Sonja Lagström

Somatic STAT3 mutations in large granular lymphocytic leukemia.

BACKGROUND T-cell large granular lymphocytic leukemia is a rare lymphoproliferative disorder characterized by the expansion of clonal CD3+CD8+ cytotoxic T lymphocytes (CTLs) and often associated with autoimmune disorders and immune-mediated cytopenias. METHODS We used next-generation exome sequencing to identify somatic mutations in CTLs from an index patient with large granular lymphocytic leukemia. Targeted resequencing was performed in a well-characterized cohort of 76 patients with this disorder, characterized by clonal T-cell-receptor rearrangements and increased numbers of large granular lymphocytes. RESULTS Mutations in the signal transducer and activator of transcription 3 gene (STAT3) were found in 31 of 77 patients (40%) with large granular lymphocytic leukemia. Among these 31 patients, recurrent mutational hot spots included Y640F in 13 (17%), D661V in 7 (9%), D661Y in 7 (9%), and N647I in 3 (4%). All mutations were located in exon 21, encoding the Src homology 2 (SH2) domain, which mediates the dimerization and activation of STAT protein. The amino acid changes resulted in a more hydrophobic protein surface and were associated with phosphorylation of STAT3 and its localization in the nucleus. In vitro functional studies showed that the Y640F and D661V mutations increased the transcriptional activity of STAT3. In the affected patients, downstream target genes of the STAT3 pathway (IFNGR2, BCL2L1, and JAK2) were up-regulated. Patients with STAT3 mutations presented more often with neutropenia and rheumatoid arthritis than did patients without these mutations. CONCLUSIONS The SH2 dimerization and activation domain of STAT3 is frequently mutated in patients with large granular lymphocytic leukemia; these findings suggest that aberrant STAT3 signaling underlies the pathogenesis of this disease. (Funded by the Academy of Finland and others.).

Discovery of somatic STAT5b mutations in large granular lymphocytic leukemia

Large granular lymphocytic (LGL) leukemia is characterized by clonal expansion of cytotoxic T cells or natural killer cells. Recently, somatic mutations in the signal transducer and activator of transcription 3 (STAT3) gene were discovered in 28% to 40% of LGL leukemia patients. By exome and transcriptome sequencing of 2 STAT3 mutation-negative LGL leukemia patients, we identified a recurrent, somatic missense mutation (Y665F) in the Src-like homology 2 domain of the STAT5b gene. Targeted amplicon sequencing of 211 LGL leukemia patients revealed 2 additional patients with STAT5b mutations (N642H), resulting in a total frequency of 2% (4 of 211) of STAT5b mutations across all patients. The Y665F and N642H mutant constructs increased the transcriptional activity of STAT5 and tyrosine (Y694) phosphorylation, which was also observed in patient samples. The clinical course of the disease in patients with the N642H mutation was aggressive and fatal, clearly different from typical LGL leukemia with a relatively favorable outcome. This is the first time somatic STAT5 mutations are discovered in human cancer and further emphasizes the role of STAT family genes in the pathogenesis of LGL leukemia.

Comparison of targeted next-generation sequencing (NGS) and real-time PCR in the detection of EGFR, KRAS, and BRAF mutations on formalin-fixed, paraffin-embedded tumor material of non-small cell lung carcinoma-superiority of NGS.

The development of tyrosine kinase inhibitor treatments has made it important to test cancer patients for clinically significant gene mutations that influence the benefit of treatment. Targeted next‐generation sequencing (NGS) provides a promising method for diagnostic purposes by enabling the simultaneous detection of multiple mutations in various genes in a single test. The aim of our study was to screen EGFR, KRAS, and BRAF mutations by targeted NGS and commonly used real‐time polymerase chain reaction (PCR) methods to evaluate the feasibility of targeted NGS for the detection of the mutations. Furthermore, we aimed to identify potential novel mutations by targeted NGS. We analyzed formalin‐fixed, paraffin‐embedded (FFPE) tumor tissue specimens from 81 non‐small cell lung carcinoma patients. We observed a significant concordance (from 96.3 to 100%) of the EGFR, KRAS, and BRAF mutation detection results between targeted NGS and real‐time PCR. Moreover, targeted NGS revealed seven nonsynonymous single‐nucleotide variations and one insertion‐deletion variation in EGFR not detectable by the real‐time PCR methods. The potential clinical significance of these variants requires elucidation in future studies. Our results support the use of targeted NGS in the screening of EGFR, KRAS, and BRAF mutations in FFPE tissue material.

