1 Cytogenetics and molecular genetics in oncology Karla Plevova, MSc, PhD 25.11.2021 Outline of the presentation 1. Differences in molecular genetics and cytogenetics of congenital disorders vs cancer 2. Application of molecular-genetic and cytogenetic findings in oncology 3. Material sources and material used 4. Methods used and practical examples MG: molecular genetics CG: cytogenetics2 Cancer as a genetic disease Two levels: Cancer hereditary syndromes – germline mutations Genetic alterations gained during a lifetime - somatic https://siteman.wustl.edu/ 3 Why molecular genetics and cytogenetics in oncology can be interesting for a dentist? Acute myeloid leukemia (AML) manifesting by blast infiltration in gums.4 1. Differences in MG and CG of congenital disorders vs cancer Characteristics Congenital disorders Cancer Prevailing origin of genetic defects Germline Somatic Extent of genetic abnormalities Single or small number of changes Variable, typically higher Type of abnormalities One/two types present per case Combination of all types Mosaicism Rare Common 5 2. Application of MG and CG findings in oncology a. Hereditary predisposition assessment b. Establishing and refining diagnosis c. Disease prognostication d. Treatment optimization e. Disease activity monitoring f. Disease complication diagnostics 6 2. Requirements on the techniques ⎼ High specificity and high sensitivity, limit of detection ⎼ Fast processing – range of few hour to few days ⎼ Tools for data analysis (bioinformatics for NGS) ⎼ Standardization and validation ⎼ Availability of reference material (positive/negative controls), reference sequences ⎼ Regular quality assessment ⎼ Compliance with legislation regulations 7 a. Hereditary predisposition assessment Tawana et al, Blood 2015 ⎼ Cases of cancer accumulated in families ⎼ Autosomal dominant and recessive inheritance ⎼ Typical onset at young age ⎼ Genetic counseling ⎼ Screening for causative variants Breast-Cancer Susceptibility Loci and Genes. Foulkes, NEJM 2008 8 a. Hereditary predisposition assessment Döhner et al, Blood 2017 Gomy & Diz, Genet Mol Biol 20169 b. Establishing and refining diagnosis ⎼ Diagnosis confirmation based on detection of specific (marker) gene or chromosomal abnormalities ⎼ Incorporation of genetic/cytogenetic markers in WHO classification ⎼ Resolving ambiguous cases ⎼ Markers specific for the whole diagnostic entity or only for a subset of a disease (implications for treatment) ⎼ Examples ⎼ Mantle cell lymphoma – translocation t(11;14) ⎼ Hairy cell leukemia - BRAF V600E mutation 10 b. Establishing and refining diagnosis Clonality ⎼ Typical characteristics of lymphoid (but also other) malignancies ⎼ Analysis of antigen receptor rearrangements, translocations and gene mutations ⎼ Monoclonal vs polyclonal picture – distiguishing of malignant vs reactive conditions ⎼ Quantification of tumor load 0 1000 0 2000 0 3000 0 4000 0 5000 0 6000 0 7000 0 8000 0 9000 0 1000 00 1100 00 1200 00 1300 00 1400 00 1500 00 1600 00 1700 00 1800 00 1900 00 2000 00 100 150 200 250 300 350 400 450 2 7 9 -6 -0 6 .G 0 7 _ 0 9 0 4 2 0 0 8 0 E Size (nt) DyeSignal 0 5 0 0 0 1 0 0 0 0 1 5 0 0 0 2 0 0 0 0 2 5 0 0 0 3 0 0 0 0 3 5 0 0 0 4 0 0 0 0 4 5 0 0 0 5 0 0 0 0 5 5 0 0 0 6 0 0 0 0 6 5 0 0 0 1 0 0 1 5 0 2 0 0 2 5 0 3 0 0 3 5 0 4 0 0 4 5 0 5 2 3 .F 0 2 _ 1 0 0 1 2 5 0 9 W A S ize (n t) DyeSignal 11 c. Disease prognostication ⎼ Genetic and cytogenetic markers associated with certain disease features ⎼ Risk assessment at time of diagnosis ⎼ Genetic markers of various types – gene mutations, chromosomal abnormalities, a type of antigen receptor rearrangement, … ⎼ Prognostic vs predictive markers del13q 133 m +12 114 m del11q 79 m del17p 32 m Döhner et al, Blood 2000 12 c. Disease prognostication AML:CLL: Hamblin et al, Blood 1999 Dohner et al, Blood 2017 13 d. Treatment optimization ⎼ Concept of personalized treatment – tailored for individual patients ⎼ Treatment response related to genetic abnormalities detected in cancer cells ⎼ Targeted treatment – blocking the growth and spread of cancer by interfering with specific molecules ("molecular targets") ⎼ Synthetic lethality – blocking or inactivation of two genes leads to cell death Nijman, FEBS Lett 201114 d. Treatment optimization - examples Diagnosis Genetic defect Treatment option Chronic myeloid leukemia BCR/ABL fusion gene Tyrosine kinase inhibitors (imatinib, dasatinib etc.) Breast cancer BRCA1 mutations PARP inhibitor olaparib Non-small cell lung cancer EGFR mutations EGFR inhibitors (erlotinib, afatinib etc.) Non-small cell lung cancer ALK gene rearrangements ALK inhibitors (crizotinib, ceritinib etc.) Melanoma R/R hairy-cell leukemia BRAF mutations BRAF inhibitors (dabrafenib, vemurafenib etc.) Colorectal cancer KRAS mutations Contraindication for targeting EGFR by cetuximab and panitumumab Chronic lymphocytic leukemia BTK mutations Contraindication for ibrutinib administration 15 e. Disease activity monitoring Buckley SA, et al. Bone Marrow Transpl. 