US 12,060,616 B2Granted

Method for assisting determination of hematological stage of childhood acute lymphoblastic leukemia

AssigneeOtsuka Pharmaceutical Co., Ltd.

InventorsYoshihide Miyazaki, Yukari Umehara, Osamu Kosaka, Kimiyoshi Sudou

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Abstract

Provided are a method for assisting in determining the hematological stage of childhood acute lymphoblastic leukemia (child ALL), and an in-vitro diagnostic pharmaceutical product usable in the method. The method for assisting in determining the hematological stage of child ALL comprises the steps of (1) obtaining the mRNA level of Wilms' tumor-1 gene (WT1) in at least one of the biological samples of peripheral blood and bone marrow fluid from a test subject; (2) obtaining the GAPDH mRNA level in the biological sample; and (3) calculating an index value necessary for assisting in the determination based on the ratio of the WT1 mRNA level obtained in step (1) to the GAPDH mRNA level obtained in step (2).

Claims15

Dependent claims

A method for making a determination with respect to a hematological stage of childhood acute lymphoblastic leukemia (child ALL), the method comprising the steps of:

(1) obtaining an mRNA level of Wilms' tumor-1 gene (WT1) in a biological sample of a test subject, wherein said test subject is a patient with child ALL or suspected child ALL;
(2) obtaining an mRNA level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the biological sample;
(3) calculating an index value by normalizing the mRNA level of the WT1 obtained in step (1) to the mRNA level of GAPDH obtained in step (2);
(4) comparing the index value calculated in step (3) with a predetermined cutoff value, wherein the predetermined cutoff value distinguishes between an average of index values calculated by normalizing mRNA levels of WT1 mRNA to mRNA levels of GAPDH mRNA of children affected by child ALL and an average of index values calculated by normalizing mRNA levels of WT1 mRNA to mRNA levels of GAPDH of children in remission phase of child ALL and/or children who are not affected by child ALL;
and
(5) making a determination that
(a) said test subject is in the remission phase when said test subject is a patient with child ALL, and when the index value obtained in step (3) is smaller than said predetermined cutoff value;
(b) said test subject is in the non-remission phase when said test subject is a patient with child ALL, and when the index value obtained in step (3) is equal to or larger than said predetermined cutoff value;
(c) said test subject is not affected by child ALL when said test subject is a patient with suspected child ALL, and when the index value obtained in step (3) is smaller than said predetermined cutoff value;
or
(d) said test subject is affected by child ALL when said test subject is a patient with suspected child ALL, and when the index value obtained in step (3) is equal to or larger than said predetermined cutoff value;
and
(6) performing one or more of the following steps:
(a) displaying the determination result on a display;
(b) printing the determination result;
(c) recording the determination result on a recording medium;
(d) storing the determination result on a storage unit or on an auxiliary storage unit;
and
(e) providing the determination result to an output unit;
wherein the mRNA level in step (1) or (2) is measured using RT-PCR, in situ RT-PCR, or next-generation sequencing.

17 more elements

A method for making a determination with respect to a hematological stage of childhood acute lymphoblastic leukemia (child ALL), the method comprising the steps of:

(1) obtaining an mRNA level of Wilms' tumor-1 gene (WT1) in a biological sample of a test subject, wherein said test subject is a patient with child ALL or suspected child ALL;
(2) obtaining an mRNA level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the biological sample;
(3) calculating an index value by normalizing the mRNA level of the WT1obtained in step (1) to the mRNA level of GAPDH obtained in step (2);
(4) comparing the index value calculated in step (3) with a predetermined cutoff value;
and
(5) making a determination that
(a) said test subject is in the remission phase when said test subject is a patient with child ALL, and when the index value obtained in step (3) is smaller than said predetermined cutoff value;
(b) said test subject is in the non-remission phase when said test subject is a patient with child ALL, and when the index value obtained in step (3) is equal to or larger than said predetermined cutoff value;
(c) said test subject is not affected by child ALL when said test subject is a patient with suspected child ALL, and when the index value obtained in step (3) is smaller than said predetermined cutoff value;
or
(d) said test subject is affected by child ALL when said test subject is a patient with suspected child ALL, and when the index value obtained in step (3) is equal to or larger than said predetermined cutoff value;
and
(6) performing one or more of the following steps:
(a) displaying the determination result on a display;
(b) printing the determination result;
(c) recording the determination result on a recording medium;
(d) storing the determination result on a storage unit or on an auxiliary storage unit;
and
(e) providing the determination result to an output unit;
wherein the mRNA level in step (1) or (2) is measured using RT-PCR, in situ RT-PCR, or next-generation sequencing;
wherein the normalization is performed by multiplying the ratio of the WT1 mRNA level to the GAPDH mRNA level (WT1 mRNA level/GAPDH mRNA level) by 2.7×107 copies/ug RNA, which is an average value of GAPDH mRNA levels contained in 1 μg RNA of healthy adults.

18 more elements

A method for making a determination with respect to a hematological stage of childhood acute lymphoblastic leukemia (child ALL), the method comprising the steps of:

(1) obtaining an mRNA level of Wilms' tumor-1 gene (WT1) in a biological sample of a test subject, wherein said test subject is a patient with child ALL or suspected child ALL;
(2) obtaining an mRNA level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the biological sample;
(3) calculating an index value by normalizing the mRNA level of the WT1 obtained in step (1) to the mRNA level of GAPDH obtained in step (2);
(4) comparing the index value calculated in step (3) with a predetermined cutoff value;
and
(5) making a determination that
(a) said test subject is in the remission phase when said test subject is a patient with child ALL, and when the index value obtained in step (3) is smaller than said predetermined cutoff value;
(b) said test subject is in the non-remission phase when said test subject is a patient with child ALL, and when the index value obtained in step (3) is equal to or larger than said predetermined cutoff value;
(c) said test subject is not affected by child ALL when said test subject is a patient with suspected child ALL, and when the index value obtained in step (3) is smaller than said predetermined cutoff value;
or
(d) said test subject is affected by child ALL when said test subject is a patient with suspected child ALL, and when the index value obtained in step (3) is equal to or larger than said predetermined cutoff value;
and
(6) performing one or more of the following steps:
(a) displaying the determination result on a display;
(b) printing the determination result;
(c) recording the determination result on a recording medium;
(d) storing the determination result on a storage unit or on an auxiliary storage unit;
and
(e) providing the determination result to an output unit;
wherein the mRNA level in step (1) or (2) is measured using RT-PCR, in situ RT-PCR, or next-generation sequencing;
wherein the determination is to distinguish between the remission phase and the non-remission phase of child ALL;
and the cutoff value is determined by statistical analysis of both groups of patients with child ALL in a non-remission phase (a group in a non-remission phase) and patients with child ALL in a remission phase (a group in a remission-phase), and
wherein the biological sample is selected from the group consisting of peripheral blood and bone marrow fluid;
and wherein the cutoff value is 220 copies/ug RNA if the biological sample is peripheral blood, and 1,820 copies/ug RNA if the biological sample is bone marrow fluid.

21 more elements

Dependencies are inferred from the claim text.

Facts

Application: 16/319,024Filed 18 Jul 2017
Priority: JP 2016-14179319 Jul 2016
Published: US 2019/0161810 A130 May 2019
Granted: 13 Aug 2024
Term ends: Not estimatedThe source records don’t support an estimate.

Classification15 codes

C12Q 1/6886
For diseases caused by alterations of genetic material: for cancer
C12Q 1/68
Measuring or testing processes involving enzymes, nucleic acids or microorganisms ; Compositions therefor; Processes of preparing such…
C12Q 1/686
Nucleic acid amplification reactions: polymerase chain reaction [PCR]
G16H 50/20
ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or…: for…