Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

Identification of an ADAM17 cleavage region in human CD16 (FcγRIII) and the engineering of a non-cleavable version of the receptor in NK cells.

CD16a and CD16b are IgG Fc receptors expressed by human natural killer (NK) cells and neutrophils, respectively. Both CD16 isoforms undergo a rapid down-regulation in expression by ADAM17-mediated proteolytic cleavage upon cell activation by various stimuli. We examined soluble CD16 released from activated NK cells and neutrophils by mass spectrometric analysis, and identified three separate cleavage sites in close proximity at P1/P1' positions alanine195/valine196, valine196/serine197, and threonine198/isoleucine199, revealing a membrane proximal cleavage region in CD16. Substitution of the serine at position 197 in the middle of the cleavage region for a proline (S197P) effectively blocked CD16a and CD16b cleavage in cell-based assays. We also show that CD16a/S197P was resistant to cleavage when expressed in the human NK cell line NK92 and primary NK cells derived from genetically-engineered human induced pluripotent stem cells. CD16a is a potent activating receptor and despite blocking CD16a shedding, the S197P mutation did not disrupt IgG binding by the receptor or its activation of NK92 cells by antibody-treated tumor cells. Our findings provide further characterization of CD16 cleavage by ADAM17 and they demonstrate that a non-cleavable version of CD16a can be expressed in engineered NK cells

Identification of an ADAM17 cleavage region in human CD16 (FcγRIII) and the engineering of a non-cleavable version of the receptor in NK cells.

CD16a and CD16b are IgG Fc receptors expressed by human natural killer (NK) cells and neutrophils, respectively. Both CD16 isoforms undergo a rapid down-regulation in expression by ADAM17-mediated proteolytic cleavage upon cell activation by various stimuli. We examined soluble CD16 released from activated NK cells and neutrophils by mass spectrometric analysis, and identified three separate cleavage sites in close proximity at P1/P1' positions alanine195/valine196, valine196/serine197, and threonine198/isoleucine199, revealing a membrane proximal cleavage region in CD16. Substitution of the serine at position 197 in the middle of the cleavage region for a proline (S197P) effectively blocked CD16a and CD16b cleavage in cell-based assays. We also show that CD16a/S197P was resistant to cleavage when expressed in the human NK cell line NK92 and primary NK cells derived from genetically-engineered human induced pluripotent stem cells. CD16a is a potent activating receptor and despite blocking CD16a shedding, the S197P mutation did not disrupt IgG binding by the receptor or its activation of NK92 cells by antibody-treated tumor cells. Our findings provide further characterization of CD16 cleavage by ADAM17 and they demonstrate that a non-cleavable version of CD16a can be expressed in engineered NK cells

Location of ectodomain cleavage sites in human CD16.

<p>(A) Tryptic peptides of soluble CD16 immunoprecipitated from the cell supernatant of PMA activated human NK cells or neutrophils were subjected to mass spectrometry analysis. Four high confidence peptides with non-tryptic C-termini were identified; 1 peptide from soluble CD16 released by NK cells (Peptide #1) and 3 peptides from soluble CD16 released by neutrophils (Peptides #2–4). (B) Illustration of Peptides #1–4 (underlined) and putative cleavage sites (arrowheads) in CD16a and CD16b. The red amino acids indicate residues that distinguish CD16a and CD16b in the identified peptides. Blue amino acids indicate predicted signal sequences of CD16a and CD16b and the transmembrane region of CD16a. Amino acid numbering begins with methionine in the signal sequence. The amino acid sequences of CD16a and CD16b are from the NCBI reference sequences NM_000569.6 and NM_000570.4, respectively.</p

Schematic illustration of CD16 ectodomain shedding, the cleavage region, and the engineered serine197 to proline mutation.

<p>CD16a and CD16b undergo ectodomain shedding by ADAM17 within a membrane proximal region, as indicated. The CD16 cleavage region within the membrane proximal region is based on mass spectrometry analysis that revealed three distinct cleavage sites in close proximity (arrowheads). Site-directed mutagenesis was performed to exchange serine at position 197 for a proline (S197P), as indicated in red font.</p

Effects of the engineered S197P mutation on CD16a function.

