| CPC C12Q 1/6874 (2013.01) [C12Q 1/6806 (2013.01); C12Q 1/6883 (2013.01); C12Q 1/6886 (2013.01); G16B 20/00 (2019.02); G16B 25/20 (2019.02); G16B 30/10 (2019.02); C12Q 2600/106 (2013.01); C12Q 2600/118 (2013.01); C12Q 2600/156 (2013.01); G16B 30/00 (2019.02)] | 15 Claims |
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1. A method of diagnosing and therapeutically treating cancer in a subject which comprises:
(A) diagnosing the subject with cancer when a level of minimal residual disease (MRD) in the subject is >0, wherein the MRD is quantified by:
(a)—amplifying by polymerase chain reaction using a PCR instrument and primers, at least one nucleotide sequence comprised in an amount, D, of genomic DNA of a biological sample obtained from said subject after therapeutic treatment for said disease, wherein the genomic DNA has an average weight, k, per diploid cell of said biological sample, wherein said primers comprise a locus-specific forward primer and a locus-specific reverse primer, and said primers identify one specific variant of a nucleotide sequence present in said biological sample, wherein said one variant or the absence of said one variant is indicative of disease; and
sequencing said at least one nucleotide sequence on a massively parallel sequencing platform to obtain at least one first list of characters reading from left to right, wherein said sequencing is massively parallel sequencing;
(b)—amplifying by polymerase chain reaction using a PCR instrument and the same locus-specific forward primer and the same locus-specific reverse primer as in step (a), at least one nucleotide sequence in a biological sample obtained from said subject prior to therapeutic treatment for said disease; and
sequencing said at least one nucleotide sequence on a massively parallel sequencing platform to obtain at least one second list of characters reading from left to right, wherein said sequencing is massively parallel sequencing;
(c) determining a degree of similarity by comparing the at least one first list of characters obtained in step (a) with the at least one second list of characters obtained in step (b), wherein the degree of similarity, DS, of the first list of characters obtained in step (a) with the second list of characters obtained in step (b) is determined using at least one computer program product, either by:
(i) selecting the character or longest continuous sequence of characters which are the same in the first list of characters and the second list of characters, wherein when two or more characters or two or more longest continuous sequences of the same length are selected, only the character or longest continuous sequence of characters which is most to the right in the lists of characters is selected;
(ii) excluding the character or longest continuous sequence of characters selected in step (i) from subsequent steps of selecting a character or longest continuous sequence of characters which is the same in the first list of characters and the second list of characters;
(iii)—selecting the character or longest continuous sequence of characters which is located to the left of the character or longest continuous sequence of characters excluded in step (ii) which are the same in the first list of characters and the second list of characters, wherein when two or more characters or two or more longest continuous sequences of the same length are selected, only the character or longest continuous sequence of characters which is most to the right in the lists of characters is selected; and
selecting the character or longest continuous sequence of characters which is located to the right of the character or longest continuous sequence of characters excluded in step (ii) which are the same in the first list of characters and the second list of characters, wherein when two or more characters or two or more longest continuous sequences of the same length are selected, only the character or longest continuous sequence of characters which is most to the left in the lists of characters is selected;
(iv) excluding each character and/or each longest continuous sequence of characters selected in step (iii) from subsequent steps of selecting a character or longest continuous sequence of characters which is the same in the first list of characters and the second list of characters;
(v)—selecting the character or longest continuous sequence of characters which is located in the continuous sequence of characters to the immediate left of each character or each longest continuous sequence of characters excluded in the previous step which are the same in the first list of characters and the second list of characters, wherein when two or more characters or two or more longest continuous sequences of the same length are selected, only the character or longest continuous sequence of characters which is most to the right in the lists of characters is selected; and
selecting the character or longest continuous sequence of characters which is located in the continuous sequence of characters to the immediate right of each character or each longest continuous sequence of characters excluded in the previous step which are the same in the first list of characters and the second list of characters, wherein when two or more characters or two or more longest continuous sequences of the same length are selected, only the character or longest continuous sequence of characters which is most to the left in the lists of characters is selected;
(vi) excluding each character and/or each longest continuous sequences of characters selected in step (v) from subsequent steps of selecting a character or longest continuous sequence of characters which is the same in the first list of characters and the second list of characters;
(vii) repeating steps (v) and (vi) until no character or longest continuous sequence of characters which is the same in the first list of characters and the second list of characters is selected;
