HLA class I-restricted MYD88 L265P-derived peptides as specific targets for lymphoma immunotherapy

Genome sequencing has uncovered an array of recurring somatic mutations in different non-Hodgkin lymphoma (NHL) subtypes. If affecting protein-coding regions, such mutations may yield mutation-derived peptides that may be presented by HLA class I proteins and recognized by cytotoxic T cells. A recurring somatic and oncogenic driver mutation of the Toll-like receptor adaptor protein MYD88, Leu265Pro (L265P) was identified in up to 90% of different NHL subtype patients. We therefore screened the potential of MYD88(L265P)-derived peptides to elicit cytotoxic T cell responses as tumor-specific neoantigens. Based on in silico predictions, we identified potential MYD88(L265P)-containing HLA ligands for several HLA class I restrictions. A set of HLA class I MYD88(L265P)-derived ligands elicited specific cytotoxic T cell responses for HLA-B*07 and -B*15. These data highlight the potential of MYD88(L265P) mutation-specific peptide-based immunotherapy as a novel personalized treatment approach for patients with MYD88(L265P+) NHLs that may complement pharmacological approaches targeting oncogenic MyD88 L265P signaling

Functional loss of IKBE leads to NF-KB deregulation in aggressive chronic lymphocytic leukemia

NF-?B is constitutively activated in chronic lymphocytic leukemia (CLL); however, the implicated molecular mechanisms remain largely unknown. Thus, we performed targeted deep sequencing of 18 core complex genes within the NF-?B pathway in a discovery and validation CLL cohort totaling 315 cases. The most frequently mutated gene was NFKBIE (21/315 cases; 7%), which encodes I?B?, a negative regulator of NF-?B in normal B cells. Strikingly, 13 of these cases carried an identical 4-bp frameshift deletion, resulting in a truncated protein. Screening of an additional 377 CLL cases revealed that NFKBIE aberrations predominated in poor-prognostic patients and were associated with inferior outcome. Minor subclones and/or clonal evolution were also observed, thus potentially linking this recurrent event to disease progression. Compared with wild-type patients, NFKBIE-deleted cases showed reduced I?B? protein levels and decreased p65 inhibition, along with increased phosphorylation and nuclear translocation of p65. Considering the central role of B cell receptor (BcR) signaling in CLL pathobiology, it is notable that I?B? loss was enriched in aggressive cases with distinctive stereotyped BcR, likely contributing to their poor prognosis, and leading to an altered response to BcR inhibitors. Because NFKBIE deletions were observed in several other B cell lymphomas, our findings suggest a novel common mechanism of NF-?B deregulation during lymphomagenesis. <br/

Different spectra of recurrent gene mutations in subsets of chronic lymphocytic leukemia harboring stereotyped B-cell receptors.

We report on markedly different frequencies of genetic lesions within subsets of chronic lymphocytic leukemia patients carrying mutated or unmutated stereotyped B-cell receptor immunoglobulins in the largest cohort (n=565) studied for this purpose. By combining data on recurrent gene mutations (BIRC3, MYD88, NOTCH1, SF3B1 and TP53) and cytogenetic aberrations, we reveal a subset-biased acquisition of gene mutations. More specifically, the frequency of NOTCH1 mutations was found to be enriched in subsets expressing unmutated immunoglobulin genes, i.e. #1, #6, #8 and #59 (22-34%), often in association with trisomy 12, and was significantly different (P<0.001) to the frequency observed in subset #2 (4%, aggressive disease, variable somatic hypermutation status) and subset #4 (1%, indolent disease, mutated immunoglobulin genes). Interestingly, subsets harboring a high frequency of NOTCH1 mutations were found to carry few (if any) SF3B1 mutations. This starkly contrasts with subsets #2 and #3 where, despite their immunogenetic differences, SF3B1 mutations occurred in 45% and 46% of cases, respectively. In addition, mutations within TP53, whilst enriched in subset #1 (16%), were rare in subsets #2 and #8 (both 2%), despite all being clinically aggressive. All subsets were negative for MYD88 mutations, whereas BIRC3 mutations were infrequent. Collectively, this striking bias and skewed distribution of mutations and cytogenetic aberrations within specific chronic lymphocytic leukemia subsets implies that the mechanisms underlying clinical aggressiveness are not uniform, but rather support the existence of distinct genetic pathways of clonal evolution governed by a particular stereotyped B-cell receptor selecting a certain molecular lesion(s

Functional loss of IκBε leads to NF-κB deregulation in aggressive chronic lymphocytic leukemia

NF-κB is constitutively activated in chronic lymphocytic leukemia (CLL); however, the implicated molecular mechanisms remain largely unknown. Thus, we performed targeted deep sequencing of 18 core complex genes within the NF-κB pathway in a discovery and validation CLL cohort totaling 315 cases. The most frequently mutated gene was NFKBIE (21/315 cases; 7%), which encodes IκBε, a negative regulator of NF-κB in normal B cells. Strikingly, 13 of these cases carried an identical 4-bp frameshift deletion, resulting in a truncated protein. Screening of an additional 377 CLL cases revealed that NFKBIE aberrations predominated in poor-prognostic patients and were associated with inferior outcome. Minor subclones and/or clonal evolution were also observed, thus potentially linking this recurrent event to disease progression. Compared with wild-type patients, NFKBIE-deleted cases showed reduced IκBε protein levels and decreased p65 inhibition, along with increased phosphorylation and nuclear translocation of p65. Considering the central role of B cell receptor (BcR) signaling in CLL pathobiology, it is notable that IκBε loss was enriched in aggressive cases with distinctive stereotyped BcR, likely contributing to their poor prognosis, and leading to an altered response to BcR inhibitors. Because NFKBIE deletions were observed in several other B cell lymphomas, our findings suggest a novel common mechanism of NF-κB deregulation during lymphomagenesis

