The Illumina GA2 system generated up to 1, 000, 000 reads of 54 base pairs, covering the entire CDR3 lengths. Raw sequence data were processed to remove PCR/sequencing errors, and a nearest neighbor algorithm was used to collapse the data into unique sequences (Blood 2009, 114 :4099). Of blood samples from six SS patients, malignant clone was identified in all 6 cases by a dominant unique TCR ß CDR3 sequence. At the time of preparative regimen, 3 patients had measurable circulating Sezary cells by standard flow cytometry and pathological evaluation of peripheral blood smear. TCR ß sequencing data showed that malignant clones contributed to 69%, 81% and 68% of circulating T cells in these 3 patients, respectively. The other 3 patients did not have detectable circulating Sezary cells by standard diagnostic tools. However, 8.47%, 0.38% and 0.22% of the TCR ß sequences were from the malignant clones in these 3 cases at the time of preparative regimen. The percentage of malignant T cell clones decreased in all cases immediately after transplant. Three patients eventually achieved molecular remission. Two of them cleared the malignant clone at day+30 and the third cleared the MRD at day+270. The follow-up is still short to determine whether achieving molecular remission correlate with better clinical outcome. We have also started to perform TCR ß sequencing of skin biopsy samples to assess the MRD status in the skin of MF patients. In addition to providing highly sensitive and specific MRD quantification, deep sequencing of TCR also revealed vital information on the T cell repertoire reconstitution after allogeneic transplant. Based on our preliminary data, adaptive transfer of donor TCR repertoire clearly occurred immediately after allogeneic transplant and the TCR diversification slowly appeared months later.

In summary, we have shown a safe and effective unique preparatory regimen for non-myeloablative allogeneic transplant in patients with advanced stage MF and SS. We also demonstrated the utility of high throughput sequencing of CDR3 of TCR ß in assessing MRD status after therapy with utmost sensitivity and specificity, and for the first time, showed a therapy resulting in molecular remission in this patient population.