Compositions and methods for cancer and neoplasia assessment
The present disclosure relates to certain compositions, kits, devices, systems and methods, e.g., certain compositions, kits, devices, systems and methods for assessing cancer or neoplasia in a subject. In particular aspects, provided herein are compositions, kits, devices, systems and methods for assessing cancer or neoplasia in a subject based on assessing methylation status of selected target polynucleotide sequences, e.g., target genomic DNA sequences, from the subject.
1 . A panel of isolated polynucleotides comprising, consisting of, or consisting essentially of at least 10 isolated polynucleotides, said 10 isolated polynucleotides having polynucleotide sequences of at least Targets 60, 192, 248, 916, 936, 1349, 1374, 1392, 1435 and 1447, or a complementary sequence thereof,
wherein said isolated polynucleotides have a level or concentration from about 1 femtomolar to about 1 millimolar, and
wherein said polynucleotide sequence of said Target 60 comprises at least 10 consecutive nucleotides in a polynucleotide sequence that is set forth in, that is complementary with or that hybridizes under stringent conditions to, one or more of polynucleotide sequence(s) set forth in SEQ ID NOs: 562-571,
said polynucleotide sequence of said Target 192 comprises at least 10 consecutive nucleotides in a polynucleotide sequence that is set forth in, that is complementary with or that hybridizes under stringent conditions to, one or more of polynucleotide sequence(s) set forth in SEQ ID NOs: 1824-1833,
said polynucleotide sequence of said Target 248 comprises at least 10 consecutive nucleotides in a polynucleotide sequence that is set forth in, that is complementary with or that hybridizes under stringent conditions to, one or more of polynucleotide sequence(s) set forth in SEQ ID NOs: 2353-2362,
said polynucleotide sequence of said Target 916 comprises at least 10 consecutive nucleotides in a polynucleotide sequence that is set forth in, that is complementary with or that hybridizes under stringent conditions to, one or more of polynucleotide sequence(s) forth set in SEQ ID NOs: 8667-8676,
said polynucleotide sequence of said Target 936 comprises at least 10 consecutive nucleotides in a polynucleotide sequence that is set forth in, that is complementary with or that hybridizes under stringent conditions to, one or more of polynucleotide sequence(s) set forth in SEQ ID NOs: 8854-8863,
said polynucleotide sequence of said Target 1349 comprises at least 10 consecutive nucleotides in a polynucleotide sequence that is set forth in, that is complementary with or that hybridizes under stringent conditions to, one or more of polynucleotide sequence(s) set forth in SEQ ID NOs: 12722-12731,
said polynucleotide sequence of said Target 1374 comprises at least 10 consecutive nucleotides in a polynucleotide sequence that is set forth in, that is complementary with or that hybridizes under stringent conditions to, one or more of polynucleotide sequence(s) set forth in SEQ ID NOs: 12966-12975,
said polynucleotide sequence of said Target 1392 comprises at least 10 consecutive nucleotides in a polynucleotide sequence that is set forth in, that is complementary with or that hybridizes under stringent conditions to, one or more of polynucleotide sequence(s) set forth in SEQ ID NO: 13140-13149,
said polynucleotide sequence of said Target 1435 comprises at least 10 consecutive nucleotides in a polynucleotide sequence that is set forth in, that is complementary with or that hybridizes under stringent conditions to, one or more of polynucleotide sequence(s) set forth in SEQ ID NOs: 13560-13569, and
said polynucleotide sequence of said Target 1447 comprises at least 10 consecutive nucleotides in a polynucleotide sequence that is set forth in, that is complementary with or that hybridizes under stringent conditions to, one or more of polynucleotide sequence(s) set forth in SEQ ID NO: 13665-13674.
2 . A kit or system, which comprises a panel of claim 1 .
3 . The kit or system of claim 2 , which is configured for assessing methylation status of at least said Targets 60, 192, 248, 916, 936, 1349, 1374, 1392, 1435 and 1447.
