AUTOMATED METHOD FOR DETECTING CERVICAL CANCERS AND HIGH GRADE HYPERPLASIAS

• US 20200131565A1
• Filed: 10/30/2019
• Published: 04/30/2020
• Est. Priority Date: 10/25/2006
• Status: Active Application

• Current Assignee: Kitaru Innovations Inc
• Original Assignee: Kitaru Innovations Inc
• Inventors: Tafas, Triantafyllos P., Tsipouras, Petros, Kilpatrick, Michael, Sarkis, Gary
• Application Number:
  US16/668,476
• Publication Number:
  US 20200131565A1
• Time in Patent Office: Days
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: G01N 2021/6421 Measuring at two or more wavelengths
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G01N 33/57496 involving intracellular compounds
• CPC Class Codes: G02B 21/0004 specially adapted for specific applications
• CPC Class Codes: G02B 21/16 adapted for ultraviolet illumination ; Fluorescence microscopes G02B21/0076 takes precedence

Abstract

Automated methods for detecting cancer and related hyperplasias in biological samples.

Claims

1. An automated method of screening for the presence and/or extent of a pathology in a subject, the pathology characterized by an abnormal chromosomal component in a cell of the subject, comprising the steps of

a) contacting a biological sample comprising cell nuclei from said subject with one or more distinguishable labeled probes directed to at least one chromosomal sequence that characterizes the abnormality under conditions that promote hybridization of the one or more probes to the at least one sequence;

b) causing interrogatable information regarding said one or more distinguishable labeled probes to be associated with said biological sample;

c) automatically obtaining a digitized representation of the one or more distinguishable labels hybridized to the chromosomal sequences by

irradiating said biological sample with fluorescence exciting illumination based on said interrogatable information,

capturing one or more images of said biological sample,

digitizing each of said one or more images,

analyzing at least one of said one or more digitizations to cause adjustment of exposure parameters to form said digitized representation;

d) automatically analyzing the distribution and intensity of binding of the one or more distinguishable labels in the digitized representation to determine the presence and/or extent of an abnormal chromosomal component; and

e) automatically reporting results of the analysis of step d);

wherein steps b)-d) are carried out without intervention by a human.

2. The method described in claim 1 wherein an automated microscope system carries out steps b)-e).

3. The method described in claim 1 wherein a probe targets at least one of a single nucleotide polymorphism (SNP), a mutated sequence, a duplicated or amplified gene or portion thereof, a centromere of chromosome 3, a centromere of chromosome 7, and a sequence comprising the TERC gene or a portion thereof.

4. The method described in claim 1 further comprising contacting the sample under the hybridizing conditions with a tagged reference probe directed to a chromosomal locus known not to be abnormal, or contacting the sample with a reference stain, and referencing the representing and analysis steps to the reference probe or stain.

5. An automated method of screening for an abnormality related to a cancer, a high grade hyperplasia or a high grade dysplasia in a subject, comprising the steps of:

a) obtaining a biological sample comprising nuclei from the subject;

b) contacting the nuclei in the sample with a first probe bearing a first detectable label directed to a chromosomal sequence related to the abnormality under conditions that promote hybridization of the probes to targeted chromosomal loci;

c) contacting the sample under the hybridizing conditions with at least one of a detectably labeled reference probe directed to a chromosomal locus known not to be abnormal and a reference stain;

d) causing interrogatable information regarding the first detectable label to be associated with the biological sample;

e) automatically imaging the labels bound to the chromosomal sequences and imaging the reference stain if used, by

irradiating the biological sample with flourescense exciting illumination based on the interrogatable information,

capturing two or more images of the biological sample,

digitizing each of the two or more images,

analyzing at least two of the two or more digitizations to cause adjustment of exposure parameters to form the digitized image of the labels bound to the chromosomal sequences, and the digitized image of the reference stain;

f) automatically analyzing the two or more digitized images for the distribution and intensity of hybridized labels and stain if used; and

g) automatically reporting results of the analysis of step f);

wherein steps d)-f) are performed without intervention by a human;

thereby providing an assessment of the abnormality in the subject.

6. The method described in claim 5 wherein an automated microscope performs steps d)-g).

7. The method described in claim 5 wherein prior to the contacting step the nuclei are isolated from the biological sample and are deposited to form a layer.

8. The method described in claim 5 wherein the sample comprises one or more of a biopsy, surgical resection specimen, blood, lymph, urine, an effusion fluid, a biological fluid, an epithelial scraping, a lavage fluid, aspiration fluid, sputum, and a tissue.

9. The method described in claim 5 wherein a label is a fluorescent label.

10. The method described in claim 5 wherein a probe targets at least one of a single nucleotide polymorphism (SNP), a mutated sequence, a duplicated or amplified gene or portion thereof a centromere of chromosome 3, a centromere of chromosome 7, and a sequence comprising the TERC gene or a portion thereof.

11. An automated method for monitoring the efficacy over time of a course of therapy in the treatment of a cancer or high grade hyperplasia in a patient, said method comprising the steps of

(a) obtaining from the patient a fluid biological sample in which cells associated with the cancer or high grade hyperplasia are found;

(b) treating said fluid biological sample or a portion thereof with one or more detectably labeled chromosomal probes having a high degree of sequence similarity to one or more chromosomal loci associated with, or whose amplification is associated with, said cancer or high grade hyperplasia, under conditions sufficient to enable hybridization of said probes to chromosomes in the sample;

(c) automatically scanning said treated fluid biological sample to obtain a digitized representation of said fluid biological sample or a portion thereof and detecting said one or more labels bound to any chromosomes in said sample by

irradiating said treated fluid biological sample with fluorescence exciting illumination based on the interrogatable information,

capturing one or more images of the biological sample,

digitizing each of the one or more images,

analyzing at least one of the one or more digitizations to cause adjustment of exposure parameters to form the digitized representation;

(d) automatically detecting from the digitized representation the number of cells associated with said chromosomes hybridized to said chromosomal probes; and

(e) automatically comparing the hybridization patterns of a label and cell number results provided in steps (c) and (d) at differing times in the therapeutic treatment course, thereby evaluating the efficacy of the therapy in the treatment of the cancer or high grade hyperplasia.

12. The method of claim 11 wherein monitoring is performed at intervals of 1 day or greater.

13. The method described in claim 11 wherein the fluid biological sample comprises one or more of blood, lymph, urine, an effusion fluid, an epithelial scraping, a lavage fluid, aspiration fluid, and sputum.

14. The method described in claim 11 wherein an automated microscope system performs steps (c)-(e) without intervention by a human.

15. The method described in claim 14 wherein the automated microscope system automatically optimizes the field scanning the sample.

16. The method described in claim 14 wherein the automated microscope scans two or more planes in a field of the sample.

17. The method described in claim 11 wherein a probe targets at least one of a single nucleotide polymorphism (SNP), a mutated sequence, a duplicated or amplified gene or portion thereof, a centromere of chromosome 3, a centromere of chromosome 7, and a sequence comprising the TERC gene or a portion thereof.

18. The method described in claim 11 wherein a patient obtains samples without assistance from another human.

19. (canceled)