Diagnostic Molecular Techniques Used in Cytology- Pros and Cons. Charles Walther, MD, PhD, Lund University, SWEDEN

Transcription

1 Diagnostic Molecular Techniques Used in Cytology- Pros and Cons Charles Walther, MD, PhD, Lund University, SWEDEN

2 Background- Tumor Diagnostics Clinical examina+on Radiological evalua+on Sampling-+ssue material - Fine needle aspira+on(fna) - Core needle biopsy(cnb) - Surgical tumor biopsy(stb) - Body fluids (BF)

3 Tissue sample Morphological assessment Immunohistochemical evalua+on Gene+c/molecular evalua+on Molecular cytology

4 Molecular Cytology Clinician/ Patient Cytologist/ Diagnosis Material Selection Molecular Analyses

5 Types of Cytology Material Used Aspirates-Smears Liquid based cytology Cell blocks

6 Characteristics of the cytological material- for molecular use Quality beler than formalin fixated +ssue OPen small quanta+es Difficult but important to assess amount of neoplas+c cells Scanning +me consuming Pa+ent friendly sampling- more material!

7 Molecular Methods in Cytology in Lund Gene+c/molecular methods - Cytogene+cs - FISH (Fluorescence In Situ Hybridiza+on) - SNP Array (Single Nucleo+de Polymorphism) - PCR (Polymerase Chain Reac+on) - NGS (Next Genera+on Sequencing)

8 Cytogenetics Chromosome bands stained Structural and numerical aberra+ons Overview, cell-cell Labour intensive Resolu+on 5 Mb Tissue culturing- Fresh!

9 Cytogenetics-How to use Malignant vs Benign Specific diagnosis (e.g. Ewing, BurkiL) Gene+c overview Direct further inves+ga+on - RT-PCR - FISH

10 Cancer Genetics 204 (2011) 203e206 Chromosome banding analysis of cells from fine-needle aspiration biopsy samples from soft tissue and bone tumors: is it clinically meaningful? Charles Walther a,b, *, Henryk A. Domanski a, Fredrik Vult von Steyern c, Nils Mandahl b, Fredrik Mertens b a Department of Pathology, University and Regional Laboratories, Lund University Hospital, Lund University, Lund, Sweden; b Department of Clinical Genetics, University and Regional Laboratories, Lund University Hospital, Lund University, Lund, Sweden; c Department of Orthopedics, Clinical Sciences, Lund, Lund University and Skane University Hospital, Sweden

11 Success rate-bone & Soft Tissue 24% of FNA samples- pathological karyotype 48% of CNB samples 64% of surgical biopsies Exception-Ewing family of tumors- 56%!

12 FISH (Fluorescence In Situ Hybridization) Fluorescent probes bind complementary DNA strands Whole or parts of chromosomes Structural and numerical aberra+ons Targeted analysis Metaphase / Interphase Resolu+on 5-20 kb Fresh/Fixed

13 FISH -How to use Malignant vs Benign (aneuploidy) Specific diagnosis (e.g. Synovial sarcoma) Specific gene+c changes -Transloca+ons -Dele+ons -Amplifica+ons -Fusions

14 SNP Array (Single Nucleotide Polymorphism) Varia+on of single nucleo+des (A,T,C,G 85 mil) Varia+ons used as markers Detects amount of DNA Allele frequency heterozygous/homozygous Detailed overview not balanced aberra+ons Resolu+on 20 kb Fresh/Fixed

15 SNP Array- How to use Produces a virtual karyotype High resolu+on Loss, Gains and loss of heterozygosity Replaces cytogene+cs Warning! Not balanced changes FNA material applicable

16 Rhabdomyosarcoma Metastasis Primary 7 p21.3 p21.1 p15.3 p14.3 p14.1 p12.3 p12.1p11.2 q11.21 q11.22 q11.23 q21.11 q21.3 q22.1 q31.1 q33 q34 q35 q36.1 q36.3 Base Position 15,913,871 31,827,741 47,741,611 63,655,481 79,569,351 95,483, ,397, ,310, ,224,831 Cytogenetic Band p21.3 p p14.3 p14 p1 p1p12.1p11.2 q11.21 q11.23 q21.11 q2 q21.3 q31.1 q q3 q3 qq32 q34 q35 p2 p15.3 p15 p14.1 q22.1 q33 q36.1 q36 q36.3 No sequence data file found for this chromosome. Sequence (+) AK Per4 TMEM195 NPY CHN2 HERPUD2 NPC1L1 POM121L12 DQ TYW1B MIR548M SRI SAMD9 CNPY4 PRKAR2B TFEC PTPRZ1 FAM40B PTN TCRBV5S1A1T LOC1

17 RT-PCR (Reverse Transcriptase Polymerase Chain Reaction) Amplifies DNA converted from RNA (cdna) Analyses muta+ons and expression of genes/ fusion genes Targeted analysis Sensi+ve method false posi+ves Fresh/Formalin fixed

18 RT-PCR - How to use Useful on FNA material Small amounts of aspirate open enough Highly sensi+ve! False posi+ves! Targeted analysis Informa+ve -Fusion genes -Specific muta+ons -Loss/ Gains -Gene expression

19 Next Generation Sequencing Detects nucleo+de sequences RNA/ DNA Massive parallell sequencing - a large number of sequences analysed Complete overview- genome, exome and transcriptome Detects anomalies down to nucleo+de level Labour intensive- high amount of data, expensive, false posi+ve/ nega+ve

20 NGS - How to use Useful on FNA material Small amounts of aspirate open enough Highly sensi+ve! False posi+ves! Targeted analysis (?) Informa+ve -Fusion genes -Specific muta+ons -Loss/ Gains -Gene expression

21 Molecular Cytology- Pros and Cons Pa+ent friendly sampling- Small amounts Good DNA/RNA quality- Normal cell dilu+on what you see is what you get what you get is not what you see Diagnos+c help- Takes +me Several metods applicable- Interpreta+on

22 The Future of Molecular Cytology

23 Thank You!

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