Significance of Chromosome Changes in Hematological Disorders and Solid Tumors

Significance of Chromosome Changes in Hematological Disorders and Solid Tumors

Size of Components of Human Genome Size of haploid genome! Estimated genetic constitution! Size of average chromosome band! Size of average gene 3.3 X 10 9 DNA basepairs 30,000 genes 3 X 10 6 DNA basepairs 5 X 10 4 DNA basepairs

Mitotic events affecting chromosomes in neoplastic cells: Abnormal segregation Chromosome breakage

60,XY,+X,+Y,+2,+5,+6,+7,+8,+13,+16,+17,+19,+21,+21,+22

46,XX,t(9;22)(q34;q11.2)

48,XX,+8,+8,t(9;11)(p22;q23)

46,XX,+7,t(8;14)(q24;q32),der(13)t(1;13)(q21;p12),-19

Secondary Leukemia! Alkylating agents and/or radiation -5/del(5q) -7/del(7q)

Secondary Leukemia! Epipodophyllotoxins / Anthracyclines t(11q23) (MLL) t(21q22) (CBFA2)

Overall AML survival by cytogenetics Slovak ML et al Blood 96: 4075-83, 2000

Mrozek K et al Blood 2007: 109: 431-48 Pie chart based on 246 patients analyzed for the presence or absence of mutations in the NPM1 and CEBPA genes, FLT3- ITD, FLT3-TKD, and MLL-PTD indicating the coexistence of mutations in individual patients. WT indicates patients with only wild-type alleles of genes tested. Adapted by Kenneth X. Probst from Döhner et al with permission.

Proposed schema assigning AML patients with a normal karyotype to riskadapted postremission therapies using information on currently known prognostic markers as a basis for designing future clinical studies. SCT denotes stem-cell transplantation; ASCT, autologous SCT; and HDAC, high-dose cytarabine. Illustration drawn by Kenneth X. Probst. Mrozek K et al Blood 2007: 109: 431-48

CYTOGENETIC MECHANISMS IN SOLID TUMORS Deletions Translocations Amplifications

Solid Tumor Cytogenetics: art or science? 1. Unpredictable growth of the neoplastic cells in tissue culture.

Solid Tumor Cytogenetics: art or science? 2. Overgrowth of neoplastic cells by reactive non-neoplastic cells.

Solid Tumor Cytogenetics: art or science? 3. Destruction of tumor cultures by bacterial or fungal infection.

Solid Tumor Cytogenetics: art or science? 4. Failure of tumor cultures to grow because of non-viable tumor.

Chromosome translocations in cancer: Gene fusion Gene activation

Chromosome abnormalities in cancer Acquired Nonrandom Clonal Used for diagnosis and prognosis

Chromosome Changes in Cancer Disease specific changes: Leukemias Lymphomas Mesenchymal Tumors

Chromosome Changes in Cancer Unbalanced multiple and unspecific karyotypic abnormalities: Epithelial tumors (breast, colon, lung, prostate, ovary) Mesenchymal tumors (osteosarcoma, malignant fibrous histiocytoma) Secondary hematologic malignancies

Functional Consequences of Balanced Chromosomal Rearrangements Fröhling S, Döhner H. N Engl J Med 2008;359:722-734

Molecular Cytogenetics

Fluorescence in situ Hybridization (FISH)

Fluorescence in situ Hybridization (FISH) Hybridization of a fluorescently labeled nucleic acid probe to a target sequence within a specimen on a microscope slide

Basic FISH Procedure Prepare Slide (denature, dehydrate) Prepare Probe (denature in hyb. buffer) Hybridize Wash Counterstain (with DAPI-antifade) Visualize

Visualizing FISH Emission / Barrier Filter Dichroic Mirror Excitation Filter

FISH in the cytogenetics laboratory: Satellite sequence probes Whole chromosome paints Unique sequence probes

Molecular Cytogenetic Analysis In situ Hybridization Targets Metaphase chromosomes Interphase nuclei Formalin-fixed, paraffin-embedded tissues

FISH Overview Each probe attaches to specific target. Differently colored probes simultaneously determine both number and location of different loci. Metaphase & interphase Quantitative assessment in intact tissue, cells, nuclei or chromosomes.

