Weimin Miao

1. Fluorescence in situ hybridization (FISH):an increasingly demanded tool for biomarker research and personalized medicine Weimin Miao • Institute of Hematology and Blood Diseases Hospital, CAMS & PUMC • Tianjin key Laboratory of Blood Cell Therapy and Technology

2. Background • Potential biomarkers for cancers • and hematological diseases • Recurrent genomic defects • amplification, • insertion • deletion, • recombination • Mutation

3. personalized medicine (Precision medicine) Precision Diagnosis Precision biomarker detection

4. Detection methods for molecular biomarkers: • 1. Cutting-edge high through-put • a. Comparative Genomic Hybridization (CGH) array • b. Single Nucleotide Polymorphism (SNP) array • c. Next Generation Sequencing (NGS) • 2. Traditional low through-put • a. PCR • b. Sanger sequencing • c. Fluorescence In situ Hybridization (FISH)

5. Fluorescence in situ hybridization (FISH) FISH uses fluorescent DNA probes to target a specific chromosome location within a nucleus, resulting in coloured signals that can be detected by fluorescent microscope. 5

6. Topic 1: Fluorescence in situ hybridization (FISH) • is increasingly demanded for biomarker detection • and personalized medicine. • Advantages: • Specific and sensitive • Simple and reliable • Results are visible • Disavantages: • 1. Low through-put

7. For example, FISH detection of Her-2 gene amplification for breast cancer and gastric cancer is gold standard companion test for Heceptin targeted therapy The left fig shows a normal cell, the right fig shows a breast cancer with strong amplification of Her-2 genes (red signals) FISH has become indispensable companion tests for several cancer targeted therapies

8. Companion diagnostics The second example is FISH detection of the fusion of BCR/ABL1 genes for Chronic Myeloid leukemia (CML) diagnosis. The FISH test is used for Guiding Gleevec therapy . Translocation between chromosome 9 and 22

9. The FISH test detected two Orange-Green fusion signals in the BM cells of a CML patient.

10. Tab. Commonly used FISH probes for clinical diagnosis of hematological diseases (1)

11. Tab. Commonly used FISH probes for clinical diagnosis of hematological diseases (2)

12. Tab. Commonly used FISH probes for clinical diagnosis of hematological diseases (3)

13. Tab. Commonly used FISH probes for clinical diagnosis of solid tumors

14. Companion diagnostics FISH test of ALK translocation for Crizotinib targeted therapy of NSCLC The ALK translocation was recently found to be a driving factor associated with certain NSCLC. The Crizotinib and its companion FISH test were simutaneouly approved in 2011 by US FDA for diagnosis and treatment of ALK-driven NSCLC.

15. Topic 2. Quantitative multi-gene FISH (5-colour qmFISH) Previously，most commercial FISH detection kits comprise one probe labeled with a single fluorochrome or two probes labeled with two distinct fluorochromes. With the rapid progress in disease gene discoveries, there is a need to detect multiple genes at a time. Thus, the FISH test employing multiple probes, called qmFISH, have become popular recently. 15 15 15

16. We have prepared the FISH probes in our own laboratory

17. 1. QM-FISH can be used to detect cancer cell heterogeity

18. Ovarian cancer showed substantial heterogeneity The three subclones were found in an ascites fluid sample from a patient with progressive epithelial ovarian cancer DAPI c-myc Rb1 ChK2 p53 BRCA1 Merge

19. Tab. 1. The three subclones in an ascites fluid sample from a patient with progressive epithelial ovarian cancer Note: the numbers in Clone column indicate the coding number of the subclones; the numbers in FISH probe coloums (c-myc, Rb1, Chk2, p53 and BRCA1) indicate average number of signals of each probe within a single nucleus of the cancer cells (50 cells were counted).

