Maija Lepistö

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.).

Comparison of solution-based exome capture methods for next generation sequencing

BackgroundTechniques enabling targeted re-sequencing of the protein coding sequences of the human genome on next generation sequencing instruments are of great interest. We conducted a systematic comparison of the solution-based exome capture kits provided by Agilent and Roche NimbleGen. A control DNA sample was captured with all four capture methods and prepared for Illumina GAII sequencing. Sequence data from additional samples prepared with the same protocols were also used in the comparison.ResultsWe developed a bioinformatics pipeline for quality control, short read alignment, variant identification and annotation of the sequence data. In our analysis, a larger percentage of the high quality reads from the NimbleGen captures than from the Agilent captures aligned to the capture target regions. High GC content of the target sequence was associated with poor capture success in all exome enrichment methods. Comparison of mean allele balances for heterozygous variants indicated a tendency to have more reference bases than variant bases in the heterozygous variant positions within the target regions in all methods. There was virtually no difference in the genotype concordance compared to genotypes derived from SNP arrays. A minimum of 11× coverage was required to make a heterozygote genotype call with 99% accuracy when compared to common SNPs on genome-wide association arrays.ConclusionsLibraries captured with NimbleGen kits aligned more accurately to the target regions. The updated NimbleGen kit most efficiently covered the exome with a minimum coverage of 20×, yet none of the kits captured all the Consensus Coding Sequence annotated exons.

mtDNA Mutagenesis Disrupts Pluripotent Stem Cell Function by Altering Redox Signaling.

Summary mtDNA mutagenesis in somatic stem cells leads to their dysfunction and to progeria in mouse. The mechanism was proposed to involve modification of reactive oxygen species (ROS)/redox signaling. We studied the effect of mtDNA mutagenesis on reprogramming and stemness of pluripotent stem cells (PSCs) and show that PSCs select against specific mtDNA mutations, mimicking germline and promoting mtDNA integrity despite their glycolytic metabolism. Furthermore, mtDNA mutagenesis is associated with an increase in mitochondrial H2O2, reduced PSC reprogramming efficiency, and self-renewal. Mitochondria-targeted ubiquinone, MitoQ, and N-acetyl-L-cysteine efficiently rescued these defects, indicating that both reprogramming efficiency and stemness are modified by mitochondrial ROS. The redox sensitivity, however, rendered PSCs and especially neural stem cells sensitive to MitoQ toxicity. Our results imply that stem cell compartment warrants special attention when the safety of new antioxidants is assessed and point to an essential role for mitochondrial redox signaling in maintaining normal stem cell function.

Enrichment of rare variants in population isolates: single AICDA mutation responsible for hyper-IgM syndrome type 2 in Finland

Antibody class-switch recombination and somatic hypermutation critically depend on the function of activation-induced cytidine deaminase (AID). Rare variants in its gene AICDA have been reported to cause autosomal recessive AID deficiency (autosomal recessive hyper-IgM syndrome type 2 (HIGM2)). Exome sequencing of a multicase Finnish family with an HIGM2 phenotype identified a rare, homozygous, variant (c.416T>C, p.(Met139Thr)) in the AICDA gene, found to be significantly enriched in the Finnish population compared with other populations of European origin (38.56-fold, P<0.001). The population history of Finland, characterized by a restricted number of founders, isolation and several population bottlenecks, has caused enrichment of certain rare disease-causing variants and losses of others, as part of a phenomenon called the Finnish Disease Heritage. Accordingly, rare founder mutations cause the majority of observed Finnish cases in these mostly autosomal recessive disorders that consequently are more frequent in Finland than elsewhere. Screening of all currently known Finnish patients with an HIGM2 phenotype showed them to be homozygous for p.(Met139Thr). All the Finnish p.(Met139Thr) carriers with available data on their geographic descent originated from the eastern and northeastern parts of Finland. They were observed to share more of their genome identity by descent (IBD) than Finns in general (P<0.001), and they all carried a 207.5-kb ancestral haplotype containing the variant. In conclusion, the identified p.(Met139Thr) variant is significantly enriched in Finns and explains all thus far found AID deficiencies in Finland.

