Introduction to Cytogenetics

Transcription

1 Introduction to Cytogenetics Catherine McCarthy Pathology Qld Cytogenetic abnormalities constitutional acquired: clonal: related or unrelated non-clonal Investigating constitutional abnormalities peripheral blood bone marrow skin fibroblasts products of conception amniotic fluid chorionic villus biopsy 1

2 Investigating acquired abnormalities bone marrow lymph nodes solid tumours fluids: ascitic, pleural other malignant tissues Collection of specimens aseptic technique appropriate specimen collection vessel clotted or unclotted? transport medium time taken for transportation climate effects Culture of specimens manipulation required whole or part of specimen stimulation required? medium to optimize results additives during culture culture vessels time in culture assessment of culture re readiness for harvesting Harvesting of specimens Mitotic arrest hypotonic treatment fixation slide making staining assessment of success of harvest factors influencing successful harvesting remedying the damage 2

3 Karyotypic analysis unbanded metaphases banded metaphases counting and analysis of metaphases appropriate types of banding to use preparing the karyotype Unbanded metaphase Centromere position 3

4 4

5 Cytogenetic nomenclature + 47,XY,+21-45,XY,-7 t t(5;8)(p13;q24) inv inv(16)(p13q22) del del(7)(q22) add add(4)(p16) dup dup(6)(q13q21) ins ins(2;5)(p21;q13q31) cen dmin, hsr mat, pat inc, cp mar rea rec tel 5

6 Translocation example Inversion example Inversion example ISH/FISH 1969: Pardue & Gall 1970s: radioactively labelled probes development of molecular cloning development of non-radioactive dyes digital imaging 6

7 FISH probes indirect label direct label quality control source: commercial in house FISH Target DNA: metaphases, interphase nuclei, histological specimens hybridization counter staining microscopic examination problems FISH probes Centromere: usually α satellite DNA; sequence homology between 13 & 21 and 5 & 19 unique sequence (LSI): gene regions and telomeres whole chromosome paint (wcp): covers all of the chromosome, excluding the centromere 7

8 8

9 CGH: Comparative Genomic Hybridization used for surveying entire genomes for DNA sequence copy number variation may highlight location of important genes increase in gene dosage gene amplification hsr and dmin CGH: Comparative Genomic Hybridization Preparation of normal metaphase spreads isolation of high molecular weight genomic DNA from the tumour specimen + normal DNA labelling of normal and tumour DNA: tumour green, normal red in situ hybridization of the labelled DNA to normal metaphases CGH (cont) Fluorescence microscopy to visualize colour ratio difference of normal and tumour DNA acquisition and display of multicolour digital images quantification of copy number differences: green to red fluorescence intensity ratio profiles combine profiles from several metaphases interpretation of ratio changes CGH (cont) Detects genetic aberrations that involve loss or gain of DNA sequences balanced translocations and inversions not detected cannot distinguish diploid from triploid or tetraploid tumours fluorescence intensities decrease at telomeres isolation of tumour DNA 9

10 Other FISH techniques Spectral karyotyping: SKY and multicolor FISH: MFISH: simultaneous hybridization of 24 chromosome specific painting probes labelled with different combinations of 5 fluorochromes combined binary ratio labelling FISH: COBRA-FISH: ratio of hybridization signals intensities of 3 fluorochromes color-changing karyotyping: CCK: combinatorial labelling of chromosomes MFISH Detects interchromosomal aberrations para and peri centric inversions, small deletions and duplications not detected 10

11 Arm-specific Multicolor FISH: armfish 42 colours uses a commercially available mfish kit set of chromosome arm specific painting probes increases resolution to level of chromsome arms two step process 11

12 Constitutional Cytogenetics Prenatal amniotic fluid chorionic villus biopsy cord blood products of conception skin Postnatal infants children teenagers adults peripheral blood skin Prenatal FISH abnormal ultrasound findings increased triple test increased nuchal translucency adequate specimen volume signal uptake blood stained specimens turn around time irreversible decisions 12

13 13

14 Constitutional Cytogenetics Prenatal amniotic fluid chorionic villus biopsy cord blood products of conception skin Postnatal infants children teenagers adults peripheral blood skin 14

