Supplementary Materials & methods

Molecular genetic analyses

Genomic DNA and RNA were isolated from the bone marrow mononuclear cells using E.Z.N.A. Blood DNA Kit (Omega Bio-Tek Inc., Norcross, GA, USA) and RNeasy Mini Kit (QIAGEN, Hilden, Germany), respectively, as recommended by the manufacturer. RNA was extracted from the ovarian samples by using TRIzol® Reagent (Invitrogen, Life Technologies, Carlsbad, CA, USA). A frozen tissue sample was placed on a Petri dish in a small volume of TRIzol® and immediately homogenized with a scalpel. Homogenized tissue was transferred to Eppendorf tube and RNA extraction done according to manufacturer’s recommendations. Genomic DNA was extracted from ovarian tissue either simultaneously with RNA using the simultaneous DNA extraction option of TRIzol® Reagent or standard Proteinase K and phenol-chloroform method if there was more ample material available. Nucleic acids were quantified spectrophotometrically by the NanoDrop Instrument (Thermo Fisher Scientific, Waltham, MA,USA). RNA was reverse-transcribed to cDNA according to the Europe Against Cancer (EAC) protocol.1

Translocation-specific fusion transcripts (TEL-AML1, MLL-AF4, AML1-ETO, E2A-PBX1, BCR-ABL1) were analyzed according to the EAC protocol. Standard curves were constructed from a cDNA dilution series of patients’ diagnostic samples and the MRD results achieved and assay sensitivities calculated after normalization with the ABL1 gene expression.2 Target screening for clonal antigen receptor gene rearrangements (IgH, IgK, TCRD, TCRG, TCRB) were done by both multiplex and singleplex primer sets.3,4 PCR products were subjected to heteroduplex treatment and run on polyacrylamide gels. After ethidium bromide staining the clonal fragments were cut off from the gel and sequenced by an automated capillary electrophoresis sequencer Applied Biosystems 3500Dx (Applied Biosystems, Life Technologies, Carlsbad, CA, USA). Sequences were analyzed by comparing them to Ig and TCR sequences obtained from the IMGT database ( Allele specific oligonucleotide (ASO) primers were designed for the hypervariable junction regions and used in conjunction with the appropriate reverse primers and TaqMan probes published elsewhere.5,6,7 Like in fusion transcript assays, the DNA dilution series of the pretreatment bone marrow sample served as a quantification standard for the ovarian samples. All samples were analyzed in triplicates. Quantification of the results and for PCR-negative samples calculation of assay sensitivities were done after normalization with quantification data from the Albumin control gene. The EuroMRD definitions9 for quantitative range and sensitivity were used throughout the study. Shortly, the quantitative range is defined as the part of the standard curve in which the MRD levels can be quantified reproducibly and accurately and assay sensitivity reflects the lowest MRD level that still can be detected, although not reproducibly and accurately. Nucleophosmin gene (NPM1) exon 12 length mutation analysis was done by fragment analysis and mutation type was determined by sequencing as described.8 NPM1 MRD analysis was performed with the ASO-primer for the TCTG insertion (NPM-A) in conjunction with the germline reverse primer and TaqMan probe (primer sequences available upon request). All MRD analyses were carried out in accordance with the EuroMRD guidelines9 both in the PCR setup and interpretation of the data. MRD analyses were performed centrally at the department of molecular genetics of TYKSLAB (TurkuUniversityHospital laboratories, Turku, Finland), which is a member of the EuroMRD consortium (

References

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