20. The three subclones were obtained from a cancer stem-cell preparation for a patient with low-differentiated ovarian adenocarcinoma DAPI c-myc Rb1 ChK2 p53 BRCA1 Merge

21. Tab. 2. The three subclones obtained from a cancer stem-cell preparation for a patient with low-differentiated ovarian adenocarcinoma Note: the numbers in Clone column indicate the coding number of the subclones; the numbers in FISH probe coloums (c-myc, Rb1, Chk2, p53 and BRCA1) indicate average number of signals of each probe within a single nucleus of the cancer cells (50 cells were counted).

22. The six subclones from the bone marrow sample taken from an ALL patient

23. Tab. 3. The six subclones from the bone marrow sample taken from an ALL patient Note: the numbers in Clone column indicate the coding number of the subclones; the numbers in FISH probe coloums (TEL, AML1, PAX5, p16 and IKZF1) indicate average number of signals of each probe within a single nucleus of the cancer cells (50 cells were counted).

24. The clonal components of bone marrow samples taken from an ALL patient upon the initial diagnosis and post chemotherapy

25. Tab. 4. The clonal components of bone marrow samples taken from an ALL patient upon the initial diagnosis and post chemotherapy Note: the numbers in FISH probe coloums (TEL, AML1, PAX5, p16 and IKZF1) indicate average number of signals of each probe within a single nucleus of the cancer cells (50 cells were counted).

26. 2. QM-FISH can be used to study cancer genetic architecture and clonal evolution

27. Detection of heterogeneity and evolution of subclones in t(8;21) AML by QM-FISH

28. The most obvious genetic changes in leukemia is the specific chromosomal translocation; • Early in 1972, Dr.Janet Rowley discovered the first translocation between chromosomes 8 and 21 in acute myeloid leukemia (AML)—t(8;21), generating AML1-ETOfusion gene; • Clinically, the t(8;21) translocation represents the most frequent chromosomal abnormality in AML, occurring in approximately 4%～12% of adult and 12%～30% of pediatric patients.

30. 36 newly diagnosed primary AML with t(8;21) cases and 1 paired relapsed case were detected; • Genomic copy number alterations (CNA) of AML1, ETO, WT1, p27 and c-kit were investigated by qmFISH; • The clonal heterogeneity, subclonal architecture andputativeancestral evolution in AML1-ETO+ AML was assembled; • The evolution of dominant clones at diagnosis and relapse were speculated.

31. Diagnosis Relapse 10 months 1 F 1 ETO 1 AML1 2 WT1 2 p27 2 c-Kit 1 F 1 ETO 1 AML1 2 WT1 2 p27 2 c-Kit 1 F 1 ETO 1 AML1 2 WT1 3 p27 2 c-Kit 1 F 1 ETO 1 AML1 2 WT1 3 p27 2 c-Kit 1 F 1 ETO 1 AML1 2 WT1 2 p27 3 c-Kit 1 F 1 ETO 1 AML1 2 WT1 2 p27 3 c-Kit 1 F 1 ETO 1 AML1 3 WT1 2 p27 2 c-Kit 1 F 1 ETO 1 AML1 3 WT1 2 p27 2 c-Kit Evolution of dominant clones at diagnosis and relapse AML1 ETO WT1 P27 C-Kit 1 F 1 ETO 1 AML1 2 WT1 3 p27 3 c-kit 1 F 1 ETO 1 AML1 2 WT1 3 p27 3 c-kit 1 F 1 ETO 1 AML1 3 WT1 3 p27 2 c-Kit 1 F 1 ETO 1 AML1 2 WT1 5 p27 3 c-Kit 1 F 1 ETO 1 AML1 2 WT1 4 p27 2 c-Kit 1 F 1 ETO 1 AML1 2 WT1 4 p27 2 c-Kit 1 F 1 ETO 1 AML1 2 WT1 4 p27 3 c-Kit

