US 20180044722A1 ( 19) United States ( 12 ) Patent Application Publication (10 ) Pub. No. : US 2018 /0044722 A1 Ruvolo et al. (43 ) Pub . Date : Feb . 15 , 2018 ( 54 ) TRI- COLOR PROBES FOR DETECTING ( 52 ) U . S . CI. MULTIPLE GENE REARRANGEMENTS IN A CPC .. .. C120 1 / 6841 (2013 .01 ) ; C120 1 /6886 FISH ASSAY ( 2013 .01 ) ; C12Q 2600 / 156 ( 2013 .01 ) ( 57 ) ABSTRACT ( 71 ) Applicant: Agilent Technologies , Inc. , Santa Clara , A probe system is provided . In some embodiments , the CA (US ) probe system may comprise : a first labeled probe that hybridizes to one side of a potential translocation breakpoint ( 72 ) Inventors : Michael Ruvolo , San Jose , CA (US ) ; in a first locus ; a second labeled probe that hybridizes to the Jimmy Jin , Fremont, CA (US ) other side of the potential translocation breakpoint in the first locus ; a third labeled probe that hybridizes to one side of a potential translocation breakpoint in a second locus; a (21 ) Appl . No. : 15 / 236 , 264 fourth labeled probe that hybridizes to the other side of the potential translocation breakpoint in the second locus ; and a ( 22 ) Filed : Aug . 12, 2016 fifth labeled probe that hybridizes to both sides of the potential translocation breakpoint in either the first locus or the second locus, but not both . The fifth probe is distin guishably labeled from the first , second , third and fourth Publication Classification probes . Methods for detecting a chromosomal rearrange (51 ) Int. Ci. ment in the first and second loci using the probe system are C12Q 1/ 68 ( 2006 . 01) also provided . Po R + - K -- 2020 - C 16 Patent Application Publication Feb . 15 , 2018 Sheet 1 of 3 US 2018 /0044722 A1 PR- Zci- - - 20 ven L 16 FIG . 1 Patent Application Publication Feb . 15 , 2018 Sheet 2 of 3 US 2018 /0044722 A1 Breakpoint 896 , 89 116 $ ,godt 17 . 980 , 9801 12 , 480 , 488 ) 119 , 00 , $ cal 112 508 , 985 (1984 , tools .' Chrow as localiza 69 FIM kuona XXXXX Kom ons Berk , oc , WW , W 006 EKKER rest 16001 K4001, KAKS KOGU N KI Cerca . .. Mb . 5 .: 29 , 509 , 000 30 , 99 , 900 ETU A . ALSO. Ursor : 3rrrrrrrrrr 98 Re Chromosome Bands Localized by FISH Mapp mmmm UOSO Genes (Refsed , Genbank ) , Rram , ORNAS &R o ROYAX A : CLIP4 - - HII TERA SDYR I FAM1790 ALM ? - - : : : : CLIP4 III . CB FAMI79AHIRMUK 988 . SÈDIA & FAM2790 ALK HMW - * SFDYA C2017101 AN ) . ID • * * 2 : 4 = > - < 3 : FIG . 2 Patent Application Publication Feb . 15 , 2018 Sheet 3 of 3 US 2018 /0044722 A1 27 25 Ban FIG . 3 . .. .; ' . : ' . 9 : . ... : .. 038 1280 ( ong Merge FIG . 4 US 2018 /0044722 A1 Feb . 15 , 2018 TRI- COLOR PROBES FOR DETECTING publication with color drawing ( s ) will be provided by the MULTIPLE GENE REARRANGEMENTS IN A U . S . Patent and Trademark Office upon request and payment FISH ASSAY of the necessary fee . [ 0007 ] FIG . 1 schematically illustrates certain features of BACKGROUND the present probe system . [0008 ] FIG . 2 is a diagram showing the position of the [0001 ] Fluorescence in situ hybridization ( FISH ) is a constituent probes relative to their target genes , ROS1 (panel cytogenetic technique that uses fluorescent probes that bind A ), and ALK (panel B ) . to only those parts of the chromosome with a high degree of [0009 ] FIG . 3 shows results obtained using a ROS1 rear sequence complementarity . FISH has long been used to ranged sample , showing ROS1 probe signal pattern and the detect chromosomal rearrangements and , because chromo ALK probe signal pattern in the same cell . A ) Co -localiza somal rearrangements are quite common in human cancer, tion of Cy3 and Aqua but no FITC signal indicates that this FISH is often used to diagnose or assess a malignancy . cell has a ROS1 rearrangement. B ) Co - localization of cy3 [ 0002 ] Because chromosomal rearrangements are thought and FITC signal with lack of Aqua signal indicates non to initiate many human cancers , detection of such rearrange rearranged ALK in this sample . ments can provide a rationale for treating patients in a [ 0010 ] FIG . 4 shows results obtained using ALK rear particular way . For example , if a non -small - cell lung cancer ranged sample showing lone red and green signals . The is associated with a translocation in ALK or ROS1 , then the non - rearranged ROS1 gene shows a red / green /blue signal patient can potentially be treated with the same therapy , e . g ., pattern . crizotinib , which inhibits both ALK and ROS1 ( see , gener ally, Soloman et al J . Clin . Onc . 