FISH Probe Design & Patterns for Detection of Chromosome Translocations & Inversions Dual Color, Single Fusion Dual Color, ES (extra signal) Dual Color, Break-Apart Dual Color, Dual Fusion

Dual Color, Break-apart: Both Probes Flank One Breakpoint 14q32.33 IGH Locus Switch 5 3 Variable Segments D J Constant Features: Cutoff for probe design~1-3 %* Best design for intact sections Useful for multiple translocation partners: IGH, MLL, BCL6, MYC, ALK * Cutoff values for probe types from Dewald et al, Chap 32,Clinical Laboratory Medicine, K. McClatchey Ed., 2002 2F Normal 1O1G1F Abnormal

Gleevec

ES Design: One Probe Flanks, Other Probe Spans Breakpoint Cen 22 BCR Cen 9 ASS ABL Normal 2O2G Cen der(22) Fused signal Cen der(9) ASS Features: Lower cutoff for probe design ~3 %* Random chance abnormal pattern less likely Del cen to ABL breakpoint - Single Fusion *Cutoff values for probe types from Dewald et al, Chap 32, Clinical Laboratory Medicine, K. McClatchey Ed., 2002! ES signal CML nucleus Abnormal 2O1G1F

BCR/ABL Dual Fusion BCR/ABL Dual Fusion

LSI PML/RARA dual color dual fusion

LSI RARA BA

LSI MLL dual color BA

CMYC

Breast cancer! HER2/Neu amplification

Science 310: 644-648, 2005

Bladder Cancer: Epidemiology and Overview

Epidemiology #4 most common CA in Men #2 most common CA of GU Tract! 600,000 in North America living with bladder UC 57,400 new cases/year 12,500 deaths/year! Highest recurrence rate of any cancer (~70%) 30% of recurrent tumors progress American Cancer Society 2005

Urine Cytology

Cytology High Specificity Low Sensitivity Frustration high for US urologists Atypical cellularity: Recommend clinical correlation or further testing.

UroVysion Intended Use The UroVysion Bladder Cancer Kit (UroVysion Kit) is designed to detect aneuploidy for chromosomes 3, 7, 17, and loss of the 9p21 locus via fluorescence in situ hybridization (FISH) in urine specimens from persons with hematuria suspected of having bladder cancer.! Results from the UroVysion Kit are intended for use, in conjunction with and not in lieu of current standard diagnostic procedures, as an aid for:! 1. Initial diagnosis of bladder carcinoma in patients with hematuria suspected of having BCa and subsequent! 2. Monitoring for tumor recurrence in patients previously diagnosed with bladder cancer.!!!

UroVysion (p16 gene)

FISH Results CEP 3 (red = 2) CEP 7 (green = 2) CEP 17 (aqua = 2) LSI 9p21 (gold = 2)

Positive FISH DAPI CEP 3 (red = 8) CEP 7 (green = 4) CEP 17 (aqua = 3) LSI 9p21 (gold = 1)

LUNG CANCER ALK (anaplastic lymphoma kinase) 2p23.2 EML4 (echinoderm microtubule associated protein-like 4) 2p21! Diverse small inversions on 2p Non-small cell lung cancer Crizotinib

NSCLCs hybridized with the ALK break-apart FISH probe (Abbott Molecular). Camidge D R et al. Clin Cancer Res 2010;16:5581-5590 2010 by American Association for Cancer Research

Strengths & Limitations of FISH Strengths Wide variety of tissues Metaphase & interphase Specimen structure remains intact. Cell-by-cell analysis Accommodates breakpoint variation Detects cytogenetically cryptic aberrations Contamination from previous tests not an issue Limitations Can not detect very small genetic lesions Lacks very low level detection of PCR Not as quick as rapid-cycle real-time PCR Not yet as automated as some other molecular techniques.

Spectral Karyotyping (SKY)

SKY Procedure Overview

WEB References! http://www.atlasgeneticsoncology.org! http://cgap.nci.nih.gov/chromosomes/mitelman