15 15

16 Postnatal Constitutional Cytogenetics perinatal period infants children puberty fertility reproductive loss 16

17 17

18 Subtelomeric deletions MR occurs in 2-3% of population Chromosome abnormalities in 4-28% of MR depending on patient selection 7.4% of children with moderate-severe MR 0.5% of children with mild MR Deletion and translocation of >2-3 Mb usually visible microscopically Deletion of 4p, 5p, 13q, 18p Subtelomeric deletions 1pter deletion: 1p36.3: growth and MR, seizures, visual problems, large anterior fontanelle, asymmetrical and low set dysplastic ears, deep set eyes, depressed nasal bridge, pointed chin, fifth finger clinodactyly 1qter deletion: growth and MR, severe microcephaly, corpus callosum abnormalities, cardiac abnormalities, hypospadias, characteristic facies 22q deletion: hypotonia, developmental delay, absence of speech, overgrowth Subtelomeric deletions Family history of MR prenatal onset growth retardation postnatal growth abnormalities 2 facial dysmorphic features 1 non-facial dysmorphic feature and/or congenital abnormalities 18

19 Constitutional Cytogenetics Puberty sex chromosome aneuploidy X/i Xq; X/XX/XXX; XX/ del Xp XXY; XXXY XYY translocations 19

20 Constitutional Cytogenetics Adults miscarrying couples Balanced translocations Sex chromosome abnormalities aneuploidy structural Infertility Balanced translocations Sex chromosome abnormalities aneuploidy structural assisted reproduction PGD 20

21 Malignancy Cytogenetics leukaemias lymphomas other solid tumours fluids FNAs 21

22 Cytogenetic Abnormalities in Malignancy numerical: monosomy trisomy polyploidy structural: translocations (unbalanced and balanced) deletions duplications inversions Occurrence of Cytogenetic Abnormalities at diagnosis: assist in classification guide to prognosis identify new primary abnormalities during treatment: residual disease impending relapse therapy resistant clones at relapse: same/further abnormalities new abnormalities Acute Leukaemia in adults: 80% myeloid 20% lymphoid in children: 85% lymphoid 15% myeloid in infants: biphenotypic (rare) 30% myeloid (monocytic and M7) 70% lymphoid Cytogenetic Abnormalities in Classification of Acute Leukaemia in myeloid disease: t(15;17)(q22;q21) in M3 t(8;21)(q22;q22) n M2, M4, M1 t(6;9)(p23;q34) in M2, M4, M1, MDS t(9;11)(p22;q23) in M5a, M5b, M4 inv(16)(p13q22) in M4Eo inv(3)(q21q26) in M6, MDS t(8;16)(p11;p13) in M5 t(12;22)(p13;q12) in M7, M0, M4 22

23 Cytogenetic Abnormalities in Classification of Acute Leukaemia: WHO (2008) Acute myeloid leukaemia with recurrent genetic abnormalities t(8;21)(q22;q22): RUNX1-RUNX1T1 inv(16)(p13.1q22) or t(16;16)(p13.1;q22);cbfb-myh11 t(15;17)(q22;q12); PML-RARA t(9;11)(p22;q23);mllt3-mll t(6;9)(p23;q34); DEK-NUP214 inv(3)(q21q26.2) or t(3;3)(q21;q26.2); RPN1-EVI1 t(1;22)(p13;q13); RBM15-MKL1 Provided by Owen Walker, Pathology Qld Cytogenetics Unit 23

24 24

25 25

26 4 26

27 27

28 t(3;3)(q21;q26.2) EVI1 28

29 Prognostic Value of Cytogenetic Abnormalities MYELOID: GOOD: t(8;21) t(15;17) inv(16) INTERMEDIATE: POOR: t(9;11)(p22;q23) +8 t(9;22) t/del of 11q23 del/monosomy 5q and 7q 3q21q26.2 Cytogenetic Abnormalities in Classification of Acute Leukaemia: WHO (2008) AML with gene mutations KIT FLT3-ITD CEBPA NPM1 WT1 BAALC ERG MN1 Cytogenetic Abnormalities in the Classification of Acute Leukaemia in lymphoid disease: t(8;14) / t(8;22) / t(2;8) in L3 t(1;19)(q23;p13) in L1, L2 t(12;21)(p13;q22) in L1, l2 in biphenotypic disease: t(11;19)(q23;p13) t(4;11)(q21;q23) t(9;22)(q34;q11.2) Cytogenetic Abnormalities in Classification of Acute Leukaemia: WHO (2008) B lymphoblastic leukaemia/lymphoma with recurrent genetic abnormalities t(9;22)(q34;q11.2); BCR-ABL1 t(v;11q23); MLL rearranged t(12;21)(p13;q22); ETV6-RUNX1 hyperdiploidy hypodiploidy t(5;14)(q31;q32); IL9-IGH t(1;19)(q23;p13.3); TCF3-PBX1 29

