A novel class of somatic mutations in blood detected preferentially in CD8+ cells.

Somatic mutations have a central role in cancer but their role in other diseases such as autoimmune disorders is poorly understood. Earlier work has provided indirect evidence of rare somatic mutations in autoreactive T-lymphocytes in multiple sclerosis (MS) patients but such mutations have not been identified thus far. We analysed somatic mutations in blood in 16 patients with relapsing MS and 4 with other neurological autoimmune disease. To facilitate the detection of somatic mutations CD4+, CD8+, CD19+ and CD4-/CD8-/CD19- cell subpopulations were separated. We performed next-generation DNA sequencing targeting 986 immune-related genes. Somatic mutations were called by comparing the sequence data of each cell subpopulation to other subpopulations of the same patient and validated by amplicon sequencing. We found non-synonymous somatic mutations in 12 (60%) patients (10 MS, 1 myasthenia gravis, 1 narcolepsy). There were 27 mutations, all different and mostly novel (67%). They were discovered at subpopulation-wise allelic fractions of 0.2%-4.6% (median 0.95%). Multiple mutations were found in 8 patients. The mutations were enriched in CD8+ cells (85% of mutations). In follow-up after a median time of 2.3years, 96% of the mutations were still detectable. These results unravel a novel class of persistent somatic mutations, many of which were in genes that may play a role in autoimmunity (ATM, BTK, CD46, CD180, CLIP2, HMMR, IKFZF3, ITGB3, KIR3DL2, MAPK10, CD56/NCAM1, RBM6, RORA, RPA1 and STAT3). Whether some of this class of mutations plays a role in disease is currently unclear, but these results define an interesting hitherto unknown research target for future studies.

Telomere shortening correlates with leukemic stem cell burden at diagnosis of chronic myeloid leukemia.

Telomere length (TL) in peripheral blood (PB) cells of patients with chronic myeloid leukemia (CML) has been shown to correlate with disease stage, prognostic scores, response to therapy, and disease progression. However, due to considerable genetic interindividual variability, TL varies substantially between individuals, limiting its use as a robust prognostic marker in individual patients. Here, we compared TL of BCR-ABL-, nonleukemic CD34+CD38- hematopoietic stem cells (HSC) in the bone marrow of CML patients at diagnosis to their individual BCR-ABL+ leukemic stem cell (LSC) counterparts. We observed significantly accelerated telomere shortening in LSC compared with nonleukemic HSC. Interestingly, the degree of LSC telomere shortening was found to correlate significantly with the leukemic clone size. To validate the diagnostic value of nonleukemic cells as internal controls and to rule out effects of tyrosine kinase inhibitor (TKI) treatment on these nontarget cells, we prospectively assessed TL in 134 PB samples collected in deep molecular remission after TKI treatment within the EURO-SKI study (NCT01596114). Here, no significant telomere shortening was observed in granulocytes compared with an age-adjusted control cohort. In conclusion, this study provides proof of principle for accelerated telomere shortening in LSC as opposed to HSC in CML patients at diagnosis. The fact that the degree of telomere shortening correlates with leukemic clone's size supports the use of TL in leukemic cells as a prognostic parameter pending prospective validation. TL in nonleukemic myeloid cells seems unaffected even by long-term TKI treatment arguing against a reduction of telomere-mediated replicative reserve in normal hematopoiesis under TKI treatment.

Aggressive natural killer-cell leukemia mutational landscape and drug profiling highlight JAK-STAT signaling as therapeutic target.

Aggressive natural killer-cell (NK-cell) leukemia (ANKL) is an extremely aggressive malignancy with dismal prognosis and lack of targeted therapies. Here, we elucidate the molecular pathogenesis of ANKL using a combination of genomic and drug sensitivity profiling. We study 14 ANKL patients using whole-exome sequencing (WES) and identify mutations in STAT3 (21%) and RAS-MAPK pathway genes (21%) as well as in DDX3X (29%) and epigenetic modifiers (50%). Additional alterations include JAK-STAT copy gains and tyrosine phosphatase mutations, which we show recurrent also in extranodal NK/T-cell lymphoma, nasal type (NKTCL) through integration of public genomic data. Drug sensitivity profiling further demonstrates the role of the JAK-STAT pathway in the pathogenesis of NK-cell malignancies, identifying NK cells to be highly sensitive to JAK and BCL2 inhibition compared to other hematopoietic cell lineages. Our results provide insight into ANKL genetics and a framework for application of targeted therapies in NK-cell malignancies.

Crosstalk between tongue carcinoma cells, extracellular vesicles, and immune cells in in vitro and in vivo models.

The crosstalk between immune cells, cancer cells, and extracellular vesicles (EVs) secreted by cancer cells remains poorly understood. We created three-dimensional (3D) cell culture models using human leiomyoma discs and Myogel to study the effects of immune cells on highly (HSC-3) and less (SCC-25) invasive oral tongue squamous cell carcinoma (OTSCC) cell lines. Additionally, we studied the effects of EVs isolated from these cell lines on the cytotoxicity of CD8+ T and NK cells isolated from three healthy donors. Our analysis included the effects of these EVs on innate immunity in zebrafish larvae. Activated immune cells significantly decreased the proliferation of both OTSCC cell lines and associated with a diminished invasion area of HSC-3 cells. In general, EVs from SCC-25 increased the cytotoxic activity of CD8+ T and NK cells more than those from HSC-3 cells. However, this effect varied depending on the source and the immune and cancer cell subgroups. In zebrafish, the amount of IL-13 mRNA was decreased by SCC-25 EVs. This study describes promising in vitro and in vivo models to investigate interactions between immune cells, cancer cells, and EVs.