2013. ⎼ Minimal residual disease (MRD) – cancer cells remaining after therapy ⎼ Need for MRD marker identification before therapy ⎼ Monitoring of MRD markers after therapy ⎼ Design of patient-specific and sensitive assays ⎼ Typical markers: ⎼ Gene rearrangements ⎼ Fusion genes ⎼ Gene mutations 16 f. Disease complication diagnostics Infection complications related to cancer treatment ⎼ Opportunistic infections – otherwise common pathogen causing severe symptoms ⎼ Related to bone marrow (BM) and peripheral blood stem cell (PBSC) transplantation and other cancerspecific treatment (e.g. alemtuzumab) Molecular diagnostics – typing of pathogens according to their DNA/RNA sequence ⎼ (multiplex) PCR, real-time PCR, NGS ⎼ Quantification and monitoring of pathogen load Young et al, Biol Blood Marrow Transplant 2016 17 3. Materials used and material sources ⎼ Types of samples ⎼ Cells ⎼ DNA ⎼ RNA ⎼ cfDNA 18 3. Materials used and material sources ⎼ Sampling ⎼ Peripheral blood ⎼ Bone marrow ⎼ Liquid biopsies ⎼ Aspirates ⎼ Fine-needle biopsies ⎼ Fresh tissue ⎼ Formalin-fixed paraffin-embedded (FFPE) tissue ⎼ Swabs (buccal, NPh, …) 19 Hematooncology – easy access to malignant cells ⎼ Peripheral blood, bone marrow ⎼ EDTA or heparin collection tubes ⎼ Different cell population used according to the application ⎼ Leukocytes, Mononuclear cells, Granulocytes, Lymphocytes, Specific cell subpopulations ⎼ Utilization of separation methods 20 Why to perform cell separation? 21 Solid tumors – tissues ! Invasive ! Biopsies, fine-needle biopsies ! Fresh frozen vs FFPE tissue ! Decreased DNA and RNA quality (fragmented, chemically modified in case of FFPE material) 22 Liquid biopsies Zeng et al, Cancer Comm 2019 Very low amount of material Sources: ! Plasma / serum ! Urine ! Joint fluid ! Cerebrospinal fluid ! … 23 cfDNA 177 361 532 Snyder et al, Cell 2016 24 cfDNA cHL, classical Hodgkin lymphoma; DLBCL, diffuse large B-cell lymphoma; MRD, minimal residual disease; PCNSL, primary nervous system lymphoma. Rossi et al, Haematologica 2019 Applications ⎼ Diagnosis, early detection ⎼ Genotyping ⎼ Disease risk stratification ⎼ Treatment selection ⎼ Treatment response assessment ⎼ Disease monitoring FDA approved assays for gene mutation detection Solid tumors and hematological malignancies 25 4. Methods used and practical examples a. Cytogenomics methods ! Chromosome banding techniques ! Fluorescence in situ hybridization ! Genomic arrays b. Amplification-based methods ! PCR and real-time PCR ! Droplet digital PCR c. Sequencing techniques ! Sanger sequencing ! Next-generation sequencing ! Amplicon sequencing ! Panel sequencing ! Whole exome sequencing ! Whole genome sequencing 26 a. Classical cytogenetics – chromosome banding techniques 27 a. Classical cytogenetics – chromosome banding techniques Resolution >10 Mbp Applications ! Detection of typical abnormalities ! Complex karyotype assessment (≥3 or ≥5 abnormalities) 28 a. Molecular cytogenetics ! Fluorescent in situ hybridization (FISH) ! Targets specific regions based on DNA sequence ! Detection of chromosomal abnormalities with diagnostic, prognostic and predictive value Probe types: centromeric genewhole chromosome 29 a. Molecular cytogenetics mFISH mBAND ! FISH methods for genome-wide analysis 30 a. Genomic arrays ! Molecular cytogenetic technique for detection of genomic gains and losses ! Detection of copy-neutral loss of heterozygosity ! Not possible to detect balanced rearrangements ! Precise breakpoint localization, identification of affected genes ! High resolution, genome-wide ! No need for viable cells arrayCGH & SNP array 31 Schoumans J et al, 2016 Aneu- ploidy CNA Poly- ploidy Clonal heterogeneity Focal amplification Balanced rearrangements Unbalanced rearrangements