Limits...
Whole exome sequencing identifies a recurrent RQCD1 P131L mutation in cutaneous melanoma.

Wong SQ, Behren A, Mar VJ, Woods K, Li J, Martin C, Sheppard KE, Wolfe R, Kelly J, Cebon J, Dobrovic A, McArthur GA - Oncotarget (2015)

Bottom Line: Compared to tumors without the mutation, the P131L mutant positive tumors were associated with increased thickness (p = 0.02), head and neck (p = 0.009) and upper limb (p = 0.03) location, lentigo maligna melanoma subtype (p = 0.02) and BRAF V600K (p = 0.04) but not V600E or NRAS codon 61 mutations.Mutant RQCD1 was predicted to bind strongly to HLA-A0201 and HLA-Cw3 MHC1 complexes.From thirteen patients with mutant RQCD1, an anti-tumor CD8⁺ T cell response was observed from a single patient's peripheral blood mononuclear cell population stimulated with mutated peptide compared to wildtype indicating a neoantigen may be formed.

View Article: PubMed Central - PubMed

Affiliation: Division of Cancer Research, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia.

ABSTRACT
Melanoma is often caused by mutations due to exposure to ultraviolet radiation. This study reports a recurrent somatic C > T change causing a P131L mutation in the RQCD1 (Required for Cell Differentiation1 Homolog) gene identified through whole exome sequencing of 20 metastatic melanomas. Screening in 715 additional primary melanomas revealed a prevalence of ~4%. This represents the first reported recurrent mutation in a member of the CCR4-NOT complex in cancer. Compared to tumors without the mutation, the P131L mutant positive tumors were associated with increased thickness (p = 0.02), head and neck (p = 0.009) and upper limb (p = 0.03) location, lentigo maligna melanoma subtype (p = 0.02) and BRAF V600K (p = 0.04) but not V600E or NRAS codon 61 mutations. There was no association with nodal disease (p = 0.3). Mutually exclusive mutations of other members of the CCR4-NOT complex were found in ~20% of the TCGA melanoma dataset suggesting the complex may play an important role in melanoma biology. Mutant RQCD1 was predicted to bind strongly to HLA-A0201 and HLA-Cw3 MHC1 complexes. From thirteen patients with mutant RQCD1, an anti-tumor CD8⁺ T cell response was observed from a single patient's peripheral blood mononuclear cell population stimulated with mutated peptide compared to wildtype indicating a neoantigen may be formed.

Show MeSH

Related in: MedlinePlus

CD8-T cell response to challenge with 13mer peptides representing the mutant or wildtype RQCD1 sequencesPercentage of TNFα positive CD8 T cells from a PBMC sample either unstimulated (Unstim., left panel) or treated with an immunogenic peptide pool (FEC) as positive control (right panel). Cells were gated on live and CD3/CD8 double positive cells. Rectangle represents TNFα positive fraction. (B). PBMCs from patient P11076 were stimulated with the indicated peptides (wild type = WT and mutant = MT) alone or as pool and percentage of TNFα positive CD8+ T cells evaluated by flow-cytometry. The gate for positivity was set on untreated control from the same patient.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4359221&req=5

Figure 4: CD8-T cell response to challenge with 13mer peptides representing the mutant or wildtype RQCD1 sequencesPercentage of TNFα positive CD8 T cells from a PBMC sample either unstimulated (Unstim., left panel) or treated with an immunogenic peptide pool (FEC) as positive control (right panel). Cells were gated on live and CD3/CD8 double positive cells. Rectangle represents TNFα positive fraction. (B). PBMCs from patient P11076 were stimulated with the indicated peptides (wild type = WT and mutant = MT) alone or as pool and percentage of TNFα positive CD8+ T cells evaluated by flow-cytometry. The gate for positivity was set on untreated control from the same patient.

Mentions: Of the three RQCD1 P131L positive cases from the discovery set used for exome sequencing, one patient displayed a BRAF V600E mutation (LM-MEL-73) and the other two were BRAF and NRAS wild-type (Figure 1). All three patients shared a common HLA type (HLA-A0201), and in silico prediction analysis of the mutant RQCD1 protein suggested strong binding affinity towards HLA-A0201 and HLA-Cw3 of epitopes harboring the mutation (Table 5). While strong MHC binding is thought to often predict immunogenicity, analysis of the mutant versus wildtype peptides using an immunogenicity predicting algorithm (http://tools.iedb.org/immunogenicity) showed no clear evidence for enhanced immune-recognition of the mutant protein (Supplementary Table 2). To address the immunogenic potential and the conflicting predictions for the recurrent RQCD1 mutation, 13mer peptides with shifted positioning of the exchanged amino acid (Supplementary Table 3) were synthesized and used to stimulate peripheral blood mononuclear cells (PBMCs), where available, from patients (n = 13) harboring melanomas with RQCD1 P131L. In one out of the 13 tested patients we could detect a clear induction of TNFα-positive CD8+ T cells using the mutant peptides when compared to their wild type counterparts (Figure 4). The percentages of TNFα positive CD8+ T cells rose to 7.62% or 14% for the mutant peptides B and C respectively. Neither of the other patients PBMCs nor a wildtype control sample gave any clear indication of enhanced immunogenicity of the mutant peptide when compared to the wild type. The HLA-status of mutant RQCD1 patients can be found in Supplementary Table 4 with results for HLA-A and -C alleles, all frequencies were in keeping with the frequencies reported in literature [19].


