fluorescent in situ hybridization (fish) to identify genetic changes in fine needle biopsy of lung...

Fluorescent In Situ Hybridization (FISH) to Identify Genetic Changes in Fine

Needle Biopsy of Lung Lesions

Prepared by Jin JenNCI

Why the Need?

• Spiral CT screening is highly sensitive for detecting lung cancers in patients at high risk.

• Spiral CT plus fine needle biopsy increases the diagnostic rate of accurate diagnoses but a portion of the cases are undetermined.

• Molecular markers might be used to assist clinical diagnosis by detecting the presence of specific chromosomal amplifications.

How it is Possible?

• Tumors are highly genetically unstable.

• Tumor related changes often are reflected at the chromosome level and are, by definition, specific to tumors.

• Genetic analyses can be done in very small tissues and are not subject to the method of detection.

Minimal Chromosome Probes That Can Detect Most Lung Cancers (n = 84)

ChromosomeAmplifications

# Detectable by Based on Any Alteration (n =84) Percentage

1q, 3q, 5p, 8q 83 98.8%

1q, 5q, 8q 80 95.2%

1q, 3q, 8q 78 92.3%

1q, 3q, 5p 77 91.7%

3q, 5p, 8q 75 89.3%

Method to Identify Genetic Changes in FNAs by FISH

Identify and prepare BAC probes unique to specific chromosomal regions

Protease digestion of biopsy samples and isolate nuclei

Hybridization with fluorescently labeled BAC probes

Visualize under fluorescent microscope for Ch. copy number changes

Correlate copy number changes with cytological/pathological findings and clinical outcome

Detecting Lung Cancer in FNA-obtained Spiral CT Identified Lung Lesions

• Probes Labeling: 1q and 5q (green)

8q and 3q (red)

nuclei (blue)

Samples: 20 paraffin embedded FNA samples 20 formalin fixed biopsies.

• All samples have pathology and clinical information.– 20 malignant– 10 benign specific– 10 benign non-specific

Sample 503 (Normal Lung)

Green: 5pRed: 8q

Normal samples often show 2 signals/cells representing their diploid status.

Sample 577 (Normal Lung)

Green: 5p, 2 signals/cellRed: 8q, 2 signals/cell

Cornell Sample: JJ001

Green: 1q, 5 signalsRed: 3q, 4 signals

Green: 5p, 9 signals

Sample: JJ003

Green: 1q, 6 signalsRed: 3q, 4 signals

A case is considered neoplastic or tumor when 5 or more signals were observed in a cell by one or more probes.

Sample: JJ005

Green: 1q, 8 signalsRed: 3q, 12 signals

Sample JJ006

Green: 1q, 4 signalsRed: 3q, 5 signals

Green: 5p, 4 signalsRed: 8q, 5 signals

Sample: JJ010

Red: 8q, 6 signalsGreen: 5p, 5 signals

Sample: JJ011

Green: 1q, 2 signalsRed: 3p, 6 signals

Sample: JJ012

Green: 1q, 2 signalsRed: 3p, 2 signals

Green: 5p, 2 signalsRed: 8q, 2 signals

Sample: JJ014

Green: 1q, >7 signalsRed: 3p, 2 signals

Sample: JJ017

Green: 5p, 10 signalsRed: 8q, 7 signals

Sample: JJ018

Green: 5p, 3 signalsRed: 8q, 6 signals

Green: 5p, 6 signalsRed: 8q, 9 signals