cn-LOH Classical cytogenetics +++ + +++ +++ ++ +++ +++ Interphase FISH +++ ++ + +++ +++ +++ ++ ArrayCGH +++ ++ - + +++ - ++ - CGH+SNP array +++ +++ + + +++ - ++ ++ SNP array +++ +++ ++ ++ +++ - ++ +++ CNA – copy number alteration, gains or lossess of genetic material cn-LOH – copy neutral loss of heterozygozity a. Comparison of sensitivity of cytogenetic techniques 32 b. Polymerase Chain Reaction (PCR) ! Amplification of region of interest using specific primers ! Cycling reaction condition ! Used for marker quantification ! Input for further analyses (restriction, Sanger sequencing, fragment analysis, NGS, …) 33 b. PCR-based quantification methods www.bio-rad.com Droplet digital PCR (ddPCR) ! Alternative method for marker absolute quantification ! Highly precise ! Need for specific instrumentation Real-time PCR ! Quantitative method – fluorescent detection of generated products ! Need for specific primers and probes ! Relative vs absolute quantification test sample 34 b. Real-time PCR applications ! Quantification of minimal residual disease after therapy – detection of tumor specific markers (fusion genes, antigen receptor rearrangements etc.) ! Gene expression analysis ! Pathogen detection and pathogen load quantification V N D N J HCDR3F primer R primer probe 35 c. Sanger sequencing Modification of PCR ! single primer extension ! Incorporation of dNTPs and ddNTPs https://www.sigmaaldrich.com/technical- documents/articles/biology/sanger-sequencing.html Applications ! Basic method for sequence variant detection (mutations, breakpoint localization) ! Fragment analysis – modification of the method for detection of fragment length variation 36 c. NGS - principles and targeted regions Next-generation sequencing (NGS) ~ masively parallel sequencing (MPS) ! PCR amplification of DNA fragments or direct sequencing of individual fragments (single molecule sequencing) ! The most common approach – sequencing by synthesis (Illumina sequencers) ! Milions of fragments are amplified simultaneously (vs capillary sequencer max 96 reactions) ! Short reads (tens to hundreds basepairs) 37 c. NGS - principles and targeted regions Illumina machines and their capacity HiSeq 4000 12 genomes/run, 1.5 TB/run NextSeq 500 1 genome/run, 120 GB/run MiSeq 0.15 genome/run, 15 GB/run NovaSeq 48 genomes/run, 6 TB/run MiniSeq 0.07 genome/run, 7.5 GB/run iSeq 0.01 genome/run, 1.2 GB/run 38 c. NGS – regions of interest genome exome selected genes or loci 3 200 000 000 bp 30 x read depth 20 000 genes 100 x read depth < 100 genes ≥ 1000 x read depth 39 c. Amplicon sequencing Application: TP53 mutation analysis ⎼ NGS with high coverage (limit of detection 0.1 % of variant allele) ⎼ treatment response prediction PCR products Transposome s ~300 bp Tagmentation Reduced cycle PCR amplification Read 1 Sequencing primer Read 2 Sequencing primer Sequencing-Ready Fragment Index 1 Index 2 P5 P7 40 c. Panel sequencing ⎼ Sets of selected regions of interest ⎼ Target enrichment by amplification or hybridization Jennings LJ et al. J Mol Diagn. 2017 Application: LYNX panel ⎼ diagnostics of molecular markers in lymphoid malignancies 41 c. Whole exome sequencing (WES) ⎼ Mainly experimental approach for exploring unknown variants ⎼ Used in ⎼ genetic counseling for identification of causative variants ⎼ discovery of novel genetic markers ⎼ searching for treatment targets 42 c. WES – case report of Shwachman-Diamond syndrome ⎼ a multisystem autosomal recessive disorder ⎼ clinical features: pancreatic exocrine insufficiency, hematologic dysfunction, and skeletal abnormalities ⎼ haematological malignancies (e.g. myelodysplastic syndrome and acute myeloid leukemia) occur in one third of patients ⎼ homozygous or compound heterozygous variations in SBDS gene 43 c. Whole genome sequencing (WGS) ⎼ Mainly experimental method for exploring unknown variants ⎼ Applications similar to WES, additional information about non-coding regions and chromosomal abnormalities ⎼ Typicall sequencing coverage ~ 30–100x – detection of clonal or germline mutations ⎼ Shallow sequencing (~ 0.5-10x coverage) – genome-wide detection of chromosomal abnormalities, low yield of mutation detection ⎼ In clinical practise a potential benefit of combination of shallow and panel sequencing 8 11 13 18 44 The end… Contact: karla.plevova@mail.muni.cz or plevova.karla@fnbrno.cz Thank you for your attention! 45