Somatic mutations in clonally expanded cytotoxic T lymphocytes in patients with newly diagnosed rheumatoid arthritis

Somatic mutations contribute to tumorigenesis. Although these mutations occur in all proliferating cells, their accumulation under non-malignant conditions, such as in autoimmune disorders, has not been investigated. Here, we show that patients with newly diagnosed rheumatoid arthritis have expanded CD8+ T-cell clones; in 20% (5/25) of patients CD8+ T cells, but not CD4+ T cells, harbour somatic mutations. In healthy controls (n=20), only one mutation is identified in the CD8+ T-cell pool. Mutations exist exclusively in the expanded CD8+ effector-memory subset, persist during follow-up, and are predicted to change protein functions. Some of the mutated genes (SLAMF6, IRF1) have previously been associated with autoimmunity. RNA sequencing of mutation-harbouring cells shows signatures corresponding to cell proliferation. Our data provide evidence of accumulation of somatic mutations in expanded CD8+ T cells, which may have pathogenic significance for RA and other autoimmune diseases.

Abstract 3175: Genomic and transcriptomic data integration in chronic myelomonocytic leukemia reveals a novel fusion gene involving onco-miR-125b-2

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Chronic myelomonocytic leukemia (CMML) is a rare malignancy characterized by increased peripheral monocytosis and dysplasia in a single- or multilineage fashion. Gene mutations so far reported in CMML include TET2, CBL, NRAS, KRAS, RUNX1 and EZH2 but their pathogenic role and driver status in the disease remains unclear. Altered expression of the microRNA miR-125b has been implicated in the pathogenesis of many types of cancers, including myeloid leukemias and Down syndrome-associated acute myeloid leukemia (DS-AML). In addition, this miRNA has been shown to play an important role in hematopoiesis and the regulation of immune cell response. Here, integration of data from next-generation transcriptome sequencing, exome sequencing and array-CGH in a CMML patient (trisomy 21 by cytogenetics) led to the identification of a novel gene fusion event involving the nuclear receptor interacting protein NRIP1 gene and the open reading frame C21orf34 (both at 21q21 approximately 1 MB apart). The fusion was validated by capillary sequencing and found to involve two copy number transition breaks, inversion of the intervening region and the upregulation of the 3′ end of C21orf34. This intronic region harbors a cluster of three miRNAs: miR-let7c, miR-99a, and miR-125b-2. Based on genomic breakpoint analysis, the gene fusion took place just upstream of miR-125b-2. Consistent with this, only miR-125b-2 was highly expressed in the sample, and was processed to a mature miRNA. By RT-PCR, increased expression of miR-125b-2 was also observed in four other CMML patients and five CML patients when compared to healthy bone marrow controls. In contrast, five AML cases studied showed expression levels similar to or lower than that of controls. Interestingly, one AML patient with trisomy 21 had very high levels of miR-125b-2. We found the NRIP1-C21orf34 fusion only in our index patient and therefore other mechanisms of miRNA deregulation at 21q21 in CMML/CML and AML+21 will also exist. In conclusion, we describe for the first time a fusion gene involving miR-125b-2 in CMML, a previously recognized and well-studied onco-miR, which is known to impact on self-renewal of hematopoietic cell precursors. We also detected overexpression of miR-125b-2 in all CMML samples studied suggesting a key pathogenetic driver gene role for this micro-RNA. The assessment of miR-125b-2 levels could potentially be applied to the diagnosis and follow-up of patients with CMML. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3175. doi:1538-7445.AM2012-3175

Abstract 5067: Exome sequencing reveals both DNA sequence and copy number changes in AML: Potential driver changes and mechanisms of drug resistance revealed from serial samples from the same patients

Despite significant advances in characterizing the molecular genetics of AML, the clonal evolution of leukemic cells and the dynamic impact of genomic changes on the development of the disease and progression to drug resistance are not well understood. Here, we applied next-generation sequencing to quantify aberrant tumor subclones carrying specific mutant alleles of key cancer genes and developed a method to extract quantitative high-resolution copy number changes across the genome using exome sequencing data from matching cancer and normal DNA. Serial bone marrow (BM) samples collected from diagnosis to relapse to post-treatment drug resistance in a patient-centric manner made it possible to trace the clonal evolution of AML and to identify variants potentially involved in drug resistance. Exome sequencing from AML blast cells and normal skin biopsies was performed as part of the Finnish Hematology Registry and Biobanking (FHRB) effort. Consecutive paired samples from different patients revealed unique genetic patterns of clonal evolution and cancer progression in each patient. In a pre-resistant sample of one AML M5 patient, we identified four closely spaced insertions in the Wilm9s Tumor (WT1) suppressor gene, none of which appear on the same sequence reads. This suggests the presence of multiple distinct leukemic subclones even before treatment resistance and underscores the strong selective advantage conferred by WT1 mutations. After relapse, one of the subclones was lost, and another one significantly increased suggesting that the relapse arose from the expansion of a pre-existing resistant subclone. In this patient, recurrent clones otherwise featured similar copy number changes and the same fusion genes as the primary diagnostic sample. In another AML patient developing recurrence an opposite pattern was observed: The relapsed, drug-resistant cells displayed an enormous increase of small microdeletions compared to the diagnostic, pre-treatment sample, while almost all sequence-level alterations in potential cancer genes were the same between the two samples. This suggests that a distinct type of DNA repair deficiency may have contributed to the drug resistant clone in this patient. Conclusions: Exome sequencing from paired samples of AML cells before and after relapse makes it possible to trace the clonal evolution of the disease and study the impact of therapy both at the level of sequence alterations of key cancer genes and simultaneously at the level of copy number changes inferred from exome sequence data. This analysis has highlighted multiple genomic patterns by which resistance may evolve in vivo during cancer treatment. Refined bioinformatic analysis and interpretation of exome-seq data provides a rich resource to identify genetic biomarkers of drug response and minimal residual disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5067. doi:1538-7445.AM2012-5067

