Application of advanced cytometric and molecular technologies to minimal residual disease monitoring

摘要

Abstract: Minimal residual disease monitoring presents a numberof theoretical and practical challenges. Recently ithas been possible to meet some of these challenges bycombining a number of new advanced biotechnologies. Tomonitor the number of residual tumor cells requirescomplex cocktails of molecular probes that collectivelyprovide sensitivities of detection on the order of oneresidual tumor cell per million total cells.Ultra-high-speed, multi parameter flow cytometry iscapable of analyzing cells at rates in excess of100,000 cells/sec. Residual tumor selection markercocktails can be optimized by use of receiver operatingcharacteristic analysis. New data minimizing techniqueswhen combined with multi variate statistical or neuralnetwork classifications of tumor cells can moreaccurately predict residual tumor cell frequencies. Thecombination of these techniques can, under at leastsome circumstances, detect frequencies of tumor cellsas low as one cell in a million with an accuracy ofover 98 percent correct classification. Detection ofmutations in tumor suppressor genes requires insolationof these rare tumor cells and single-cell DNAsequencing. Rare residual tumor cells can be isolatedat single cell level by high-resolution single-cellcell sorting. Molecular characterization of tumorsuppressor gene mutations can be accomplished using acombination of single- cell polymerase chain reactionamplification of specific gene sequences followed by TAcloning techniques and DNA sequencing. Mutations assmall as a single base pair in a tumor suppressor geneof a single sorted tumor cell have been detected usingthese methods. Using new amplification procedures andDNA micro arrays it should be possible to extend thecapabilities shown in this paper to screening ofmultiple DNA mutations in tumor suppressor and othergenes on small numbers of sorted metastatic tumorcells. !23