2015 33 : 972 - 4 ) . DEFINITIONS [0003 ] Current FISH methods for detecting particular rear [0011 ] The term “ nucleic acid” and “ polynucleotide ” are rangements are limited in that they only able to reliably used interchangeably herein to describe a polymer of any detect such rearrangements one -by -one (see , e . g . , Bergethon length , e . g . , greater than about 2 bases , greater than about 10 et al J Clin Oncol 2012 30 : 863 - 870 ) . This can be problem bases, greater than about 100 bases , greater than about 500 atic in situations where there is limited sample , which is bases , greater than 1000 bases, up to about 10 , 000 or more often the case . Further , performing two separate assays bases composed of nucleotides , e . g . , deoxyribonucleotides doubles the time to reach a diagnosis , which can lead to a or ribonucleotides, and may be produced enzymatically or delay in diagnosis and treatment. Multiplex detection of two synthetically ( e . g . , PNA as described in U . S . Pat . No . or more distinct chromosomal rearrangements by FISH 5 , 948 , 902 and the references cited therein ) which can would greatly aid in cancer prognostics , diagnostics and hybridize with naturally occurring nucleic acids in a theranostics and , as such , are highly desirable . sequence specific manner analogous to that of two naturally occurring nucleic acids , e . g . , can participate in Watson SUMMARY Crick base pairing interactions . Naturally -occurring nucleo [ 0004 ] Among other things , a probe system is provided . In tides include guanine , cytosine , adenine and thymine (G , C , some embodiments , the probe system may comprise: a first A and T , respectively ) . labeled probe that hybridizes to one side of a potential [0012 ] The term “ oligonucleotide ” as used herein denotes translocation breakpoint in a first locus; a second labeled a single stranded multimer of nucleotide of about 2 to 200 probe that hybridizes to the other side of the potential or more , up to about 500 nucleotides or more. Oligonucle translocation breakpoint in the first locus ; a third labeled otides may be synthetic or may be made enzymatically, and , probe that hybridizes to one side of a potential translocation in some embodiments , are less than 10 to 50 nucleotides in breakpoint in a second locus ; a fourth labeled probe that length . Oligonucleotides may contain ribonucleotide mono hybridizes to the other side of the potential translocation mers ( i. e ., may be oligoribonucleotides ) or deoxyribonucle breakpoint in the second locus; and a fifth labeled probe that otide monomers. Oligonucleotides may be 10 to 20 , 11 to 30 , hybridizes to both sides of the potential translocation break 31 to 40 , 41 to 50 , 51 - 60 ,61 to 70 , 71 to 80 , 80 to 100 , 100 point in either the first locus or the second locus, but not to 150 or 150 to 200 nucleotides in length , for example . both . The first and second probes are distinguishably [0013 ]. The term “ sequence - specific oligonucleotide ” as labeled , the third and fourth probes are distinguishably used herein refers to an oligonucleotide that only binds to a labeled , and the fifth probe is distinguishably labeled from single site in a haploid genome. In certain embodiments , a the first , second , third and fourth probes. “ sequence - specific ” oligonucleotide may hybridize to a [ 0005 ] A method for detecting a chromosomal rearrange complementary nucleotide sequence that is unique in a ment using the probe system is also provided . In some sample under study . embodiments , this method may comprise: ( a ) hybridizing [0014 ] The term “ complementary ” as used herein refers to the probe system with a chromosome in situ to produce to a nucleotide sequence that base -pairs by non - covalent bonds produce a labeled sample ; ( b ) reading the labeled sample to to a target nucleic acid of interest . In the canonical Watson detect hybridization of the labeled probes ; and ( c ) determin Crick base pairing , adenine ( A ) forms a base pair with ing whether the sample contains a rearrangement in the first thymine ( T ) , as does guanine (G ) with cytosine ( C ) in DNA . In RNA , thymine is replaced by uracil ( U ) . As such , A is or second locus using the results obtained from the reading complementary to T and G is complementary to C . In RNA , step ( b ) . A is complementary to U and vice versa . Typically, “ comple mentary ” refers to a nucleotide sequence that is fully BRIEF DESCRIPTION OF THE FIGURES complementary to a target of interest such that every nucleo 10006 ] The patent or application file contains at least one tide in the sequence is complementary to every nucleotide in drawing executed in color . Copies of this patent application the target nucleic acid in the corresponding positions . In US 2018 /0044722 A1 Feb . 15 , 2018 certain cases , a nucleotide sequence may be partially a nucleic acid to a complementary nucleic acid in an complementary to a target , in which not all nucleotide is interphase or metaphase cell that contains relatively intact complementary to every nucleotide in the target nucleic acid chromosomes ( some fragmentation occurs during the pro in all the corresponding positions. cess ) . Suitable in situ hybridization conditions may include [0015 ] The terms “ determining ” , “ measuring ” , “ evaluat both hybridization conditions and optional wash conditions , ing ” , “ assessing ” , “ analyzing ” , and “ assaying ” are used which include temperature , concentration of denaturing interchangeably herein to refer to any form of measurement, reagents , salts , incubation time, etc .