32. 50.0% 65.5% Diagnosis Relapse 10 months 1 F 1 ETO 1 AML1 2 WT1 2 p27 2 c-Kit 1 F 1 ETO 1 AML1 2 WT1 2 p27 2 c-Kit 2.59% 9.0% 5.6% 9.0% 3.02% 2.0% 27.59% 1.72% 1 F 1 ETO 1 AML1 2 WT1 3 p27 2 c-Kit 1 F 1 ETO 1 AML1 2 WT1 3 p27 2 c-Kit 1 F 1 ETO 1 AML1 2 WT1 2 p27 3 c-Kit 1 F 1 ETO 1 AML1 2 WT1 2 p27 3 c-Kit 1 F 1 ETO 1 AML1 3 WT1 2 p27 2 c-Kit 1 F 1 ETO 1 AML1 3 WT1 2 p27 2 c-Kit 2.0% 2.5% 2.5% 1.29% Evolution of dominant clones at diagnosis and relapse 1 F 1 ETO 1 AML1 2 WT1 3 p27 3 c-kit 1 F 1 ETO 1 AML1 2 WT1 3 p27 3 c-kit 1 F 1 ETO 1 AML1 3 WT1 3 p27 2 c-Kit 1 F 1 ETO 1 AML1 2 WT1 4 p27 2 c-Kit 1 F 1 ETO 1 AML1 2 WT1 4 p27 3 c-Kit 1 F 1 ETO 1 AML1 2 WT1 4 p27 2 c-Kit 1 F 1 ETO 1 AML1 2 WT1 5 p27 3 c-Kit 1.72%

33. Summary 1. Copy number alterations can be identified in all t(8;21) AML patients by 5-color qmFISH; 2. The genetic architectures observed in t(8;21) were very diverse, there is a marked subclonal heterogeneity, and the subclones could be aligned in a linear or branching ancestral tree; 3. The dominant subclone in relapse is originated from a major clone at diagnosis, continues to genetically diversify and acquires genetic lesions to generate secondary dominant subclone.

34. Topic 3. Development of a Sequential FISH strategy up to 20 genes can be detected at the single-cell level

35. Background (1) While FISH is useful for detecting certain chromosomal abnormalities, the number of genes that can be simultaneously detected is limited. Even with qmFISH, the number of detected genes is usually less than 5 due to the capacity of currently available fluorescence filter sets. (2) Multi-gene detection at the single-cell level is desirable to enable more precise genotyping of heterogeneous hematology and oncology samples

36. Sequential qmFISH strategy Five fluorochromes were used to label each set of FISH gene probes, and 5 genes were detected each time using a five-color FISH protocol. After the first hybridization, the previous FISH probe set was stripped, and a second set of five-color FISH probes was used for rehybridization. After each hybridization, the fluorescence signals were recorded in 6 fluorescence filter channels that included DAPI, Spectrum Green™, Cy3™ v1, Texas Red, Cy5, and PF-415. A digital automatic relocation procedure was used to ensure that exactly the same microscopic field was studied in each stripping and hybridization cycle. Therefore, up to 20 genes can be detected within a single nucleus

37. sequential qmFISH strategy Hybridization Strip wash Rehybridization z z z y y y Five-colour fluorescence signals: X X X

38. b a Probes: c-myc, Rb1, MDM2, CCND1, p53 ChK2, CKS1B, STK6, PAX5, CD27 Strip wash Strip wash c d Probes: p18, hCDC14a, PTEN, p16, BRCA1 LATS, Her-2, TER, IKZF1, PIK3CA Strip wash

39. This method can be used for more precise molecular subtyping or for clonal evolution studies of various types of diseases.

40. Future direction: 1. Discovery of novel cancer biomarkers 2. Development of novel FISH tests for personalized medicine 3. We welcome any kind of collaborations

41. Acknowledgement Co-workers： Linping Hu Chengwen Li Jing Ge Jiangman Sun Weixia Xue Xinchun Zhang Collaborators: Tao Cheng, M.D. Zetterberg Anders, M.D., Ph.D. Wang Jianxiang, M.D. Kun Ru, MD. Ph.D. Ling Shi, Ph.D.