Whole exome sequencing identifies a recurrent RQCD1 P131L mutation in cutaneous melanoma.

Wong SQ, Behren A, Mar VJ, Woods K, Li J, Martin C, Sheppard KE, Wolfe R, Kelly J, Cebon J, Dobrovic A, McArthur GA - Oncotarget (2015)

CD8-T cell response to challenge with 13mer peptides representing the mutant or wildtype RQCD1 sequencesPercentage of TNFα positive CD8 T cells from a PBMC sample either unstimulated (Unstim., left panel) or treated with an immunogenic peptide pool (FEC) as positive control (right panel). Cells were gated on live and CD3/CD8 double positive cells. Rectangle represents TNFα positive fraction. (B). PBMCs from patient P11076 were stimulated with the indicated peptides (wild type = WT and mutant = MT) alone or as pool and percentage of TNFα positive CD8+ T cells evaluated by flow-cytometry. The gate for positivity was set on untreated control from the same patient.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4359221&req=5

Figure 4: CD8-T cell response to challenge with 13mer peptides representing the mutant or wildtype RQCD1 sequencesPercentage of TNFα positive CD8 T cells from a PBMC sample either unstimulated (Unstim., left panel) or treated with an immunogenic peptide pool (FEC) as positive control (right panel). Cells were gated on live and CD3/CD8 double positive cells. Rectangle represents TNFα positive fraction. (B). PBMCs from patient P11076 were stimulated with the indicated peptides (wild type = WT and mutant = MT) alone or as pool and percentage of TNFα positive CD8+ T cells evaluated by flow-cytometry. The gate for positivity was set on untreated control from the same patient.
Mentions: Of the three RQCD1 P131L positive cases from the discovery set used for exome sequencing, one patient displayed a BRAF V600E mutation (LM-MEL-73) and the other two were BRAF and NRAS wild-type (Figure 1). All three patients shared a common HLA type (HLA-A0201), and in silico prediction analysis of the mutant RQCD1 protein suggested strong binding affinity towards HLA-A0201 and HLA-Cw3 of epitopes harboring the mutation (Table 5). While strong MHC binding is thought to often predict immunogenicity, analysis of the mutant versus wildtype peptides using an immunogenicity predicting algorithm (http://tools.iedb.org/immunogenicity) showed no clear evidence for enhanced immune-recognition of the mutant protein (Supplementary Table 2). To address the immunogenic potential and the conflicting predictions for the recurrent RQCD1 mutation, 13mer peptides with shifted positioning of the exchanged amino acid (Supplementary Table 3) were synthesized and used to stimulate peripheral blood mononuclear cells (PBMCs), where available, from patients (n = 13) harboring melanomas with RQCD1 P131L. In one out of the 13 tested patients we could detect a clear induction of TNFα-positive CD8+ T cells using the mutant peptides when compared to their wild type counterparts (Figure 4). The percentages of TNFα positive CD8+ T cells rose to 7.62% or 14% for the mutant peptides B and C respectively. Neither of the other patients PBMCs nor a wildtype control sample gave any clear indication of enhanced immunogenicity of the mutant peptide when compared to the wild type. The HLA-status of mutant RQCD1 patients can be found in Supplementary Table 4 with results for HLA-A and -C alleles, all frequencies were in keeping with the frequencies reported in literature [19].

Bottom Line: Compared to tumors without the mutation, the P131L mutant positive tumors were associated with increased thickness (p = 0.02), head and neck (p = 0.009) and upper limb (p = 0.03) location, lentigo maligna melanoma subtype (p = 0.02) and BRAF V600K (p = 0.04) but not V600E or NRAS codon 61 mutations.Mutant RQCD1 was predicted to bind strongly to HLA-A0201 and HLA-Cw3 MHC1 complexes.From thirteen patients with mutant RQCD1, an anti-tumor CD8⁺ T cell response was observed from a single patient's peripheral blood mononuclear cell population stimulated with mutated peptide compared to wildtype indicating a neoantigen may be formed.

View Article: PubMed Central - PubMed

Affiliation: Division of Cancer Research, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia.

ABSTRACT
Melanoma is often caused by mutations due to exposure to ultraviolet radiation. This study reports a recurrent somatic C > T change causing a P131L mutation in the RQCD1 (Required for Cell Differentiation1 Homolog) gene identified through whole exome sequencing of 20 metastatic melanomas. Screening in 715 additional primary melanomas revealed a prevalence of ~4%. This represents the first reported recurrent mutation in a member of the CCR4-NOT complex in cancer. Compared to tumors without the mutation, the P131L mutant positive tumors were associated with increased thickness (p = 0.02), head and neck (p = 0.009) and upper limb (p = 0.03) location, lentigo maligna melanoma subtype (p = 0.02) and BRAF V600K (p = 0.04) but not V600E or NRAS codon 61 mutations. There was no association with nodal disease (p = 0.3). Mutually exclusive mutations of other members of the CCR4-NOT complex were found in ~20% of the TCGA melanoma dataset suggesting the complex may play an important role in melanoma biology. Mutant RQCD1 was predicted to bind strongly to HLA-A0201 and HLA-Cw3 MHC1 complexes. From thirteen patients with mutant RQCD1, an anti-tumor CD8⁺ T cell response was observed from a single patient's peripheral blood mononuclear cell population stimulated with mutated peptide compared to wildtype indicating a neoantigen may be formed.

Show MeSH
Related in: MedlinePlus