class: center, middle, inverse, title-slide # High allele frequency of somatic mutation ### Jun Kang ### 2019 11 1 --- # Question 1 > Can we determine whether the variant is germline or somatic using allele frequency and tumor percentage in tumor only test? --- # BRCA result * BRCA1 mutation: Positive - p.Glu649Ter (c.1945G>T) * Variant allele frequency 63.84% * Tumor cell percentage: 70% * BRCA negative in blood sample --- # Question 2 > Is it possible **64% allele frequency** of somatic mutation? --- class: center # Variant allele frequency in clinical tumor sample `$$Allele \space frequency \approx Read \space count$$` ![:scale 70%](img/Allele_3.png) --- class: center # Somatic variant, Two copy, Tumor cellularity 50% ![](img/Allele_4.png) Variant allele frequency? --- class: center # Germline variant, Heterozigosity, Two copy, Tumor cellularity 25% ![](img/Allele_2.png) Variant allele frequency? class: center # Germline variant, Heterozigosity, Two copy, Tumor cellularity 50% ![](img/Allele_1.png) Variant allele frequency? --- # Hypothesis Germline, homo | Germline, hetero | Somatic :-----: | :----: | :----------------------: 100% | 50% | half of tumor cellularity, `\(\le 50\%\)` --- class: center # Germline variant, Heterozigosity, One copy, LOH, Tumor cellularity 50% ![](img/Allele_5.png) Variant allele frequency? --- class: center # Germline variant, Heterozigosity, One copy, LOH, Tumor cellularity 25% ![](img/Allele_6.png) Variant allele frequency? --- class: center # Somatic variant, One copy, LOH, Tumor cellularity 50% ![](img/Allele_8.png) Variant allele frequency? --- class: center # Somatic variant, Amplification, Tumor cellularity 25% ![:scale 70%](img/Allele_9.png) Variant allele frequency? --- # Variant allele frequency in clinical tumor sample * Germline vs somatic * Tumor cell proportion * Loss of heterozygosity * Copy number --- # Allele frequency in Somatic vs Germline in tumor only sample * BRCA1 mutation: Positive - p.Glu649Ter (c.1945G>T) * Variant allele frequency 63.84% * Tumor cell percentage: 70% --- # Allele frequency[1] `\(AF_{germline}\)` `\(=\)` `\({pV+1-p} \over {pC+2(1-p)}\)` `\(AF_{somatic}\)` `\(=\)` `\(pV \over pC+2(1-p)\)` * Given copy number (C) * Variant allele count (V) * Sample purity (p) * Variant status (somatic or germline) --- # Limited information What we know | What we don't know :-------------:|:-----------------: Tumor cell percentage | Copy number Variant allele frequency | LOH --- # Error * Tumor cell percentage * Allele frequency `$$Allele \space frequency \not\approx Read \space count$$` --- # BRCA mutation and LOH [2] ![:scale 80%](LOH.png) --- # Allele frequency * BRCA1 mutation: Positive - p.Glu649Ter (c.1945G>T) * Variant allele frequency 63.84% * Tumor cell percentage: 70% -> 80% * Somatic (supposed) * LOH (supposed) * One copy (supposed) --- class: center # Allele frequency ![:scale 70%](img/Allele_12.png) `\(AF_{somatic}\)` `\(=\)` `\(pV \over pC+2(1-p)\)` `\(= 0.67\)` --- # Allele frequency * BRCA1 mutation: Positive - p.Glu649Ter (c.1945G>T) * Variant allele frequency 63.84% * Tumor cell percentage: 70% -> 60% * Germline (supposed) * LOH (supposed) * One copy (supposed) --- class: center # Allele frequency ![:scale 60%](img/Allele_13.png) `\(AF_{germline}\)` `\(=\)` `\({pV+1-p} \over {pC+2(1-p)}\)` `\(= 0.71\)` --- # Someone ask ... >Does tumor only NGS test distinguish a variant somatic and germline? Answer 'No' >Is it possible **64% allele frequency** of somatic BRCA variant? Answer 'Yes, It is when BRCA somatic variant accompanied with LOH' --- class: center # Germline variant, One copy, LOH, Tumor cellularity 100% (Bonus1) Allele frequency 100% **hetero germline** variant ![:scale 60%](img/Allele_7.png) --- class: center # Somatic variant, Amplification, Hetero, Tumor cellularity 100% (Bonus2) Allele frequency 90% **somatic** variant ![:scale 70%](img/Allele_11.png) --- class: my-one-page-font # References [1] J. X. Sun, Y. He, E. Sanford, et al. "A computational approach to distinguish somatic vs. germline origin of genomic alterations from deep sequencing of cancer specimens without a matched normal". En. In: _PLOS Computational Biology_ 14.2 (2018), p. e1005965. ISSN: 1553-7358. [2] M. M. de Jonge, D. Ruano, R. van Eijk, et al. "Validation and Implementation of BRCA1/2 Variant Screening in Ovarian Tumor Tissue". In: _The Journal of Molecular Diagnostics_ 20.5 (9. 2018), pp. 600-611. ISSN: 1525-1578. <style type="text/css"> .remark-slide-content { font-size: 32px; padding: 1em 4em 1em 4em; } .my-one-page-font { font-size: 18px; } .beginners{ visibility: hidden; } .intermediate{ visibility: hidden; } .advanced{ visibility: visible; } { max-width: 100%; } </style>