Abstract 982: Analysis of clonal evolution of leukemia in vivo following novel targeted treatments

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Acute myeloid leukemia (AML) is a genetically heterogeneous disease characterized by significant clonal evolution. It is critical to understand clonal diversity and evolution during cancer progression and drug resistance in order to tailor curative therapies that block the growth of the multiple AML subclones in each patient. In our individualized systems medicine (ISM) initiative, we performed ex-vivo drug sensitivity and resistance testing (DSRT) with a comprehensive set of 306 cancer drugs on primary cells from 22 AML patients (Pemovska et al, Cancer Discovery, 2013). Objective clinical responses were seen in 3/6 chemorefractory patients treated based on DSRT results. Here, we applied next-generation genome and transcriptome sequencing in order to a) follow clonal progression of adult AML in patients during treatment with novel targeted drugs, b) identify putative cancer predisposing mutations in patients who later develop aggressive relapsed AML. Serial samples were obtained from each patient during diagnosis, treatment, remission and relapse. The data indicated significant clonal evolution and emergence of new subclones after relapse and drug resistance. In one such case, treatment of an AML patient with a combination of dasatinib-sunitinib-temsirolimus led to the selection of an already existing low-frequency subclone carrying ETV6-NTRK3 fusion. DSRT of the relapsed sample indicated addiction to IGF1R signalling, compatible with the NTRK3-fusion acting as a driver gene for drug resistance after this treatment. Similar trend where a low-frequency subclone was selected for in the resistance sample was seen in 3 other cases analyzed. Thereby, in depth analysis of clonal evolution from paired AML samples should facilitate the design of new combinatorial regimens to block relapse from arising. Altogether, we analyzed the genomic profile of 20 recurrent, chemorefractory AML cases. The profile was different between the individual patients and showed an overrepresentation of FLT3 and WT1 mutations, whereas previously reported recurrent mutations in primary AML such as DNMT3A, IDH1/2 and RUNX1 mutations were seen only in individual cases. We also analyzed the germline variants in the 20 cases, of which two had a positive family history. These patients were analyzed to identify putative variants predisposing these patients to refractory AMLs. The finnish population data comprising of exome sequencing data of 3700 individuals was used to filter the germline variants and assess their potential significance. This analysis indicated recurrent germline changes in multidrug resistance superfamily of genes, which suggests potential germline clues to the etiology to the chemorefractory, recurrent AML cases. Citation Format: Naga Poojitha Ojamies, Mika Kontro, Henrik Edgren, Samuli Eldfors, Pekka Ellonen, Tea Pemovska, Langstrom Sonja, Henrikki Almusa, Maija Lepisto, Tero Aittokallio, Krister Wennerberg, Caroline Heckman, Kimmo Porkka, Olli Kallioniemi. Analysis of clonal evolution of leukemia in vivo following novel targeted treatments. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 982. doi:10.1158/1538-7445.AM2014-982

Abstract 3164: Somatic mutation analysis pipeline for exome-sequencing data identifies oncogenic STAT3 mutations in T-LGL leukemia.

