Basics of Molecular Diagnostics for the Everyday Practicing Pathologist

BASICS OF MOLECULAR DIAGNOSTICS FOR THE EVERYDAY PRACTICING PATHOLOGIST

Artur Rangel, MD, PhD

Pathology Consultants of South Broward Medical Director, Clinical and Molecular Pathology - Memorial Healthcare System

Genome Structure

______Start Stop |||||||||||||||||||||||||||||||||||| ATGACGGATCAGCCGCAAGCGGAATTGGCGACATAA

TACTGCCTAGTCGGCGTTCGCCTTAACCGCTGTATT |||||||||||||||||||||||||||||||||||| Common Types of Genetic Variants

Chromosomal GAINS / LOSSES (CNV) Complete vs Partial

Large translocations Balanced vs unbalanced

1p / 19q Co-deletion Oligodendroglioma

19q 1p / 19q Co-deletion Oligodendroglioma 250,000,000 bp Technique: Karyotype

Variants GAINS / LOSSES / Rearrangements Resolution Chromosome level / Megabases Scope Genome wide Quantitative Semi ( x / 20 cells) Specimen Requires living / dividing cells Limitations Specimen / resolution Common Types of Genetic Variants

Sub-chromosomal GAINS / LOSSES (CNV) Several kilobases range Small Translocations Balanced vs unbalanced Genome Structure DNA Complementarity A <–> T

G <–> C DNA Complementarity -> hybridization

DNA Hybridization DNA Complementarity -> hybridization DNA Complementarity -> hybridization DNA Complementarity -> hybridization

Millions of Bases Thousands of Bases 22 22 Break apart probe

Karyotype FISH Karyotype FISH Technique: FISH

Variants GAINS / LOSSES / Rearrangements Resolution Sub-Chromosome / 100’s Kilobases Scope Targeted – specific probes Quantitative Yes, % affected cells Specimen FFPE or dividing cells (metaphase) Limitations Specimen / Decal / resolution Common Types of Genetic Variants

Chromosomal GAINS / LOSSES (CNV) Complete vs Partial Single Nucleotide Polymorphisms (SNP) Gene Expression Over, Under Expression profiling Microarray Applications

Comparative Genomic Hybridization - array CGH

Gene Expression Profiling

Single Nucleotide Polymorphism - SNP array Microarray Microarray CTTAACCG s Microarray CTTAACCG CTTAACCG

CTTAACCG CTTAACCG ATG TAC A ACGG T TGCC TC AG G AGCC C TCGG Patient C G AAG TTC CGG GCC AA TT TT AA GGC CCG GA CT C G ATAA TATT irary-CGH Microarray -

CTTAACCG

CTTAACCG CTTAACCG

CTTAACCG CTTAACCG A T TG AC A ACGG T TGCC TC AG A T G GCC C CGG Control C G AAG TTC CGG GCC AA TT TT AA GGC CCG GA CT C G ATAA TATT ATG TAC A ACGG T TGCC TC AG G AGCC C TCGG Patient C G AAG TTC CGG GCC AA TT TT AA GGC CCG GA CT C G ATAA TATT irary-CGH Microarray -

CTTAACCG

CTTAACCG CTTAACCG

CTTAACCG CTTAACCG A T TG AC A ACGG T TGCC TC AG A T G GCC C CGG Control C G AAG TTC CGG GCC AA TT TT AA GGC CCG GA CT C G ATAA TATT Patient DNAPatient Normal Patient Normal CTTAACCG CTTAACCG

CTTAACCG

CTTAACCG Control DNA Control Microarray - Copy Number Variation (CNV) Variation Copy Number - Microarray Normal Patient Normal

CTTAACCG

CTTAACCG CTTAACCG

CTTAACCG

CNV : GAIN +4 copies / Fold change: 3 / CNV:6

Patient DNA Control DNA G CC AA G G C TT CC CC AA G AA CC C TT C TT AA TT C +4 copies /Fold +4 copies change: 3 / CNV:6 CNV : GAIN CNV:

CTTAACCG

CTTAACCG CTTAACCG CTTAACCG CNV : LOSS -1 copy / Fold change: 0.5 / CNV:1

Patient DNA Control DNA G CC G AA CC TT C AA G TT CC C AA G G TT CC C CC G AA AA CC TT C TT AA C TT C -1 copy / Fold change: copy -1/ Fold change: 0.5/ CNV:1 CNV : LOSS CNV:

CTTAACCG CTTAACCG

CTTAACCG

CTTAACCG Microarray Variations

Comparative Genomic Hybridization - array CGH

Gene Expression Profiling

Single Nucleotide Polymorphism - SNP array Microarray Applications Expression Profiling Gene Expression

______Start ______Stop DNA |||||||||||||||||||||||||||||||||||| ATGACGGATCAGCCGCAAGCGGAATTGGCGACATAA

TACTGCCTAGTCGGCGTTCGCCTTAACCGCTGTATT |||||||||||||||||||||||||||||||||||| Gene Expression

______Start ______Stop DNA |||||||||||||||||||||||||||||||||||| ATGACGGATCAGCCGCAAGCGGAATTGGCGACATAA

UACUGCCUAGUCGGCGUUCGCCUUAACCGCUGUAUU mRNA |||||||||||||||||||||||||||||||||||| exon 1 intron exon 2 intron exon 3 spliced UACUGCCGUUCGCCUUCUGUAUU ||||||||||||||||||||||| mRNA exon 1 exon 2 exon 3 cDNA TACTGCCGTTCGCCTTCTGTATT ||||||||||||||||||||||| mRNA UACUGCCGUUCGCCUUCUGUAUU |||||||||||||||||||||||

cDNA TACTGCCGTTCGCCTTCTGTATT ||||||||||||||||||||||| cDNA Probes Microarrays Variations Gene Expression Profiling G G G CC CC CC G G G AA AA AA CC CC CC TT TT TT C C C AA AA AA G G G TT TT TT CC CC CC C C C AA AA AA G G G G G G TT TT TT CC CC CC C C C CC CC CC G G G AA AA AA AA AA AA CC CC CC TT TT TT TT TT TT C C C AA AA AA C C C TT TT TT C C C Microarray Variations

Comparative Genomic Hybridization - array CGH

Gene Expression Profiling

Single Nucleotide Polymorphism - SNP array Single Nucleotide Polymorphisms

G C A * CGTT AGA SNP array Target SNP * (A,G,T) GCA*CGTTAGA Comp. DNA CGT*GCAATCT G C AA T G C AA T G C AA T T C A C G T C G T C G T Patient Genotype at *: AA GCAACGTTAGA Patient DNA GCAACGTTAGA T C A G T C A G T C A G T G C AA C G TT G C AA C G TT G C AA T A C A A A/A C G T G C A C G T G C A C G T A G T Patient Genotype at *: AT GCAACGTTAGA Patient DNA GCATCGTTAGA T C A G T C A G T C A G T G C AA C G TT G C AA C G TT G C AA T A C T A A/T C G T G C A C G T G C A C G T A G T

Technique: Microarrays

Variants CNV, SNP, Gene Expression Resolution SNP to 10’s Kilobases Scope Targeted / genome wide Quantitative Yes, CNV /Expression Specimen Blood, Fresh, Frozen, FFPE, cells Limitations Balanced alterations / Decal Common Types of Genetic Variants

Single Nucleotide Polymorphisms (SNP) Insertions / Deletions (InDel) Fusions (translocations) CNV Gene Expression Polymerase Chain reaction - PCR

______Start ______Stop DNA |||||||||||||||||||||||||||||||||||| ATGACGGATCAGCCGCAAGCGGAATTGGCGACATAA

TACTGCCTAGTCGGCGTTCGCCTTAACCGCTGTATT |||||||||||||||||||||||||||||||||||| ______Start ______Stop |||||||||||||||||||||||||||||||||||| ATGACGGATCAGCCGCAAGCGGAATTGGCGACATAA TACTGCCTAGTCGGCGTTCGCCTT AACCGCTGTATT

for primer |||||||||||||||||||||||| ||||||||||||

______||||||| ||||||||||||||||||||||||||||| AATACAG ATGACGGATCAGCCGCAAGCGGAATTGGC rev primer TACTGCCTAGTCGGCGTTCGCCTTAACCGCTGTATT |||||||||||||||||||||||||||||||||||| ______Start ______Stop |||||||||||||||||||||||||||||||||||| ATGACGGATCAGCCGCAAGCGGAATTGGCGACATAA

