American Society of Hematology

Figure 6.

$Subclonal point mutations and genomic catastrophes of MYC are associated with refractory subclones. (A,B) Dot and line graphs of somatic point mutations in patients (A) REL#3 and (B) REL#4 according to their VAFs (y-axis, logarithmic) in plasma samples drawn at diagnosis and at relapse. Lines connect the same mutations. Labels show all the exonic mutations detected by explorative genotyping of relapse plasma samples that were not detected in pretreatment samples. Also, an MYC SNV (S197N) detected subclonally at diagnosis is annotated in (B). Swimmer plots below the dot plots demonstrate clinical courses and time points of the plasma draws. (C) IGV screenshot showing reads spanning the relapse emerged MYC SNVs phased with an ancestral MYC SNV in the relapse plasma sample of REL#4. (D) Representative figures of MYC immunohistochemical (IHC) staining in primary and relapse tissues of REL#4. Relapse tissue biopsy is concurrent with the sequenced relapse plasma sample. Inlets show fluorescent images of representative cells in the analyzed tissues hybridized with FISH break-apart probes for MYC locus. Arrowheads denote break-apart signals. (E) Dot plot of somatic point mutations according to their VAF (y-axis, logarithmic scale) in the diagnostic biopsy and pretreatment plasma ctDNA of patient REL#5. Lines connect the same mutations. Nonsynonymous mutations of genomic driver genes are labeled excluding those of BCL2. (F) Bar plot showing the sequencing depth of chromosome 8 in WGS data of REL#5 pretreatment cfDNA. Note the exceptional spikes in coverage that give away high-level amplification of these sequences. One thousand base pair binning. (G) Scaled schematic representation of the completely solved structure of 1.30 megabase amplified double minute detected with plasma pretreatment cfDNA WGS. The double minute contained DNA derived only from chromosome 8, carried 7 novel junctions (black lines), and encompassed an intact MYC gene with hybridization sequences for centrosomal MYC break-apart probe (orange color annotation). (H-J) Genomic analysis of the complex karyotype in the diagnostic biopsy of REL#5. (H) Representative G-banding analysis showing near tetraploid corresponding karyotype (n = 96). (I) Representative cells from the FISH analyses: BCL2 and BCL6 with multiple break-apart signals and MYC with tetrasomy of 8 corresponding signals without a rearrangement. (J) Fluorescent image of MYC FISH showing a microscopy field in REL#5 diagnostic tissue with 1 single cell (red dashed box, inlet) detected carrying amplified double minute corresponding additional red fluorescent signals. (K) MYC IHC in the diagnostic tissue of REL#5 with 40% tumor cell positivity. (L) Dot and line graph showing individual mutations according to their VAF (blue dots, left y-axis) and ctDNA concentration (orange line, right y-axis) in the plasma samples drawn at different therapy phases of REL#5. Red and blue text annotations denote adverse and favorable risk features in ctDNA, respectively. Swimmer plot below showing timeline of REL#5 plasma sampling and disease course. DHIT, MYC/BCL2 double-hit; MRD, minimal residual disease. Asterisk denotes end-of-therapy ctDNA concentration estimated, although MRD was negative (borderline). (M) CT-scan, coronal section of patient REL#5 at progression showing a large partially necrotic mass near the right psoas muscle. (N) MYC IHC stain in the tissue biopsy taken at the time of progression showing markedly increased MYC immunoreactivity (95% tumor cell positivity) in comparison with the diagnostic MYC IHC stain. (O) Representative archival FISH analysis of MYC locus in the tissue biopsy taken at the time of Innumerable red fluorescent signals corresponding to high-level amplified double minutes in all lymphoma cells. Inlet: representative cell.$

Subclonal point mutations and genomic catastrophes of MYC are associated with refractory subclones. (A,B) Dot and line graphs of somatic point mutations in patients (A) REL#3 and (B) REL#4 according to their VAFs (y-axis, logarithmic) in plasma samples drawn at diagnosis and at relapse. Lines connect the same mutations. Labels show all the exonic mutations detected by explorative genotyping of relapse plasma samples that were not detected in pretreatment samples. Also, an MYC SNV (S197N) detected subclonally at diagnosis is annotated in (B). Swimmer plots below the dot plots demonstrate clinical courses and time points of the plasma draws. (C) IGV screenshot showing reads spanning the relapse emerged MYC SNVs phased with an ancestral MYC SNV in the relapse plasma sample of REL#4. (D) Representative figures of MYC immunohistochemical (IHC) staining in primary and relapse tissues of REL#4. Relapse tissue biopsy is concurrent with the sequenced relapse plasma sample. Inlets show fluorescent images of representative cells in the analyzed tissues hybridized with FISH break-apart probes for MYC locus. Arrowheads denote break-apart signals. (E) Dot plot of somatic point mutations according to their VAF (y-axis, logarithmic scale) in the diagnostic biopsy and pretreatment plasma ctDNA of patient REL#5. Lines connect the same mutations. Nonsynonymous mutations of genomic driver genes are labeled excluding those of BCL2. (F) Bar plot showing the sequencing depth of chromosome 8 in WGS data of REL#5 pretreatment cfDNA. Note the exceptional spikes in coverage that give away high-level amplification of these sequences. One thousand base pair binning. (G) Scaled schematic representation of the completely solved structure of 1.30 megabase amplified double minute detected with plasma pretreatment cfDNA WGS. The double minute contained DNA derived only from chromosome 8, carried 7 novel junctions (black lines), and encompassed an intact MYC gene with hybridization sequences for centrosomal MYC break-apart probe (orange color annotation). (H-J) Genomic analysis of the complex karyotype in the diagnostic biopsy of REL#5. (H) Representative G-banding analysis showing near tetraploid corresponding karyotype (n = 96). (I) Representative cells from the FISH analyses: BCL2 and BCL6 with multiple break-apart signals and MYC with tetrasomy of 8 corresponding signals without a rearrangement. (J) Fluorescent image of MYC FISH showing a microscopy field in REL#5 diagnostic tissue with 1 single cell (red dashed box, inlet) detected carrying amplified double minute corresponding additional red fluorescent signals. (K) MYC IHC in the diagnostic tissue of REL#5 with 40% tumor cell positivity. (L) Dot and line graph showing individual mutations according to their VAF (blue dots, left y-axis) and ctDNA concentration (orange line, right y-axis) in the plasma samples drawn at different therapy phases of REL#5. Red and blue text annotations denote adverse and favorable risk features in ctDNA, respectively. Swimmer plot below showing timeline of REL#5 plasma sampling and disease course. DHIT, MYC/BCL2 double-hit; MRD, minimal residual disease. Asterisk denotes end-of-therapy ctDNA concentration estimated, although MRD was negative (borderline). (M) CT-scan, coronal section of patient REL#5 at progression showing a large partially necrotic mass near the right psoas muscle. (N) MYC IHC stain in the tissue biopsy taken at the time of progression showing markedly increased MYC immunoreactivity (95% tumor cell positivity) in comparison with the diagnostic MYC IHC stain. (O) Representative archival FISH analysis of MYC locus in the tissue biopsy taken at the time of Innumerable red fluorescent signals corresponding to high-level amplified double minutes in all lymphoma cells. Inlet: representative cell.

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