Exome-sequencing of matched tumor-normal sample pairs enables the identification of the full set of somatically acquired mutations that directly affect protein function in a given tumor. Inferring the functional significance of coding region mutations is relatively straightforward compared to mutations occurring in non-coding regions. Furthermore, exome sequencing enables sufficient sampling depth required for sensitive detection of somatic mutations in tumor samples. However, identification of novel driver mutations remains challenging due to the high levels of sequencing errors observed with current next generation sequencing technologies and due to passenger mutations which vastly out number driver mutations in any given tumor. Identification of novel driver mutations is especially challenging in the absence of cohort data. To identify driving somatic events in exome sequence data, we developed an analytical pipeline utilizing a combination of 3rd party tools for sequence read processing and mutation calling. This was coupled to an annotation framework that aids prioritization of driver mutation candidates for downstream validation experiments. The annotation framework integrates prior knowledge from reference databases on known cancer genes, sequence level evolutionary conservation and functional consequence predictions. Gene expression data from RNA-seq of the tumor sample can also be integrated in the annotations. To test this pipeline, we performed exome sequencing of a tumor-normal sample pair from an index patient diagnosed with T-cell derived large granular lymphocytic leukemia (T-LGL), which is a rare lymphoproliferative disease of previously unknown pathogenesis. The analysis pipeline identified a high-ranking candidate mutation in STAT3. Structural analysis indicated that the mutation resulted in a hydrophobic substitution at the SH2 dimerization interface, suggesting that the mutation stabilizes the active dimer form through increased hydrophobic interaction between monomers. Further validation experiments showed STAT3 SH2 hydrophobic substitutions to be recurrent in T-LGL and to result in constitutive STAT3 activation [1]. Application of the pipeline enabled identification of a novel class of oncogenic STAT3 SH2 domain mutations and established STAT3 as a key driver oncogene in T-LGL. Citation Format: Samuli Eldfors, Hanna LM Rajala, Pekka Ellonen, Emma I. Andersson, Sonja Lagstrom, Henrikki Almusa, Henrik Edgren, Maija Lepisto, Pirkko Mattila, Jonathan Knowles, Janna Saarela, Kimmo Porkka, Olli Kallioniemi, Satu Mustjoki, Caroline A. Heckman. Somatic mutation analysis pipeline for exome-sequencing data identifies oncogenic STAT3 mutations in T-LGL leukemia. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3164. doi:10.1158/1538-7445.AM2013-3164

Abstract 4580: Personalized treatment selection for therapy-resistant AML by integrating ex-vivo drug sensitivity and resistance testing (DSRT) as well as serial genomic, transcriptomic and phosphoproteomic profiling

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Samples from recurrent, treatment-refractory cancers are rarely available, but would be valuable in understanding the molecular drivers of drug resistance. In leukemias, consecutive samples are readily available during treatment. Hence, we explored here the progression of adult acute myeloid leukemias (AML) by serial sampling and by integrating data from multiple platforms. Next-generation exome and RNA sequencing, and phosphoproteomic data were combined with comprehensive 240 cancer drug sensitivity and resistance testing (DSRT) of leukemic blasts ex-vivo before and after clinical relapse. The data were generated in an experimental diagnostic setting, with intent to improve and personalize treatment of patients with recurrent AML. A 54-year old AML-M5 patient with a FLT-3-ITD mutation and a normal karyotype was monitored by serial sampling. The patient was initially refractory to three consecutive high-dose induction treatments and had limited therapy options. AML blasts from the patient were screened with the DSRT platform. Results implied that the blast cells were 710-times more sensitive to temsirolimus and other rapamycin analogs as compared to normal BM cells, and showed a 1100-fold increased sensitivity to dasatinib. Proteomic analysis showed high phosphorylation of several signaling molecules, such as the insulin receptor and mTOR. Sequencing identified WT1 mutations and a NUP98-NSD1 fusion transcript, an infrequent event associated with poor prognosis in AML. Based on the DSRT results, the patient received compassionate off-label treatment with dasatinib, sunitinib and temsirolimus, resulting in a remarkable clinical remission, normalization of blast counts and a rapid recovery of neutrophil counts as a sign of selective elimination of the leukemic cells. The patient relapsed 4 weeks later, and at this point a new DSRT assay was performed, which showed the blast cells to be completely resistant to temsirolimus and less sensitive to dasatinib ex vivo. Consistent with this drug sensitivity profile was a genomic evolution of a distinct AML subclone with new changes, such as NF1 mutation and a microdeletion of the LEF1 gene, which were not observed in the pre-treatment sample. Taken together, we have demonstrated, how molecular profiling and functional ex vivo drug sensitivity and resistance data can be used to individually optimize patient treatment. Remission was achieved in a patient with advanced, treatment-refractory AML. Serial sampling from human AML patients coupled with molecular profiling and drug sensitivity testing may shed light on clonal progression of disease, and the molecular events underlying drug response. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4580. doi:1538-7445.AM2012-4580