STAT3-mutations indicate the presence of subclinical T cell clones in a subset of aplastic anemia and myelodysplastic syndrome patients

Large granular lymphocyte leukemia (LGL) is often associated with immune cytopenias and can cooccur in the context of aplastic anemia (AA) and myelodysplastic syndromes (MDS). We took advantage of the recent description of signal transducer and activator of transcription 3 (STAT3) mutations in LGL clonal expansions to test, using sensitive methods, for the presence of these mutations in a large cohort of 367 MDS and 140 AA cases. STAT3 clones can be found not only in known LGL concomitant cases, but in a small proportion of unsuspected ones (7% AA and 2.5% MDS). In STAT3-mutated AA patients, an interesting trend toward better responses of immunosuppressive therapy and an association with the presence of human leukocyte antigen-DR15 were found. MDSs harboring a STAT3 mutant clone showed a lower degree of bone marrow cellularity and a higher frequency of developing chromosome 7 abnormalities. STAT3-mutant LGL clones may facilitate a persistently dysregulated autoimmune activation, responsible for the primary induction of bone marrow failure in a subset of AA and MDS patients.

Novel activating STAT5B mutations as putative drivers of T-cell acute lymphoblastic leukemia.

Novel activating STAT5B mutations as putative drivers of T-cell acute lymphoblastic leukemia

The analysis of clonal diversity and therapy responses using STAT3 mutations as a molecular marker in large granular lymphocytic leukemia

T-cell large granular lymphocytic leukemia and chronic lymphoproliferative disorder of natural killer cells are intriguing entities between benign and malignant lymphoproliferation. The molecular pathogenesis has partly been uncovered by the recent discovery of somatic activating STAT3 and STAT5b mutations. Here we show that 43% (75/174) of patients with T-cell large granular lymphocytic leukemia and 18% (7/39) with chronic lymphoproliferative disorder of natural killer cells harbor STAT3 mutations when analyzed by quantitative deep amplicon sequencing. Surprisingly, 17% of the STAT3-mutated patients carried multiple STAT3 mutations, which were located in different lymphocyte clones. The size of the mutated clone correlated well with the degree of clonal expansion of the T-cell repertoire analyzed by T-cell receptor beta chain deep sequencing. The analysis of sequential samples suggested that current immunosuppressive therapy is not able to reduce the level of the mutated clone in most cases, thus warranting the search for novel targeted therapies. Our findings imply that the clonal landscape of large granular lymphocytic leukemia is more complex than considered before, and a substantial number of patients have multiple lymphocyte subclones harboring different STAT3 mutations, thus mimicking the situation in acute leukemia.

Activating somatic mutations outside the SH2-domain of STAT3 in LGL leukemia

Large granular lymphocyte (LGL) leukemia is a rare clonal disease characterized by a persistent increase in the number of CD8+ cytotoxic T cells or CD16/56+ natural killer (NK) cells.1 Patients are prone to recurrent infections and often suffer from severe cytopenias and autoimmune diseases that are thought to be mediated by cytotoxic LGL lymphocytes. LGL leukemia is believed to begin as an antigen-driven immune response followed by the constitutive activation of cytotoxic T lymphocytes or NK cells. Overall, studies have highlighted the dysregulation of different apoptotic pathways (for example, sphingolipid and FAS/FAS ligand) and the activation of survival signaling pathways (for example, PI3K/AKT and RAS).1

A balanced translocation truncates Neurotrimin in a family with intracranial and thoracic aortic aneurysm

