Multiplex pcr detection of alk, ret, and ros fusions

Abstract

Provided herein are methods and compositions for multiplex detection of a large number of actionable gene fusions with very high sensitivity and specificity. The present methods and compositions can detect ALK, RET, and ROS1 gene fusions, optionally in combination with other mutations and fusions.

We claim: 
     
         1 . A method of treating an individual with cancer comprising:
 A. contacting a biological sample from the individual with a composition comprising:
 (i) at least one primer set and probe that specifically amplify and detect at least one ALK fusion gene, wherein the probe that specifically detects the at least one ALK fusion gene is labeled with at least one non-naturally occurring moiety; 
 (ii) at least one primer set and probe that specifically amplify and detect at least one RET fusion gene, wherein the probe that specifically detects the at least one RET fusion gene is labeled with at least one non-naturally occurring moiety; 
 (iii) at least one primer set and probe that specifically amplify and detect at least one ROS1 fusion gene, wherein the probe that specifically detects the at least one ROS1 fusion gene is labeled with at least one non-naturally occurring moiety; and 
 (iv) a primer and probe that specifically amplify and detect an internal control, wherein the probe that specifically detects the internal control is labeled with at least one non-naturally occurring moiety; 
   B. carrying out amplification and detection under conditions that allow formation and detection of an amplification product in the presence of at least one fusion gene in the biological sample;   C. determining that at least one fusion gene is present if a fusion gene is detected in step B; and   D. treating the individual with a kinase inhibitor therapy if at least one fusion gene is present.   
     
     
         2 . The method of  claim 1 , wherein the at least one ALK fusion gene is selected from the group consisting of: EML4 exon 13-ALK exon 20, EML4 exon 20-ALK exon 20, EML4 exon 6a/b-ALK exon 20, EML4 exon 2-ALK exon 20, EML4 exon 18-ALK exon 20, KIF5B exon 17-ALK exon 20, and KIF5B exon 24-ALK exon 20. 
     
     
         3 . The method of  claim 1 , wherein the at least one RET fusion gene is selected from the group consisting of: KIF5B exon 15-RET exon 12, KIF5B exon 16-RET exon 12, KIF5B exon 22-RET exon 12, KIF5B exon 23-RET exon 12, CCDC6 exon 1-RET exon 12, and NCOA4 exon 6-RET exon 12. 
     
     
         4 . The method of  claim 1 , wherein the at least one ROS1 fusion gene is selected from the group consisting of: CD74 exon 6-ROS1 exon 34, CD74 exon 6-ROS1 exon 32, EZR exon 10-ROS1 exon 34, TPM3 exon 8-ROS1 exon 35, SDC4 exon 4-ROS1 exon 34, SDC4 exon 2-ROS1 exon 34, SDC4 exon 2-ROS1 exon 32, SDC4 exon 4-ROS1 exon 32, SLC34A2 exon 13-ROS1 exon 34, SLC34A2 exon 13-ROS1 exon 32v2, SLC34A2 exon 4-ROS1 exon 32, SLC34A2 exon 4-ROS1 exon 35, and LRIG3 exon 16-ROS1 exon 35. 
     
     
         5 . The method of  claim 1 , wherein the biological sample includes DNA or RNA. 
     
     
         6 . The method of  claim 1 , wherein the biological sample is RNA from plasma of the individual. 
     
     
         7 . The method of  claim 1 , wherein the amplification and detection are carried out using quantitative reverse transcription polymerase chain reaction (qRT-PCR). 
     
     
         8 . The method of any one of  claim 1 , wherein the kinase inhibitor therapy is selected from the group consisting of alectinib, crizotinib, ceritinib, lorlatinib, brigatinib, cabozantinib, apatinib, vandetanib, ponatinib, lenvatinib, DS6051b, or a variant thereof. 
     
     
         9 . A method for determining the presence of at least one fusion gene in a biological sample from an individual with cancer comprising:
 A. contacting a biological sample from the individual with a composition comprising:
 (i) at least one primer set and probe that specifically amplify and detect at least one ALK fusion gene, wherein the probe that specifically detects the at least one ALK fusion gene is labeled with at least one non-naturally occurring moiety; 
 (ii) at least one primer set and probe that specifically amplify and detect at least one RET fusion gene, wherein the probe that specifically detects the at least one RET fusion gene is labeled with at least one non-naturally occurring moiety; 
 (iii) at least one primer set and probe that specifically amplify and detect at least one ROS1 fusion gene, wherein the probe that specifically detects the at least one ROS1 fusion gene is labeled with at least one non-naturally occurring moiety; and 
 (iv) a primer and probe that specifically amplify and detect an internal control, wherein the probe that specifically detects the internal control is labeled with at least one non-naturally occurring moiety; 
   B. carrying out amplification and detection under conditions that allow formation and detection of an amplification product in the presence of at least one fusion gene in the biological sample; and   C. determining the presence of at least one fusion gene if a fusion gene is detected in step B.   
     
