	US 11,781,189 B2
	Method for determining the presence or absence of minimal residual disease (MRD) in a subject who has been treated for a disease
Santiago Barrio García, Madrid (ES); Rosa María Ayala Díaz, Madrid (ES); María Inmaculada Rapado Martinez, Madrid (ES); Eva María Garrido Martín, Madrid (ES); Luis Paz-Ares Rodriguez, Madrid (ES); Maria Esther Onecha De La Fuente, Palencia (ES); and Joaquín Martínez López, Madrid (ES)
Assigned to Fundación para la Investigación Biomédica del Hospital Universitario 12 de Octubre; and Fundación del Sector Público Estatal Centro Nacional de Investigaciones Oncológicas Carlos III (F.S.P. CNIO)
Appl. No. 17/638,035
Filed by FUNDACIÓN PARA LA INVESTIGACIÓN BIOMÉDICA DEL HOSPITAL UNIVERSITARIO 12 DE OCTUBRE, Madrid (ES); and FUNDACIÓN DEL SECTOR PÚBLICO ESTATAL CENTRO NACIONAL DE INVESTIGACIONES ONCOLOGICAS CARLOS III (F.S.P. CNIO), Madrid (ES)
PCT Filed Aug. 27, 2020, PCT No. PCT/EP2020/073960 § 371(c)(1), (2) Date Feb. 24, 2022, PCT Pub. No. WO2021/037971, PCT Pub. Date Mar. 4, 2021.
Claims priority of application No. 19382730 (EP), filed on Aug. 27, 2019.
Prior Publication US 2022/0380852 A1, Dec. 1, 2022
Int. Cl. G16B 30/10 (2019.01); G16B 20/20 (2019.01); G16B 25/20 (2019.01); G16B 40/20 (2019.01); C12Q 1/6886 (2018.01); C12Q 1/6858 (2018.01); C12Q 1/6869 (2018.01)

CPC C12Q 1/6886 (2013.01) [G16B 20/20 (2019.02); G16B 25/20 (2019.02); G16B 30/10 (2019.02); G16B 40/20 (2019.02); C12Q 1/6858 (2013.01); C12Q 1/6869 (2013.01); C12Q 2600/112 (2013.01); C12Q 2600/156 (2013.01)]

20 Claims

1. A method of therapeutically treating a proliferative disease in a subject diagnosed with said disease, wherein said method comprises:

(1) administering therapy to the subject, wherein said therapy is selected from chemotherapy, immunotherapy, radiotherapy, or combinations thereof:

(2) determining the presence or absence of minimal residual disease (MRD) in the subject, wherein the determination comprises the following steps:

(A)—amplifying by polymerase chain reaction using a pair of primers comprising a locus-specific forward primer and a locus-specific reverse primer, at least one nucleotide sequence comprised in genomic DNA from a biological sample obtained from said subject prior to treatment for said disease; and

sequencing each amplified nucleotide sequence, whereby a first list of characters reading from left to right is obtained from each nucleotide sequence thus sequenced;

(B)—amplifying by polymerase chain reaction using the same locus-specific forward primer and the same locus-specific reverse primer as in step (A), at least one nucleotide sequence comprised in an amount, D, of genomic DNA from a biological sample obtained from said subject after treatment for said disease, wherein the genomic DNA has an average weight, k, per diploid cell of said biological sample; and

sequencing each amplified nucleotide sequence, whereby a second list of characters reading from left to right is obtained from each nucleotide sequence thus sequenced;

wherein each nucleotide sequence amplified in steps (A) and (B) is shorter than 400 nucleotides and is either a mutated nucleotide sequence or a non-mutated nucleotide sequence of a gene, wherein when a nucleotide sequence is mutated it is a genetic marker comprising a mutation selected from the group of a single nucleotide variant mutation, an indel mutation and somatic gene rearrangement mutation;

(C) determining, for each second list of characters obtained in step (B), a degree of similarity with each first list of characters obtained in step (A), wherein the degree of similarity, DS, of a second list of characters obtained in step (B) with a first list of characters obtained in step (A) is determined by:

(i) counting a total number of characters, C_c, in the second and first lists of characters which are the same as in the first and second lists of characters, respectively;