Background Balanced chromosomal rearrangements occasionally have strong phenotypic effects, which may be useful in understanding pathobiology. However, conventional strategies for characterising breakpoints are laborious and inaccurate. We present here a proband with a thoracic aortic aneurysm (TAA) and a balanced translocation t(10;11) (q23.2;q24.2). Our purpose was to sequence the chromosomal breaks in this family to reveal a novel candidate gene for aneurysm. Methods and results Intracranial aneurysm (IA) and TAAs appear to run in the family in an autosomal dominant manner: After exploring the family history, we observed that the probands two siblings both died from cerebral haemorrhage, and the probands parent and parents sibling died from aortic rupture. After application of a genome-wide paired-end DNA sequencing method for breakpoint mapping, we demonstrate that this translocation breaks intron 1 of a splicing isoform of Neurotrimin at 11q25 in a previously implicated candidate region for IAs and AAs (OMIM 612161). Conclusions Our results demonstrate the feasibility of genome-wide paired-end sequencing for the characterisation of balanced rearrangements and identification of candidate genes in patients with potentially disease-associated chromosome rearrangements. The family samples were gathered as a part of our recently launched National Registry of Reciprocal Balanced Translocations and Inversions in Finland (n=2575), and we believe that such a registry will be a powerful resource for the localisation of chromosomal aberrations, which can bring insight into the aetiology of related phenotypes.

Discovery of novel drug sensitivities in T-PLL by high-throughput ex vivo drug testing and mutation profiling

T-cell prolymphocytic leukemia (T-PLL) is a rare and aggressive neoplasm of mature T-cells with an urgent need for rationally designed therapies to address its notoriously chemo-refractory behavior. The median survival of T-PLL patients is <2 years and clinical trials are difficult to execute. Here we systematically explored the diversity of drug responses in T-PLL patient samples using an ex vivo drug sensitivity and resistance testing platform and correlated the findings with somatic mutations and gene expression profiles. Intriguingly, all T-PLL samples were sensitive to the cyclin-dependent kinase inhibitor SNS-032, which overcame stromal-cell-mediated protection and elicited robust p53-activation and apoptosis. Across all patients, the most effective classes of compounds were histone deacetylase, phosphoinositide-3 kinase/AKT/mammalian target of rapamycin, heat-shock protein 90 and BH3-family protein inhibitors as well as p53 activators, indicating previously unexplored, novel targeted approaches for treating T-PLL. Although Janus-activated kinase–signal transducer and activator of transcription factor (JAK-STAT) pathway mutations were common in T-PLL (71% of patients), JAK-STAT inhibitor responses were not directly linked to those or other T-PLL-specific lesions. Overall, we found that genetic markers do not readily translate into novel effective therapeutic vulnerabilities. In conclusion, novel classes of compounds with high efficacy in T-PLL were discovered with the comprehensive ex vivo drug screening platform warranting further studies of synergisms and clinical testing.

Mutated ephrin receptor genes in non-small cell lung carcinoma and their occurrence with driver mutations-targeted resequencing study on formalin-fixed, paraffin-embedded tumor material of 81 patients.

Non‐small cell lung carcinoma (NSCLC) is the most common subtype of lung cancer. The oncogenic potential of receptor tyrosine kinases (RTKs) is widely known and they are potential targets for tailored therapy. Ephrin receptors (Ephs) form the largest group of RTKs. Nevertheless, Ephs are not widely studied in NSCLC so far. The aim of our study was to investigate novel mutations of Eph genes (EPHA1–8, EPHB1–4, EPHB6) and their association with clinically relevant mutations in BRAF, EML4‐ALK, EGFR, INSR, KDR, KRAS, MET, PDGFRA, PDGFRB, PIK3, PTEN, RET, and TP53 in NSCLC patients. Targeted resequencing was conducted on 81 formalin‐fixed, paraffin‐embedded NSCLC tumor specimens. We analyzed missense and nonsense mutations harbored in the coding regions of the selected genes. We found 18 novel mutations of Ephs in 20% (16 of 81) of the patients. Nearly half of these mutations occurred in the protein kinase domain. The mutations were not mutually exclusive with other clinically relevant mutations. Our study shows that Ephs are frequently mutated in NSCLC patients, and occur together with other known mutations relevant to the pathogenicity of NSCLC.