<p>(A) NK92 cells expressing CD16a or CD16a/S197P at equivalent levels (left panel) were treated with monomeric human IgG (0–20μg/ml). As controls, cells were also treated with monomeric human IgA (20μg/ml), and NK92 parent cells were treated with IgG (20μg/ml) (black bar). Antibody binding was determined by flow cytometry, as described in Materials and Methods. The bar graph shows mean ± SD of at least 3 separate experiments. Statistical significance is indicated as *P<0.05 versus IgG (0 μg/ml), IgA, or NK92 parent cells + IgG. (B) Mock transduced NK92 cells or NK92 cells expressing CD16a or CD16a/S197P were incubated in the absence (Unstim.) or presence of Raji cells treated with or without anti-CD20 rituximab for the indicated time points at 37°C. NK92 cell activation was assessed by the up-regulation in CD107a staining by flow cytometry. For the histogram plots, the x-axis = Log 10 fluorescence and the y-axis = cell number. Data are representative of at least 3 independent experiments.</p

Effects of the engineered S197P mutation on CD16a and CD16b shedding.

<p>Transfected HEK293 (human embryonic kidney) cells separately expressed CD16b and CD16b/S197P (A) or CD16a and CD16a/S197P (B) at similar levels, as determined by flow cytometry (left panels). The different transfectants were treated with or without PMA (15ng/ml for 30 minutes at 37°C) and soluble levels of CD16 in the media supernatant were quantified by ELISA (right panels). Each treatment condition was repeated 3 times for each experiment and the data are representative of 3 independent experiments. Bar graphs show mean ± SD. Statistical significance is indicated as ***P<0.001. (C) Transfected HEK293 cells expressed L-selectin (CD62L) or L-selectin and CD16b/S197P. Surface levels of L-selectin and CD16b/S197P on transfected and mock-transfected cells were measured using flow cytometry (histogram plots). Transfectants expressing L-selectin or L-selectin and CD16b/S197P were incubated in the presence or absence of PMA for 30 minutes at 37°C, and the mean fluorescence intensity (MFI) of L-selectin staining determined (bar graph). Each treatment condition was repeated 3 times for each experiment and the data are representative of 2 independent experiments. Bar graphs show mean ± SD. Statistical significance is indicated as *P<0.05. For all histogram plots, the x-axis = Log 10 fluorescence and the y-axis = cell number.</p

Effects of the engineered S197P mutation on CD16a shedding in NK cells.

<p>NK92 cells transduced with empty vector (vector only), CD16a, or CD16a/S197P were treated without (Unstim.) or with PMA (100ng/ml) for 30 minutes at 37°C (A), with IL-12 and IL-18 (100ng/ml and 400ng/ml, respectively) for 24 hours at 37°C (B), or with Raji cells and rituximab for 60 min at 37°C (C). Cell surface levels of CD16a were determined by flow cytometry. Isotype-matched negative control antibody staining is indicated by a dotted line. (D) Parent NK92 cells and transduced cells expressing CD16a or CD16a/S197P were treated with Raji cells and rituximab in the presence or absence of the ADAM17 inhibitor BMS566394 (5μM) for 60 min at 37°C. Soluble CD16a levels were determined by ELISA. Each treatment condition was repeated 3 times and the data are representative of 3 independent experiments. Bar graphs show mean ± SD. Statistical significance is indicated as ***P<0.001. (E) NK92 cells expressing CD16a or CD16a/S197P were stained with the anti-ADAM17 mAbs M220, 623, 633, or an isotype-matched negative control antibody, as indicated. (F) CD56⁺CD45⁺ NK cells derived from mock-transduced iPSCs (left panel) or iPSCs expressing recombinant CD16a or CD16a/S197P (right panels) were incubated with or without K562 target cells for 4 hours at 37°C. For all histogram plots, the x-axis = Log 10 fluorescence, the y-axis = cell number, and the data are representative of at least 3 independent experiments.</p