(viii) adding up
the number of characters in the first list of characters which were excluded in any of the steps (i) to (vii); and
the number of characters in the second list of characters which were excluded in any of the steps (i) to (vii)
to obtain the total number of characters, Cc, in the first and second lists of characters which are the same as in the second and first lists of characters, respectively;
(ix) adding up
Cc; and
the number of characters in the first list of characters which are located between the characters and/or longest continuous sequences of characters that were excluded from the first list of characters, and which were not excluded in any of the steps (i) to (vii) of step (c); and
the number of characters in the second list of characters which are located between the characters and/or longest continuous sequences of characters that were excluded from the second list of characters, and which were not excluded in any of the steps (i) to (vii) of step (c),
to obtain the total number of characters, Ct, in the first and second lists of characters; and
(x) calculating DS according to the following formula:
DS=Cc/Ct
or by:
(xi) selecting the character or longest continuous sequence of characters which are the same in the first list of characters and the second list of characters, wherein when two or more characters or two or more longest continuous sequences of the same length are selected, only the character or longest continuous sequence of characters which is most to the right in the lists of characters is selected;
(xii) excluding the character or longest continuous sequence of characters selected in step (xi) from subsequent steps of selecting a character or longest continuous sequence of characters which is the same in the first list of characters and the second list of characters;
(xiii)—selecting the character or longest continuous sequence of characters which is located to the left of the character or longest continuous sequence of characters excluded in step (xii) which are the same in the first list of characters and the second list of characters, wherein when two or more characters or two or more longest continuous sequences of the same length are selected, only the character or longest continuous sequence of characters which is most to the right in the lists of characters is selected; and
selecting the character or longest continuous sequence of characters which is located to the right of the character or longest continuous sequence of characters excluded in step (xii) which are the same in the first list of characters and the second list of characters, wherein when two or more characters or two or more longest continuous sequences of the same length are selected, only the character or longest continuous sequence of characters which is most to the left in the lists of characters is selected;
(xiv) excluding each character and/or each longest continuous sequence of characters selected in step (xiii) from subsequent steps of selecting a character or longest continuous sequence of characters which is the same in the first list of characters and the second list of characters;
(xv) repeating steps (xiii) and (xiv) until no character or longest continuous sequence of characters which is the same in the first list of characters and the second list of characters is selected;
(xvi) adding up
the number of characters in the first list of characters which were excluded in any of the steps (xi) to (xv); and
the number of characters in the second list of characters which were excluded in any of the steps (xi) to (xv)
to obtain the total number of characters, Cc, in the first and second lists of characters which are the same as in the second and first lists of characters, respectively;
(xvii) adding up
Cc; and
the number of characters in the first list of characters which are located between the characters and/or longest continuous sequences of characters that were excluded from the first list of characters, and which were not excluded in any of the steps (xi) to (xv) of step (c); and
the number of characters in the second list of characters which are located between the characters and/or longest continuous sequences of characters that were excluded from the second list of characters, and which were not excluded in any of the steps (xi) to (xv) of step (c),
to obtain the total number of characters, Ct, in the first and second lists of characters; and
(xviii) calculating DS according to the following formula:
DS=Cc/Ct
(d) selecting using at least one computer program product, for each first list of characters obtained in step (a), the DS of highest value, DSHV;
(e) adding up using at least one computer program product, the number of first lists of characters which have a DSHV that is greater than a threshold value, T, to obtain the total number of first lists of characters, Lc, which are the same as a second list of characters;
(f) adding up, using at least one computer program product,
Lc; and
the number of first lists of characters which do not have a DSHV that is greater than T,
to obtain the total number of first lists of characters, Lt; and
(g) calculating using at least one computer program product, the level of minimal residual disease (MRD) according to any of the following formulae:
MRD=(Lc×k)/(Lt×D)
or
MRD=Lc/Lt
or
MRD=Lc×(D/k)/Lt2;
(B) administering a therapeutic amount of chemotherapy to the subject having an MRD>0, wherein the chemotherapy comprises (i) bortezomib plus melphalan and prednisone (VMP) and lenalidomide plus dexamethasone (Rd), and/or (ii) cytarabine and an anthracycline antibiotic or an anthracenedione, optionally followed by additional cytarabine; and
(C) repeating step (A), and when the level of MRD determined in step (A) is >0, repeating step (B), until the level of MRD in step (A) equals 0.
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