In addition, to identify patients for entry into defined molecular cohorts treated within the phase I study of crizotinib, we began to screen tumor biopsies from selected NSCLC patients for MET gene copy number alterations and for ALK gene rearrangements by FISH.Briefly, tumorrich regions were identified in each block of formalinfixed, paraffinembedded tissue.Tissue cores were then obtained from these regions using a microarrayer device and mm arrayer needles. T issue cores were deparaffinized by soaking in xylene overnight and subsequently digested with protease K.EGFR exons, and and KRAS exon were then amplified and sequenced using a ABI model capillary gel sequencer.For both probe sets, assays were done according to a protocol previously described. Signals were enumerated in at least tumor nuclei per core using an epifluorescence microscope with single interference filters sets for green and triple bandpass filters.In the MET FISH assay, specimens were classified as carrying gene amplification when the METCEP ratio was or when tumor cells carried clusters or copies per cell of MET signals.For documentation, images were captured using a chargecoupled device. The occurrence of an ALK rearrangement wasconc ludedif of tumorcellsshowed split red and green signals andor single red signals; otherwise, the specimen was classified as ALK FISH negative.Beginning at a selected starting corner and using thehighpower ob jective, a tumorarea was selec ted and representative tumor cells were analyzed.Then, the microscope stage was moved mm along the Y axis and another tumor area was chosen for analysis of tumor cells.A similar sequence of steps was then used to reach the third location, and so on.In the event that a selected location included nontumor cells such as lymphocytes, macrophages, or smooth muscle cells, this area was identified as N, and of these cells were analyzed.When reaching an area without tissue, the microscope stage was moved mm along the X axis and the stage was again moved mm along the Y axis, looking for tumor areas.This strategy Cefadroxil guaranteed that specimens were scanned in entirety, that no overlap occurred among different areas, and that no cell was counted in more than one area.Following this schema, from to tumor areas and from to nontumor areas were analyzed in each specimen.If the average percent of positive cells in the permutation was, we called that permutation ALK posit ive, andvice versa for ALK negative. We defined TP and TN as the tumor areas that were obtained and correctly determined as ALK positive and negative in patients who, overall, were concluded as ALK positive and negative, respectively.From this permutation test, we could calculate sensitivity for the number of highpower microscope fields and number of tumor nuclei required to determine ALK positivity.Of these, four cases could not be analyzed due to insufficient tumor material Carvacrol remaining in the biopsy specimen and three cases could not be analyzed due to assay failure, in one case due to DNA degradation following decalcification of a bone biopsy specimen.From the remaining spec imens, cases were posit ive for ALK gene rearrangements. Normal signals are frequently seen in cells that also display ALK gene rearrangements.

Leave a Reply