Somatic MED12 mutations are associated with poor prognosis markers in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults. We performed systematic database search and identified highly specific MED12 mutations in CLL patients. To study this further, we collected three independent sample series comprising over 700 CLL samples and screened MED12 exons 1 and 2 by direct sequencing. Mutations were identified at significant frequency in all three series with a combined mutation frequency of 5.2% (37/709). Positive mutation status was found to be associated with unmutated IGHV and ZAP70 expression, which are well-known poor prognosis markers in CLL. Our results recognize CLL as the first extrauterine cancer type where 5'terminus of MED12 is mutated at significant frequency. Functional analyses have shown that these mutations lead to dissociation of Cyclin C-CDK8/19 from the core Mediator and to the loss of Mediator-associated CDK kinase activity. Additional studies on the role of MED12 mutation status as a putative prognostic factor as well as mutations' exact tumorigenic mechanism in CLL are warranted.


SUPPLEMENTARY DATA Patient series
Three independent CLL patient series, two from the United States and one from Finland, were included in the study. The total number of patients was 746.

Chronic Lymphocytic Leukemia Research Consortium (CRC) sample series
Frozen peripheral-blood mononuclear cells of 278 CLL patients across the USA were obtained through the CLL Research Consortium (CRC; http://cll.ucsd. edu/), a multi-institutional program project investigating new molecular targets that have potential to lead to the development of novel and effective treatments for CLL. The CRC Tissue Core Biorepository processes and banks biospecimens according to established standardized operating procedures. A multiparameter flow cytometry is used to determine the CLL cell surface antigen phenotype. In addition, the expression of ZAP-70 and the IGHV mutation status is determined on all samples received. A more detailed description of the CRC cohort is given in Rassenti et al., 2004 and2008 [1,2]. All studies are performed with the patient's informed consent that is approved by the Institutional Review Board of each participating CRC study site, and in accordance with the Declaration of Helsinki.

The Ohio State University's Human Genetics Sample Bank sample series
DNA samples from 292 CLL cases were obtained from the Human Genetics Sample Bank at The Ohio State University (OSU), Columbus, OH, USA (http:// internalmedicine.osu.edu/genetics/research/humangenetics-sample-bank/). The samples included in this study were collected as part of ongoing studies approved by the Institutional Review Board at OSU. All patients gave informed consent for the research use of their specimens, in accordance with the Declaration of Helsinki.

Helsinki University Central Hospital clinical sample series
Finnish sample series was collected under the authority of a research project at the Helsinki University Central Hospital and the Hematology Research Unit at University of Helsinki investigating molecular pathogenesis, predisposing factors, and individualized care of hematological diseases (HRUHLAB2; http://www. hematology.fi/hruhlab2). Patients diagnosed with CLL at the Hospital District of Helsinki and Uusimaa (HUS) were identified from the Finnish Hematological Registry and Biobank project (FHRB), compiling a list of altogether 259 patients. Current status of the patients was confirmed from their medical records and patients at terminal stage or with memory disorder (n = 6) were excluded from the study. Invitation to participate in the study was sent to altogether 194 living patients, of whom 179 (92%) signed an informed consent and 15 (8%) declined from the study or did not respond to the invitation. By signing the informed consent, patients permitted the usage of their previously derived blood/bone marrow samples and the collection of information from their medical records. Authorization to use previously collected samples and clinical information of deceased patients (n = 59) was obtained from the Ethics Committee of the Helsinki University Central Hospital. Samples from altogether 176 patients (129 living and 47 deceased) were found at the hospital's archives and included in the study. No new samples were collected for this study. General information on the sample series can be found in Supplementary Table 1.

DNA extraction US samples
Genomic DNA of the CRC samples was extracted from cryopreserved peripheral-blood mononuclear cells and that of the OSU Human Genetics Sample Bank specimens from whole blood using a standard phenolchloroform isolation method, and a traditional salting out procedure [3], respectively. DNA concentration and purity were measured with Nanodrop 1000 (for the CRC samples; Nanodrop Technologies, Wilmington, DE) and 2000 (for the Sample Bank samples; Thermo Fisher Scientific, Waltham, MA, USA).

Finnish samples
Genomic DNA was extracted from the archived cytogenetic preparations (fixed and stored in 3:1 methanol/ acetic acid) with DNeasy ® Blood and Tissue Kit (Qiagen, Hilden, Germany) according to manufacturer's instructions. DNA concentration and purity were measured with Nanodrop 8000 (Thermo Fisher Scientific).

MED12 mutation screening
MED12 exon 1 and 2 mutation status was determined by direct sequencing. Previously described primers for exon 1 and primers amplifying all the observed mutation hotspots and insertion/deletions in exon 2 were used in the screening [4,5]