CTAGTCGGCGTTCGCCTTAACCGCTGTATT ||||||||||||||||||||||||||||||

______||||||||||||||||||||||||||||| ATGACGGATCAGCCGCAAGCGGAATTGGC

TACTGCCTAGTCGGCGTTCGCCTTAACCGCTGTATT |||||||||||||||||||||||||||||||||||| Polymerase Chain reaction - PCR

Typical 35 cycle PCR => 2^35

~ 34 Billion fold amplification PCR - Results

Gel electrophoresis Capillary electrophoresis Quantitative PCR Gel electrophoresis Capillary Electrophoresis

______Start ______Stop |||||||||||||||||||||||||||||||||||| ATGACGGATCAGCCGCAAGCGGAATTGGCGACATAA

TACTGC ||||||

TACTGCCTAGTCGGCGTTCGCCTTAACCGCTGTATT |||||||||||||||||||||||||||||||||||| qPCR – Quantitative PCR

Quantitative PCR ______Start ______Stop |||||||||||||||||||||||||||||||||||| ATGACGGATCAGCCGCAAGCGGAATTGGCGACATAA TACTGCCTAGTCGG CCGGTTTTCCGGCCCCTTTT |||||||||||||| |||||||||||||||||||| Technique: PCR applications

Variants typing (SNP, InDel), CNV, Expression Resolution SNP to 100’s of bases Scope Targeted Quantitative Yes: qPCR (CNV /Expression) Specimen Blood, Fresh, Frozen, FFPE, cells Limitations Targeted / Resolution / Decal ______Start ______Stop Sanger Sequencing |||||||||||||||||||||||||||||||||||| ATGACGGATCAGCCGCAAGCGGAATTGGCGACATAA TACTGCC* ||||||| TACTGCCT* |||||||| TACTGCCTA* ||||||||| TACTGCCTAG* |||||||||| TACTGCCTAGT* ||||||||||| Technique: Sanger Sequencing

Variants Novel (SNP, InDel) Resolution SNP to 100’s of bases Scope Limited Targeted Quantitative No, LOD ~20% VAF Specimen Blood, Fresh, Frozen, FFPE, cells Limitations Targeted / low throughput/ LOD / Decal Next Generation Sequencing - NGS

Not meant for humans

CNV - Gain Control Patient CNV - Loss Control Patient CNV by NGS Fusions by NGS

EWSR1 (22q12.2) WT1 (11p13) FISH Break Apart

22 22 Technique: NGS Sequencing

Variants Novel (SNP, InDel, CNV, Fusions) Resolution SNP to 100’s of bases Scope Targeted to genome wide Quantitative Yes: SNP, CNV, InDel / LOD 1-5% VAF Specimen Blood, Fresh, Frozen, FFPE, cells Limitations CNV sensitivity / Decal / Data Intense Pathologist’s Roles

CLIA validation Molec Tissue assessment AP + Molec

Test / Panel selection AP + Molec + onc Data analysis Molec Clinical report Molec Tertiary analysis - Accurate variant inclusion Somatic (tumor) mutations Read depth

Limit of detection LOD (analytical sensitivity) Variant Allele Fraction (VAF) Interpretation Read depth

20 12

● Measure of consensus accuracy ● Acceptable ~>200 ● Usually depth of coverage is in the thousands for a standard run Variant Allele Fraction (VAF) ...... Reference Human genome

6/20 = 30% VAF in sample Variant allele fraction (VAF) Pure Tumor

Gene

VAF 50% 50% Tumor

Gene Gene

VAF 25% 20% Tumor VAF 10%

10% Tumor VAF 5% LOD analytic sensitivity

5% ● Variant allele fraction at which 95% of samples would reliably be detected ● Usually 1-5% allele frequency ● "Noise" vs. true allele ○ Polymerase errors ○ Deamination ○ DNA oxidative damage

LOD 5%

55% overcall It’s All About Nuclei Pitfalls... Pitfalls: Necrosis Pitfalls: Necrosis Pitfalls: Mucinous CA Pitfalls: Mucinous CA Pitfalls: Mucinous CA

http://www.pathologyoutlines.com Pitfalls: Lymph nodes Pitfalls: Melanin

https://en.wikipedia.org/wiki/Melanoma Tumor Enrichment 4x Low Power Field Tissue Selection: Size