Quantitative in situ characterization of biological samples

Patent metadata

Field	Value
Publication number	US-9778263-B2
Application number	US-201314079347-A
Country	US
Kind code	B2
Filing date	Nov 13, 2013
Priority date	Nov 13, 2013
Publication date	Oct 3, 2017
Grant date	Oct 3, 2017

Abstract

The present disclosure relates to characterization of biological samples. By way of example, a biological sample may be contacted with a plurality of probes specific for targets in the sample, such as probes for immune markers and segmenting probes. Acquired image data of the sample may be used to segment the images into epithelial and stromal regions to characterize individual cells in the sample based on the binding of the probes. Further, the biological sample may be characterized by a distribution, location, and type of a plurality of the characterized cells.

First claim

The invention claimed is:

1. A method for determining distribution of immune cell populations in a biological sample comprising:
applying sequentially individual probes of a plurality of probes to a biological tissue sample obtained from a tumor region, each of the plurality of probes comprising a respective distinguishably detectable signal generator;
imaging each probe of the plurality of probes in a sequential manner to acquire image data of the biological sample representative of the plurality of probes bound to a respective plurality of target molecules in the biological sample based on distinguishable signals detected from each respective distinguishably detectable signal generator of the plurality of probes, wherein at least one of the plurality of probes comprises a first signal generator and is an epithelium probe, a membrane probe, a cytoplasm probe, or nuclear probe specific for a cell nucleus, wherein at least one of the plurality of probes comprises a second signal generator and is an immune probe specific for an immune marker, the plurality of target molecules comprises an epithelium target molecule, a membrane target molecule, a cytoplasm target molecule, or a nuclear target molecule and wherein the biological sample comprises the immune marker;
segmenting epithelial and stromal regions of the sample using the signals from the first signal generator to identify single cells within each region, wherein identifying single cells in the epithelial region or the stromal region comprises using image data of the first signal generator representative of the epithelium probe, the membrane probe, the cytoplasm probe, or the nuclear probe bound to at least one of the target molecules and wherein identification of single cells in the stromal region comprises segmenting the epithelial region of the sample to generate an epithelial mask and classifying regions not contained within the epithelial mask as one or more of the stromal region or background such that each cell of the single cells is assigned to either the epithelial region or the stromal region;
identifying immune cells among the single cells using signals from the second signal generator generating an immune probe signal representative of the immune probe bound to the immune marker, wherein identifying comprises reclassifying single cells in the sample as immune cells based on a signal intensity of the image data from the immune probe signal representative of the immune probe bound to the immune marker;
generating an immune marker positive epithelial fraction and an immune marker positive stromal fraction based on the identified immune cells among the single cells in the epithelial region and the stromal region;
determining a first standard deviation of the immune probe signal intensity for the immune marker in the epithelial region and a second standard deviation of the immune marker in the stromal region based on the image data of the immune probe bound to the immune marker in the epithelial region and the stromal region; and
determining a distribution, location, and type of a plurality of the immune cells in the biological sample based on the immune marker positive epithelial fraction relative to the immune marker positive stromal fraction and the first standard deviation or the second standard deviation.

2. The method of claim 1 , wherein at least one of the plurality of probes comprises an epithelial probe specific for epithelial cells and wherein a signal generated by the epithelial probe is used to determine the epithelial region.

3. The method of claim 2 , wherein the epithelial probe is used to create the epithelial mask.

4. The method of claim 3 , wherein a region not defined by the epithelial mask is used to define a stromal mask.

5. The method of claim 4 , wherein determining a distribution, location, and type of a plurality of immune cells in the biological sample comprises quantifying the immune cells within the stromal mask.

6. The method of claim 1 , wherein determining a distribution, location, and type of a plurality of immune cells in the biological sample comprises quantifying the immune cells within the epithelial mask.

7. The method of claim 1 , wherein intensity and morphology-based algorithms, generalized wavelet-based algorithms, or probabilistic-based methods are used to detect individual nuclei in the stromal region using the image data representative of the nuclear probe.

8. The method of claim 1 , comprising removing a non-specific 4′,6-diaminidino-2-phenylindole (DAPI) signal by using two-class clustering to identify positive DAPI nuclei in the stromal region.

9. The method of claim 1 , wherein the plurality of probes comprise a plurality of immune probes specific for immune markers of immune cell subtypes and respective sub-type signal signerators, wherein the immune cells are characterized by an immune probe signal intensity of the sub-type signal generators above a threshold signal intensity, and wherein threshold values associated with a positive signal are determined for the immune markers of the immune cell subtypes.

10. The method of claim 1 , wherein the immune cells are characterized by an immune probe signal intensity above a threshold signal intensity and wherein the threshold value is determined based on a box-plot or histogram or violin plot analysis of overall immune marker expression in stroma across all images for the sample set.

11. The method of claim 1 , wherein the immune cells are characterized by an immune probe signal intensity above a threshold signal intensity and wherein the threshold is determined based on a predetermined quantile for overall expression of the immune marker and one or more of the target molecules in the epithelial or stromal region selected as the threshold value for a positive signal.

12. The method of claim 11 , wherein the predetermined quantile is 80%-99%.

13. The method of claim 1 , wherein determining a distribution, location, and type of a plurality of immune cells in the biological sample comprises determining a number of each cell of the single cells positive for the immune marker.

14. The method of claim 1 , wherein the plurality of probes comprises a plurality of epithelial probes specific for epithelial markers and wherein segmenting the epithelial region of the tissue comprises using image data representative of the plurality of epithelial probes to produce a super-epithelium image.

15. The method of claim 14 , wherein segmenting the epithelial region comprises smoothing the image data representative of the nuclear probe and applying an optimal global threshold for positive signal.

16. The method of claim 15 , wherein small gaps in the super-epithelium image are filled using the image data representative of the nuclear probe.

17. The method of claim 1 , wherein determining overall distribution, location, and type of immune cells comprises determining a number of cells of the single cells positive for the immune marker in the epithelial region.

18. The method of claim 1 , comprising determining overall distribution patterns of immune cells in the epithelial region and the stromal region.

19. The method of claim 1 , comprising determining overall distribution patterns of immune cells in a combined epithelial and stromal region.

20. The method of claim 1 , comprising determining a ratio of immune cells in the epithelial region versus the stromal region.

21. The method of claim 1 , comprising determining a patient survival associated with the biological sample base

Assignees

• Gen Electric

Inventors

• Bhaumik Srabani
• Gerdes Michael John
• Pang Zhengyu
• Ginty Fiona
• Sevinsky Christopher James
• Li Qing
• Santamaria-Pang Alberto
• Sui Yunxia
• Desai Keyur Hemantkumar

Classifications

• G01N33/5759 Primary

  involving compounds localised on the membrane of tumour or cancer cells · CPC title

• G01N33/56977

  HLA or MHC typing · CPC title

• G06T2207/10024

  Color image · CPC title

• G06T2207/30024

  Cell structures in vitro; Tissue sections in vitro · CPC title

• G01N33/54393 Primary

  Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding · CPC title