Calreticulin mutant myeloproliferative neoplasms induce MHC-I skewing, which can be overcome by an optimized peptide cancer vaccine.

The majority of JAK2V617F-negative myeloproliferative neoplasms (MPNs) have disease-initiating frameshift mutations in calreticulin (CALR), resulting in a common carboxyl-terminal mutant fragment (CALRMUT), representing an attractive source of neoantigens for cancer vaccines. However, studies have shown that CALRMUT-specific T cells are rare in patients with CALRMUT MPN for unknown reasons. We examined class I major histocompatibility complex (MHC-I) allele frequencies in patients with CALRMUT MPN from two independent cohorts. We observed that MHC-I alleles that present CALRMUT neoepitopes with high affinity are underrepresented in patients with CALRMUT MPN. We speculated that this was due to an increased chance of immune-mediated tumor rejection by individuals expressing one of these MHC-I alleles such that the disease never clinically manifested. As a consequence of this MHC-I allele restriction, we reasoned that patients with CALRMUT MPN would not efficiently respond to a CALRMUT fragment cancer vaccine but would when immunized with a modified CALRMUT heteroclitic peptide vaccine approach. We found that heteroclitic CALRMUT peptides specifically designed for the MHC-I alleles of patients with CALRMUT MPN efficiently elicited a CALRMUT cross-reactive CD8+ T cell response in human peripheral blood samples but not to the matched weakly immunogenic CALRMUT native peptides. We corroborated this effect in vivo in mice and observed that C57BL/6J mice can mount a CD8+ T cell response to the CALRMUT fragment upon immunization with a CALRMUT heteroclitic, but not native, peptide. Together, our data emphasize the therapeutic potential of heteroclitic peptide-based cancer vaccines in patients with CALRMUT MPN.

High frequencies of circulating memory T cells specific for calreticulin exon 9 mutations in healthy individuals.

Mutations in exon 9 of the calreticulin gene (CALR) frequently occur in patients with chronic myeloproliferative neoplasms (MPN). Patients exhibit spontaneous cellular immune responses to epitopes derived from the mutant CALR C-terminus, and CALR-mutant-specific T cells recognize autologous CALR-mutant malignant cells. This study investigated whether CALR-mutant-specific T cells occur naturally in CALRwt MPN-patients and in healthy individuals. Specific immune responses against epitopes in the mutant CALR peptide sequence were detected in both CALRwt MPN-patients and in healthy individuals. Healthy donors displayed more frequent and stronger CALR-mutant specific T-cell responses compared to the responses identified in CALR-mutant MPN-patients. Several T-cell responses were identified in healthy donors directly ex vivo. Importantly, by running functional analyses on live-sorted immune cells from healthy donors, we showed that circulating CALR-mutant-specific immune cells are T-memory cells. These findings suggest, that healthy individuals acquire a CALR exon 9 mutation, but the immune system reacts and clears the mutant cells, and during this reaction generates CALR-mutant specific T-memory cells. We believe that these findings provide the evidence for tumor immune surveillance in MPN.

Differential Dynamics of CALR Mutant Allele Burden in Myeloproliferative Neoplasms during Interferon Alfa Treatment.

Discovery of somatic mutations in the calreticulin gene (CALR) has identified a subgroup of Philadelphia-negative chronic myeloproliferative neoplasms (MPN) with separate haematological characteristics and prognosis. CALR mutations serve as novel markers both of diagnostic value and as targets for monitoring molecular responses during therapy. Interferon-α (IFN) selectively targets the malignant clone in a subset of MPN patients and can induce both haematological and molecular remissions in CALR mutated essential thrombocythemia (ET) patients. We investigated the response to IFN in a cohort of 21 CALR mutated MPN patients including ET, prefibrotic primary myelofibrosis (pre-PMF), and primary myelofibrosis (PMF) with a median follow-up of 31 months. For evaluation of a molecular response, we developed highly sensitive quantitative PCR (qPCR) assays for monitoring the mutant allele burden of the two most prevalent CALR mutations (type 1 and type 2). Thirteen patients (62%) experienced a decrease in the mutant allele burden with a median decline of 29% from baseline. However, only four patients, including patients with ET, pre-PMF, and PMF diagnosis, achieved molecular responder (MR) status with >50% reduction in mutant allele burden according to European LeukemiaNet (ELN) guidelines. MR patients displayed significant differences in the dynamics of the CALR mutant load with regard to time to response and dynamics in mutant allele burden after discontinuation of IFN treatment. Furthermore, we highlight the prognostic value of the CALR mutant allele burden by showing a close association with leucocyte- and platelet counts, hemoglobin concentration, in addition to plasma lactate dehydrogenase (LDH) irrespective of molecular response and treatment status.

The Danish National Multiple Myeloma Registry.

AIM: The Danish National Multiple Myeloma Registry (DMMR) is a population-based clinical quality database established in January 2005. The primary aim of the database is to ensure that diagnosis and treatment of plasma cell dyscrasia are of uniform quality throughout the country. Another aim is to support research. Patients are registered with their unique Danish personal identification number, and the combined use of DMMR, other Danish National registries, and the Danish National Cancer Biobank offers a unique platform for population-based translational research. STUDY POPULATION: All newly diagnosed patients with multiple myeloma (MM), smoldering MM, solitary plasmacytomas, and plasma cell leukemia in Denmark are registered annually; ~350 patients. Amyloid light-chain amyloidosis, POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes syndrome), monoclonal gammopathy of undetermined significance and monoclonal gammopathy of undetermined significance with polyneuropathy have been registered since 2014. MAIN VARIABLES: The main registered variables at diagnosis are patient demographics, baseline disease characteristics, myeloma-defining events, clinical complications, prognostics, first- and second-line treatments, treatment responses, progression free, and overall survival. DESCRIPTIVE DATA: Up to June 2015, 2,907 newly diagnosed patients with MM, 485 patients with smoldering MM, 64 patients with plasma cell leukemia, and 191 patients with solitary plasmacytomas were registered. Registration completeness of new patients is ~100%. A data validation study performed in 2013-2014 by the Danish Myeloma Study Group showed >95% data correctness. CONCLUSION: The DMMR is a population-based data validated database eligible for clinical, epidemiological, and translational research.