RESUMEN
BACKGROUND: A recurrent breast cancer patient received high-dose chemotherapy, a transplant of multidrug resistance 1 (MDR1)-transduced cells and four different protocols of post-transplantation chemotherapy. We report the analysis of MDR1-transduced cells in this patient. METHODS: MDR1 transgene levels in the peripheral blood mononuclear cells of the patient were evaluated by polymerase chain reaction (PCR). Retroviral integration sites of the MDR1-transduced cells were identified by linear amplification-mediated (LAM)-PCR. RESULTS: Twelve days after transplantation, approximately 1% of the peripheral blood mononuclear cells were MDR1 transgene-positive. The transgene levels decreased quickly, and were at low levels until day 504. A remarkable increase in MDR1 transgene-positive cells was observed on day 532, during combination chemotherapy with mitomycin C and methotrexate. Using LAM-PCR, 31 MDR1-transduced clones were identified, and eight of these were long-life clones that survived for more than 500 days. Among the 31 clones, ten had a retroviral integration site near genes listed in the Retroviral Tagged Cancer Gene (RTCG) Database. Two long-life clones, N-30 and N-31, had retroviral integration sites within the MDS1-EVI1 locus. Another two long-life clones had integration sites close to PRDM16 or CUEDC1. CONCLUSIONS: These results suggest that MDR1-transduced cells were enriched in vivo by an MDR1 substrate, mitomycin C. The possible activation of EVI1 or other RTCGs by retroviral insertion may have affected the survival and persistence of a proportion of the transduced cells.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Trasplante de Células Madre Hematopoyéticas , Metotrexato/uso terapéutico , Mitomicina/uso terapéutico , Transducción Genética , Proliferación Celular/efectos de los fármacos , Células Clonales , Proteínas de Unión al ADN/genética , Femenino , Genes Relacionados con las Neoplasias/genética , Humanos , Proteína del Locus del Complejo MDS1 y EV11 , Metotrexato/farmacología , Mitomicina/farmacología , Reacción en Cadena de la Polimerasa , Proto-Oncogenes/genética , Retroviridae/genética , Factores de Transcripción/genética , Transgenes/genética , Integración Viral/efectos de los fármacosRESUMEN
A clinical study of an MDR1 gene therapy protocol targeting metastatic breast cancer has been conducted in which the patients received high-dose chemotherapy, a transplant of MDR1-transduced autologous CD34(+) cells, and docetaxel. We herein report the molecular results of a 6-year follow-up of an individual in this study (patient 1). HaMDR-transduced cells, which had been initially detected in the peripheral blood of this individual, were found to have gradually decreased. After 10 cycles of docetaxel (days 71-316), MDR1 transgene levels were found to have increased, and then decreased to undetectable levels by day 1461. Thirty-eight MDR1-transduced clones were identified in patient 1, of which 11 showed a retroviral integration in close proximity to genes listed in the Retrovirus Tagged Cancer Gene Database (RTCGD). Four short-life clones in this group were found to harbor retroviral integrations close to the ZFHX1B, NOTCH1, BMI1, or HHEX gene; these genes have been frequently reported in the RTCGD. In addition, a long-lived RTCGD-hit clone, L-34, had a retroviral integration at a position 179 kb upstream of the EVI1 gene. L-34 was detectable on days 327-1154, but became undetectable 3 years after the docetaxel treatments had ceased. An additional three docetaxel-induced long-life clones showed comparable polymerase chain reaction profiles, which were also similar to that of the total MDR1-transduced cells. Our results thus show that docetaxel may have been effective in promoting the expansion of several MDR1-transduced clones in patient 1, but that they persist in the peripheral blood for only a few years.