(ii) counting a total number of characters, C_t, in the first and second lists of characters; and

(iii) calculating DS according to the following formula:

DS=C_c/C_t

(D) selecting, for each second list of characters obtained in step (B), the DS of highest value, DS_HV;

(E) adding up the number of second lists of characters which have a DS_HVthat is greater than a threshold value, T, to obtain the total number of second lists of characters, L_c, which are the same as a first list of characters;

(F) adding up

(i) L_c; and

(ii) the number of second lists of characters which do not have a DS_HVthat is greater than T,

to obtain the total number of second lists of characters, L_t; and

(G) calculating the level of minimal residual disease, MRD, according to any of the following formulae:

MRD=(L_c×k)/(L_t×D)

MRD=L_c/L_t

MRD=g×L_c×(D/k)/L_t²

wherein g is the number of gene copies per cell, D is in units of ng and k is in units of ng/cell;

(H) determining:

(i) a minimum variant read frequency, min VRF, of said genetic marker, wherein min VRF is calculated according to the following formula:

min VRF=k/D

wherein D and k are as defined above; and

(ii) a limit of detection, D-limit, of said genetic marker, by:

(a) obtaining a first composition by diluting one part of a solution of genomic DNA comprising said genetic marker with 10 parts of a solution of genomic DNA which does not comprise said genetic marker;

(b) determining the level of MRD of said genetic marker in said first composition;

(c) obtaining a second composition by diluting one part of said first composition with 10 parts of a solution of genomic DNA which does not comprise said genetic marker;

(d) determining the level of MRD of said genetic marker in said second composition;

(e) obtaining a third composition by diluting one part of said second composition with 10 parts of a solution of genomic DNA which does not comprise said genetic marker;

(f) determining the level of MRD of said genetic marker in said third composition;

(g) obtaining a fourth composition by diluting one part of said third composition with 10 parts of a solution of genomic DNA which does not comprise said genetic marker;

(h) determining the level of MRD of said genetic marker in said fourth composition;

(i) calculating:

an average logarithm of the level of MRD, av log MRD1, of said genetic marker in the first, second and third compositions and an average logarithm of the concentration, av log C1, of said genetic marker in the first, second and third compositions; and

an average logarithm of the level of MRD, av log MRD2, of said genetic marker in the second, third and fourth compositions and an average logarithm of the concentration, av log C2, of said genetic marker in the second, third and fourth compositions;

(j) calculating:

a difference, D1A, between the logarithm of the level of MRD of said genetic marker in the first composition and the av log MRD1;

a difference, D1B, between the logarithm of the level of MRD of said genetic marker in the second composition and the av log MRD1;

a difference, D1C, between the logarithm of the level of MRD of said genetic marker in the third composition and the av log MRD1;

a difference, D1D, between the logarithm of the concentration of said genetic marker in the first composition and the av log C1;

a difference, D1E, between the logarithm of the concentration of said genetic marker in the second composition and the av log C1;

a difference, D1F, between the logarithm of the concentration of said genetic marker in the third composition and the av log C1;

a difference, D2A, between the logarithm of the level of MRD of said genetic marker in the second composition and the av log MRD2;

a difference, D2B, between the logarithm of the level of MRD of said genetic marker in the third composition and the av log MRD2;

a difference, D2C, between the logarithm of the level of MRD of said genetic marker in the fourth composition and the av log MRD2;

a difference, D2D, between the logarithm of the concentration of said genetic marker in the second composition and the av log C2;

a difference, D2E, between the logarithm of the concentration of said genetic marker in the third composition and the av log C2; and

a difference, D2F, between the logarithm of the concentration of said genetic marker in the fourth composition and the av log C2;

(k) calculating:

R1 by multiplying D1A and D1D;

R2 by multiplying D1B and D1E;

R3 by multiplying D1C and D1F;

R4 by multiplying D1A by D1A;

R5 by multiplying D1B by D1B;

R6 by multiplying D1C by D1C;

R7 by multiplying D2A and D2D;

R8 by multiplying D2B and D2E;

R9 by multiplying D2C and D2F;

R10 by multiplying D2A by D2A;

R11 by multiplying D2B by D2B;

R12 by multiplying D2C by D2C;

(l) calculating:

S1 using the following formula:

S1=(R1+R2+R3)/(R4+R5+R6)

S2 using the following formula:

S2=(R7+R8+R9)/(R0+R11+R12);

(m) comparing S1 and S2, whereby:

when S2 is at least 30% lower than S1, the concentration of the third composition is the D-limit; and

when S2 is equal to S1 or less than 30% lower than S1, steps (H)(ii)(a) to (H)(ii)(1) are repeated using said first composition in place of said solution of genomic DNA comprising said genetic marker; and

(iii) an average mutation noise, avMut, when said mutation is a single nucleotide variant mutation, by

(a) amplifying by polymerase chain reaction using the same locus-specific forward primer and the same locus-specific reverse primer as in step (A), at least one nucleotide sequence of genomic DNA from a biological sample obtained from a subject without said disease and without said genetic marker;

(b) sequencing each amplified nucleotide sequence, whereby a third list of characters reading from left to right is obtained from each nucleotide sequence thus sequenced;

(c) repeating steps (H)(iii)(a) and (H)(iii)(b) in m subjects without said disease and without said genetic marker, wherein m is at least 9; and

(d) calculating an average fraction of third lists of characters which are identical to that obtained from sequencing said genetic marker, wherein said average fraction is avMut; and

(iv) an average position noise, avPos, when said mutation is a single nucleotide variant mutation, by calculating a variant read frequency, VRF, for each nucleotide sequence that is identical to said genetic marker and to said non-mutated sequence, but wherein the nucleotide responsible for said single nucleotide variant mutation in said genetic marker is different from that in said genetic marker and said non-mutated sequence, wherein the mean of said VRF values is avPos;

(I) determining an experimental sensitivity, ES, wherein ES is:

(i) the greater of min VRF, D-limit, avMut and avPos, as calculated in step (H), when said mutation is a single nucleotide variant mutation; or

(ii) the greater of min VRF and D-limit, as calculated in step (H), when said mutation is an indel mutation or somatic gene rearrangement mutation;

and

(J) determining the presence or absence of minimal residual disease in said subject by either:

(i) comparing the value of the level of minimal residual disease, MRD, calculated in step (G) with the value of the experimental sensitivity, ES, determined in step (I), wherein

(a) when said level of MRD value is equal to or greater than said ES value, minimal residual disease is present in said subject; and

(b) when said level of MRD value is less than said ES value, minimal residual disease is absent from said subject;

(ii) when said mutation is a single nucleotide variant mutation, comparing the value of the level of minimal residual disease, MRD, calculated in step (G) with the value of min VRF calculated in step (H), wherein

(a) when said level of MRD value is equal to or greater than said min VRF value, minimal residual disease is present in said subject; and

comparing the value of the level of minimal residual disease, MRD, calculated in step (G) with the value of avMut calculated in step (H) when said level of MRD value is less than said min VRF value, wherein

(b) when said level of MRD value is equal to or greater than said avMut value, minimal residual disease is present in said subject; and

comparing the value of the level of minimal residual disease, MRD, calculated in step (G) with the value of avPos calculated in step (H) when said level of MRD value is less than said avMut value, wherein

(c) when said level of MRD value is equal to or greater than said avPos value, minimal residual disease is present in said subject; and

comparing the value of the level of minimal residual disease, MRD, calculated in step (G) with the value of D-limit calculated in step (H) when said level of MRD value is less than said avPos value, wherein

(d) when said level of MRD value is equal to or greater than said D-limit value, minimal residual disease is present in said subject; and

(e) when said level of MRD value is less than said min VRF, avMut, avPos and D-limit values, minimal residual disease is absent from said subject;

(iii) when said mutation is an indel mutation or somatic gene rearrangement mutation, comparing the value of the level of minimal residual disease, MRD, calculated in step (G) with the value of min VRF calculated in step (I), wherein

(f) when said level of MRD value is equal to or greater than said min VRF value, minimal residual disease is present in said subject; and

(g) when said level of MRD value is equal to or greater than said D-limit value, minimal residual disease is present in said subject; and

(h) when said level of MRD value is less than said min VRF and D-limit values, minimal residual disease is absent from said subject; and

(3) repeating steps (1) and (2) until minimal residual disease is determined to be absent in said subject.