4 . A kit or system, which comprises reagents for assessing methylation status of at least Targets 60, 192, 248, 916, 936, 1349, 1374, 1392, 1435 and 1447,
wherein said reagents comprise, consist essentially of, or consist of probes or primers configured for hybridizing with each of said at least Targets 60, 192, 248, 916, 936, 1349, 1374, 1392, 1435 and 1447, and said probes or primers have a level of concentration from about 1 femtomolar to about 1 millimolar, and
wherein said polynucleotide sequence of said Target 60 comprises at least 10 consecutive nucleotides in a polynucleotide sequence that is set forth in, that is complementary with or that hybridizes under stringent conditions to, one or more of polynucleotide sequence(s) set forth in SEQ ID NOs: 562-571,
said polynucleotide sequence of said Target 192 comprises at least 10 consecutive nucleotides in a polynucleotide sequence that is set forth in, that is complementary with or that hybridizes under stringent conditions to, one or more of polynucleotide sequence(s) set forth in SEQ ID NOs: 1824-1833,
said polynucleotide sequence of said Target 248 comprises at least 10 consecutive nucleotides in a polynucleotide sequence that is set forth in, that is complementary with or that hybridizes under stringent conditions to, one or more of polynucleotide sequence(s) set forth in SEQ ID NOs: 2353-2362,
said polynucleotide sequence of said Target 916 comprises at least 10 consecutive nucleotides in a polynucleotide sequence that is set forth in, that is complementary with or that hybridizes under stringent conditions to, one or more of polynucleotide sequence(s) forth set in SEQ ID NOs: 8667-8676,
said polynucleotide sequence of said Target 936 comprises at least 10 consecutive nucleotides in a polynucleotide sequence that is set forth in, that is complementary with or that hybridizes under stringent conditions to, one or more of polynucleotide sequence(s) set forth in SEQ ID NOs: 8854-8863,
said polynucleotide sequence of said Target 1349 comprises at least 10 consecutive nucleotides in a polynucleotide sequence that is set forth in, that is complementary with or that hybridizes under stringent conditions to, one or more of polynucleotide sequence(s) set forth in SEQ ID NOs: 12722-12731,
said polynucleotide sequence of said Target 1374 comprises at least 10 consecutive nucleotides in a polynucleotide sequence that is set forth in, that is complementary with or that hybridizes under stringent conditions to, one or more of polynucleotide sequence(s) set forth in SEQ ID NOs: 12966-12975,
said polynucleotide sequence of said Target 1392 comprises at least 10 consecutive nucleotides in a polynucleotide sequence that is set forth in, that is complementary with or that hybridizes under stringent conditions to, one or more of polynucleotide sequence(s) set forth in SEQ ID NO: 13140-13149,
said polynucleotide sequence of said Target 1435 comprises at least 10 consecutive nucleotides in a polynucleotide sequence that is set forth in, that is complementary with or that hybridizes under stringent conditions to, one or more of polynucleotide sequence(s) set forth in SEQ ID NOs: 13560-13569, and
said polynucleotide sequence of said Target 1447 comprises at least 10 consecutive nucleotides in a polynucleotide sequence that is set forth in, that is complementary with or that hybridizes under stringent conditions to, one or more of polynucleotide sequence(s) set forth in SEQ ID NO: 13665-13674.
5 . The kit or system of claim 4 , which further comprises a computer readable medium that is configured for obtaining a methylation metric in the form of average methylation frequency, methylation haplotype load, unmethylation haplotype load, percent discordant reads, or a combination thereof.
6 . The kit or system of claim 4 , which is configured for assessing cancer or neoplasia in a subject.
7 . A method for obtaining methylation status of target polynucleotides in a sample of a subject, which method comprises:
a) providing a sample from a subject containing at least 10 target polynucleotides of said subject, said at least 10 target polynucleotides having polynucleotide sequences of at least Targets 60, 192, 248, 916, 936, 1349, 1374, 1392, 1435 and 1447, or a complementary sequence thereof; and
b) assessing methylation status of said at least 10 target polynucleotides; and
wherein said step b) is conducted using a panel of isolated polynucleotides comprising, consisting of, or consisting essentially of at least 10 isolated polynucleotides, said 10 isolated polynucleotides having polynucleotide sequences of at least Targets 60, 192, 248, 916, 936, 1349, 1374, 1392, 1435 and 1447, or a complementary sequence thereof, and said isolated polynucleotides have a level or concentration from about 1 femtomolar to about 1 millimolar, and
wherein said polynucleotide sequence of said Target 60 comprises at least 10 consecutive nucleotides in a polynucleotide sequence that is set forth in, that is complementary with or that hybridizes under stringent conditions to, one or more of polynucleotide sequence(s) set forth in SEQ ID NOs: 562-571,