30 Provided by Owen Walker, Pathology Qld Cytogenetics Unit 30

31 ETV6/RUNX1 ES 31

32 Prognostic Value of Cytogenetic Abnormalities LYMPHOID: GOOD: >50 INTERMEDIATE: t(1;19) del 9p POOR: t(9;22) t(4;11) t(8;14) / t(8;22) / t(2;8) MDS / 2 o AML MDS / 2 o AML de novo or secondary del 5q / monosomy 5 del 7q / monosomy 7 t/del 11q23 trisomies 8, 11, 13, 21 trisomy 1q Probes available: D7Z1/7q31; 7q22/7q35 EGR1 (5q31) MLL D20S108 p53; RARA D8Z1,D11Z1 RUNX1 RB1 32

33 33

34 Lymphomas Follicular t(14;18)(q32;q21) Burkitt t(8;14) / t(8;22) / t(2;8) Mantle cell t(11;14)(q13;q32) Diffuse large B cell Additional abnormalities Double hit lymphoma 34

35 Gene amplification dmin:double minutes hsr: homogeneously staining region RUNX1 ABL1 MYC NMYC MLL 35

36 amplification amplification in double minutes 36

37 Myeloma Culture methods Prognosis Probes available: IGH, p53, D13S319 (13q14.3)/13q34 IGH/CCND1 IGH/MAF IGH/FGFR3 CLL Culture methods Prognosis Probes available: p53 LSI ATM D13S319/13q34 CEP12 Limitations of conventional Cytogenetics BM case with 300 bphs Conventional karyotyping:resolution of >10Mb quite reliably (</= 550 bphs) High resolution banding techniques 3-5 Mb deletions or duplications can be visualised ( bphs) FISH - <3Mb 100 Kb (submicroscopic) 37

38 Prenatal case with bphs Peripheral blood (stimulated with mitogen) with 700 bphs FISH Limitations of chromosome studies Resolution is limited Labour intensive and subjective Minimum of 2 days required for culture and urgents can be turned around in 2-4 days 38

39 Array CGH/microarrays - Principle Arrayed DNA on a solid surface acts as targets for hybridisation. Arrayed DNA could be cdna, oligonucleotides or SNPs Sample to be tested only or test and reference samples are labeled with different fluorochromes and hybridised to the arrayed surface Array is then imaged and relative fluorescence intensities are captured and an intensity ratio is established The intensity ratio is the DNA copy number difference - objective interpretation Interpretation of Results Diagnostic Yield ~10% abnormality rate on karyotype in developmental delay cases Increase in detection rates to ~20% using array technology Am J Hum Genet ; Chromosomal Microarray is a First-Tier Clinical Diagnostic Test for Individuals with Developmental Disabilities or Congenital Anomalies 39

40 Microarrays Microdeletion syndromes Autism Psychiatric disorders Malignancy Further applications of this technology Targetted/custom arrays for oncology (AML profiler, ALL profiler, MM) Products of conception 40

41 Further applications of this technology Targetted/custom arrays for oncology (AML profiler, ALL profiler, MM) Products of conception BoBs HD and LD chips 41

42 KARYOLITE TM BoBs TM BACs-on-Beads Prenatal applications Amniotic fluid Products of conception Detects aneuploidies, gains and losses on all 24 chromosomes in a single assay Summary Appropriate test for specimen type Turnaround time Clinical urgency Discovery of new regions of interest The world is getting smaller! Acknowledgements Pathology Qld Cytogenetics Unit staff Dr Bronwyn Williams Dr Adayapalam Nandini Cinical Haematologists Pathology Qld 42