Consistent B Cell Receptor Immunoglobulin Features Between Siblings in Familial Chronic Lymphocytic Leukemia

Key processes in the onset and evolution of chronic lymphocytic leukemia (CLL) are thought to include chronic (antigenic) activation of mature B cells through the B cell receptor (BcR), signals from the microenvironment, and acquisition of genetic alterations. Here we describe three families in which two or more siblings were affected by CLL. We investigated whether there are immunogenetic similarities in the leukemia-specific immunoglobulin heavy (IGH) and light (IGL/IGK) chain gene rearrangements of the siblings in each family. Furthermore, we performed array analysis to study if similarities in CLL-associated chromosomal aberrations are present within each family and screened for somatic mutations using paired tumor/normal whole-genome sequencing (WGS). In two families a consistent IGHV gene mutational status (one IGHV-unmutated, one IGHV-mutated) was observed. Intriguingly, the third family with four affected siblings was characterized by usage of the lambda IGLV3-21 gene, with the hallmark R110 mutation of the recently described clinically aggressive IGLV3-21R110 subset. In this family, the CLL-specific rearrangements in two siblings could be assigned to either stereotyped subset #2 or the immunogenetically related subset #169, both of which belong to the broader IGLV3-21R110 subgroup. Consistent patterns of cytogenetic aberrations were encountered in all three families. Furthermore, the CLL clones carried somatic mutations previously associated with IGHV mutational status, cytogenetic aberrations and stereotyped subsets, respectively. From these findings, we conclude that similarities in immunogenetic characteristics in familial CLL, in combination with genetic aberrations acquired, point towards shared underlying mechanisms behind CLL development within each family

Targeted next-generation sequencing in chronic lymphocytic leukemia: a high-throughput yet tailored approach will facilitate implementation in a clinical setting

Next- generation sequencing has revealed novel recurrent mutations in chronic lymphocytic leukemia, particularly in patients with aggressive disease. Here, we explored targeted re- sequencing as a novel strategy to assess the mutation status of genes with prognostic potential. To this end, we utilized HaloPlex targeted enrichment technology and designed a panel including nine genes: ATM, BIRC3, MYD88, NOTCH1, SF3B1 and TP53, which have been linked to the prognosis of chronic lymphocytic leukemia, and KLHL6, POT1 and XPO1, which are less characterized but were found to be recurrently mutated in various sequencing studies. A total of 188 chronic lymphocytic leukemia patients with poor prognostic features ( unmutated IGHV, n= 137; IGHV3- 21 subset # 2, n= 51) were sequenced on the HiSeq 2000 and data were analyzed using well- established bioinformatics tools. Using a conservative cutoff of 10% for the mutant allele, we found that 114/ 180 ( 63%) patients carried at least one mutation, with mutations in ATM, BIRC3, NOTCH1, SF3B1 and TP53 accounting for 149/ 177 ( 84%) of all mutations. We selected 155 mutations for Sanger validation ( variant allele frequency, 10- 99%) and 93% ( 144/ 155) of mutations were confirmed; notably, all 11 discordant variants had a variant allele frequency between 11- 27%, hence at the detection limit of conventional Sanger sequencing. Technical precision was assessed by repeating the entire HaloPlex procedure for 63 patients; concordance was found for 77/ 82 ( 94%) mutations. In summary, this study demonstrates that targeted next- generation sequencing is an accurate and reproducible technique potentially suitable for routine screening, eventually as a stand- alone test without the need for confirmation by Sanger sequencing

Consistent B Cell Receptor Immunoglobulin Features Between Siblings in Familial Chronic Lymphocytic Leukemia

Key processes in the onset and evolution of chronic lymphocytic leukemia (CLL) are thought to include chronic (antigenic) activation of mature B cells through the B cell receptor (BcR), signals from the microenvironment, and acquisition of genetic alterations. Here we describe three families in which two or more siblings were affected by CLL. We investigated whether there are immunogenetic similarities in the leukemia-specific immunoglobulin heavy (IGH) and light (IGL/IGK) chain gene rearrangements of the siblings in each family. Furthermore, we performed array analysis to study if similarities in CLL-associated chromosomal aberrations are present within each family and screened for somatic mutations using paired tumor/normal whole-genome sequencing (WGS). In two families a consistent IGHV gene mutational status (one IGHV-unmutated, one IGHV-mutated) was observed. Intriguingly, the third family with four affected siblings was characterized by usage of the lambda IGLV3-21 gene, with the hallmark R110 mutation of the recently described clinically aggressive IGLV3-21(R110) subset. In this family, the CLL-specific rearrangements in two siblings could be assigned to either stereotyped subset #2 or the immunogenetically related subset #169, both of which belong to the broader IGLV3-21(R110) subgroup. Consistent patterns of cytogenetic aberrations were encountered in all three families. Furthermore, the CLL clones carried somatic mutations previously associated with IGHV mutational status, cytogenetic aberrations and stereotyped subsets, respectively. From these findings, we conclude that similarities in immunogenetic characteristics in familial CLL, in combination with genetic aberrations acquired, point towards shared underlying mechanisms behind CLL development within each family.&lt;/p&gt