said polynucleotide sequence of said Target 192 comprises at least 10 consecutive nucleotides in a polynucleotide sequence that is set forth in, that is complementary with or that hybridizes under stringent conditions to, one or more of polynucleotide sequence(s) set forth in SEQ ID NOs: 1824-1833,
said polynucleotide sequence of said Target 248 comprises at least 10 consecutive nucleotides in a polynucleotide sequence that is set forth in, that is complementary with or that hybridizes under stringent conditions to, one or more of polynucleotide sequence(s) set forth in SEQ ID NOs: 2353-2362,
said polynucleotide sequence of said Target 916 comprises at least 10 consecutive nucleotides in a polynucleotide sequence that is set forth in, that is complementary with or that hybridizes under stringent conditions to, one or more of polynucleotide sequence(s) forth set in SEQ ID NOs: 8667-8676,
said polynucleotide sequence of said Target 936 comprises at least 10 consecutive nucleotides in a polynucleotide sequence that is set forth in, that is complementary with or that hybridizes under stringent conditions to, one or more of polynucleotide sequence(s) set forth in SEQ ID NOs: 8854-8863,
said polynucleotide sequence of said Target 1349 comprises at least 10 consecutive nucleotides in a polynucleotide sequence that is set forth in, that is complementary with or that hybridizes under stringent conditions to, one or more of polynucleotide sequence(s) set forth in SEQ ID NOs: 12722-12731,
said polynucleotide sequence of said Target 1374 comprises at least 10 consecutive nucleotides in a polynucleotide sequence that is set forth in, that is complementary with or that hybridizes under stringent conditions to, one or more of polynucleotide sequence(s) set forth in SEQ ID NOs: 12966-12975,
said polynucleotide sequence of said Target 1392 comprises at least 10 consecutive nucleotides in a polynucleotide sequence that is set forth in, that is complementary with or that hybridizes under stringent conditions to, one or more of polynucleotide sequence(s) set forth in SEQ ID NO: 13140-13149,
said polynucleotide sequence of said Target 1435 comprises at least 10 consecutive nucleotides in a polynucleotide sequence that is set forth in, that is complementary with or that hybridizes under stringent conditions to, one or more of polynucleotide sequence(s) set forth in SEQ ID NOs: 13560-13569, and
said polynucleotide sequence of said Target 1447 comprises at least 10 consecutive nucleotides in a polynucleotide sequence that is set forth in, that is complementary with or that hybridizes under stringent conditions to, one or more of polynucleotide sequence(s) set forth in SEQ ID NO: 13665-13674.
8 . The method of claim 7 , which further comprises isolating the target polynucleotides from a sample.
9 . The method of claim 7 , which further comprises preparing a library of the target polynucleotides.
10 . The method of claim 7 , which further comprises amplifying the target polynucleotides.
11 . The method of claim 7 , which further comprises purifying the target polynucleotides, a library of the target polynucleotides, amplified target polynucleotides or a library of amplified target polynucleotides.
12 . The method of claim 7 , wherein the methylation status of target polynucleotides is assessed using mass spectrometry, methylation-specific PCR (MSP), methylation-sensitive sequencing, e.g., bisulphite sequencing, the HpaII tiny fragment Enrichment by ligation-mediated PCR assay (HELP Assay), Glal hydrolysis and ligation adapter dependent PCR assay (GLAD-PCR assay), restriction landmark genomic scanning (RLGS), methylated DNA immunoprecipitation (MeDIP or mDIP), pyrosequencing, molecular break light assay for DNA adenine methyltransferase activity, methyl sensitive Southern blotting or high resolution Melt (HRM) analysis.
13 . The method of claim 7 , wherein the methylation status of target polynucleotides is assessed using methylation-sensitive sequencing, e.g., bisulphite sequencing.
14 . The method of claim 13 , which further comprises, prior to the methylation-sensitive sequencing, e.g., bisulphite sequencing, obtaining a library of linear, single-stranded ligation products, each of the linear, single-stranded ligation products comprises of a linear, single-stranded target polynucleotide linked to an adaptor comprising a unique molecular identifier (UMI) sequence that earmarks the single-stranded target polynucleotide to which the adaptor is ligated.
15 . The method of claim 7 , wherein the methylation status of each of the target polynucleotides is assessed to obtain a methylation metric, e.g., in the form of average methylation frequency, methylation haplotype load, unmethylation haplotype load, percent discordant reads, or a combination thereof.
16 . The method of claim 15 , wherein a numerical methylation matrix is computed using a classification algorithm from the methylation metrics from each of the target polynucleotides to assess methylation status of a sample.
17 . The method of claim 7 , which is used for treatment of cancer or neoplasia in a subject.
18 . The method of claim 17 , wherein cancer or neoplasia in multiple subjects are treated.