     
         10 . The method of  claim 9 , wherein the at least one ALK fusion gene is selected from the group consisting of: EML4 exon 13-ALK exon 20, EML4 exon 20-ALK exon 20, EML4 exon 6a/b-ALK exon 20, EML4 exon 2-ALK exon 20, EML4 exon 18-ALK exon 20, KIF5B exon 17-ALK exon 20, and KIF5B exon 24-ALK exon 20. 
     
     
         11 . The method of  claim 9 , wherein the at least one RET fusion gene is selected from the group consisting of: KIF5B exon 15-RET exon 12, KIF5B exon 16-RET exon 12, KIF5B exon 22-RET exon 12, KIF5B exon 23-RET exon 12, CCDC6 exon 1-RET exon 12, and NCOA4 exon 6-RET exon 12. 
     
     
         12 . The method of  claim 9 , wherein the at least one ROS1 fusion gene is selected from the group consisting of: CD74 exon 6-ROS1 exon 34, CD74 exon 6-ROS1 exon 32, EZR exon 10-ROS1 exon 34, TPM3 exon 8-ROS1 exon 35, SDC4 exon 4-ROS1 exon 34, SDC4 exon 2-ROS1 exon 34, SDC4 exon 2-ROS1 exon 32, SDC4 exon 4-ROS1 exon 32, SLC34A2 exon 13-ROS1 exon 34, SLC34A2 exon 13-ROS1 exon 32v2, SLC34A2 exon 4-ROS1 exon 32, SLC34A2 exon 4-ROS1 exon 35, and LRIG3 exon 16-ROS1 exon 35. 
     
     
         13 . The method of  claim 9 , wherein the biological sample includes DNA or RNA. 
     
     
         14 . The method of  claim 9 , wherein the biological sample is RNA from plasma of the individual. 
     
     
         15 . The method of  claim 9 , wherein the amplification and detection are carried out using quantitative reverse transcription polymerase chain reaction (qRT-PCR). 
     
     
         16 . The method of  claim 1 , wherein:
 (i) the at least one primer set that specifically amplifies the at least one ALK fusion gene comprises a forward primer comprising SEQ ID NO: 1 and a reverse primer comprising SEQ ID NO:52, and the probe that specifically detects the at least one ALK fusion gene comprises SEQ ID NO: 182;   (ii) the at least one primer set that specifically amplifies the at least one RET fusion gene comprises a forward primer comprising SEQ ID NO: 83 and a reverse primer comprising SEQ ID NO: 161, and the probe that specifically detects the at least one RET fusion gene comprises SEQ ID NO: 189; and   (iii) the at least one primer set that specifically amplifies the at least one ROS1 fusion gene comprises a forward primer comprising SEQ ID NO: 195 and a reverse primer comprising SEQ ID NO: 213, and the probe that specifically detects the at least one ROS1 fusion gene comprises SEQ ID NO: 227.   
     
     
         17 . The method of  claim 9 , wherein:
 (i) the at least one primer set that specifically amplifies the at least one ALK fusion gene comprises a forward primer comprising SEQ ID NO: 1 and a reverse primer comprising SEQ ID NO:52, and the probe that specifically detects the at least one ALK fusion gene comprises SEQ ID NO: 182;   (ii) the at least one primer set that specifically amplifies the at least one RET fusion gene comprises a forward primer comprising SEQ ID NO: 83 and a reverse primer comprising SEQ ID NO: 161, and the probe that specifically detects the at least one RET fusion gene comprises SEQ ID NO: 189; and   (iii) the at least one primer set that specifically amplifies the at least one ROS1 fusion gene comprises a forward primer comprising SEQ ID NO: 195 and a reverse primer comprising SEQ ID NO: 213, and the probe that specifically detects the at least one ROS1 fusion gene comprises SEQ ID NO: 227.