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The CALR exon 9 mutations are shared neoantigens in patients with CALR mutant chronic myeloproliferative neoplasms

Leukemia volume 30, pages 2413–2416 (2016)Cite this article

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Mutations in exon 9 of the calreticulin (CALR) gene are found in ~60% of patients with JAK2wt essential thrombocythemia (ET) or primary myelofibrosis (PMF).1, 2 CALR exon 9 mutations result in a 1-bp frameshift mutation, which alters the C terminus of the CALR protein.1, 2 So far, more than 50 mutations have been described, and they all share a 34-amino acid consensus sequence in the C terminus. This novel C terminus is a potential tumor-associated antigen. Such antigens are believed to be important for the immune system to obtain tumor control, as the immune system allegedly targets the tumor-associated antigens.3 Thus, the CALR exon 9 mutations might be an ideal target for cancer immune therapies. This study aimed to describe the spontaneous T-cell responses against the CALR exon 9 mutations in patients with CALR-mutated chronic myeloproliferative neoplasms (MPN). These T-cell responses were directed against two overlapping peptides that spanned the mutated CALR C terminus.

After the promising responses in the first two patients, we analyzed PBMC from additional 29 CALR-mutated MPN patients. Some of the patients’ PBMC showed low viability; consequently, we were able to analyze responses against CALRLong1 in 18 patients and against CALRLong2 in 24 patients. Of the 24 evaluable patients, 10 had ET and 14 had MF; the latter group comprised 11 patients with PMF, 2 with post-ET MF and 1 with prefibrotic MF. Analyses showed that 9 patients (50%) responded to CALRLong1 (Figure 1c) and 10 patients (42%) to CALRLong2 (Figure 1d); among these patients, 6 (32%) responded to both CALRLong1 and CALRLong2. To confirm these results, we performed an additional ELISPOT in samples from 5 of the 9 patients that showed responses against CALRLong1, and the response was confirmed in 4 patients. Likewise, we performed a supplementary ELISPOT in 5 of the 10 patients that showed responses against CALRLong2, and we confirmed the response in 4 patients. Furthermore, we examined PBMC from two patients in a tumor necrosis factor alpha (TNF-α) ELISPOT. Interestingly, a significant TNF-α response was detected in one of these patients (data not shown).

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Acknowledgements

We thank laboratory technician Merete Jonassen for outstanding help learning M.O.H to perform the immune cell assays, and the secretaries, nurses and laboratory technicians at University Hospital Zealand for organizing blood draws from patients. This study was supported in part by grant from Danish Cancer Society to H.C.H (Grant Number R90-A6143-14-S2) and by grants from Region Sjællands Sundhedsvidenskabelige Forskningsfond to M.O.H (Grant Numbers 12-000095 and 15-000342) in addition to support from Herlev Hospital.

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Authors and Affiliations

  1. University Hospital Zealand, Roskilde, Denmark

    M O Holmström, C H Riley & H C Hasselbalch

  2. Department of Hematology, Center for Cancer Immune Therapy, Copenhagen University Hospital at Herlev, Herlev, Denmark

    M O Holmström, I M Svane & M H Andersen

  3. Department of Immunology and Microbiology, University of Copenhagen, Copenhagen, Denmark

    M H Andersen

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  1. M O Holmström
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Correspondence to M H Andersen.

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No authors have conflicts of interest to disclose. However, it should be noted that Morten Orebo Holmström, Hans Carl Hasselbalch and Mads Hald Andersen have filed a patent regarding the CALR exon 9 mutations as a target for cancer immune therapy. The patent has been transferred to University Hospital Zealand, Zealand Region and Copenhagen University Hospital at Herlev, Capital Region according to the Danish Law concerning inventions made at public research institutions.

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Holmström, M., Riley, C., Svane, I. et al. The CALR exon 9 mutations are shared neoantigens in patients with CALR mutant chronic myeloproliferative neoplasms. Leukemia 30, 2413–2416 (2016). https://doi.org/10.1038/leu.2016.233

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