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• W2885429207 abstract "Multiple myeloma (MM) is a genetically heterogeneous disease with a diverse clinical outcome. Copy number alterations (CNAs), including whole chromosome and subchromosomal gains and losses, are common contributors of the pathogenesis and have demonstrated prognostic impact in MM. We tested the performance of digital multiplex ligation-dependent probe amplification (digitalMLPA), a novel technique combining MLPA and next-generation sequencing, to detect disease-related CNAs. Copy number status at 371 genomic loci was simultaneously analyzed in 56 diagnostic bone marrow samples, which were also examined by conventional MLPA and interphase fluorescence in situ hybridization (iFISH). On average, digitalMLPA identified 4.4 subchromosomal CNAs per patient. The increased number of probes compared with conventional MLPA allowed a detailed mapping of CNAs, especially on chromosome 1, where 24 different patterns were observed in 38 patients harboring loss(1p) and/or gain(1q). iFISH, MLPA, and digitalMLPA results at loci investigated by multiple methods showed a congruency of 95%. Besides precise characterization of hyperdiploid karyotypes not efficiently achievable by iFISH or MLPA, digitalMLPA unraveled 156 CNAs not detected by the other two methods in 45 patients (80%). In addition, we provide proof of principle that digitalMLPA can detect known point mutations, in this case the BRAFV600E. Our study demonstrates the robustness of digitalMLPA to profile CNAs and to screen point mutations in MM, which could efficiently be used in myeloma diagnostics. Multiple myeloma (MM) is a genetically heterogeneous disease with a diverse clinical outcome. Copy number alterations (CNAs), including whole chromosome and subchromosomal gains and losses, are common contributors of the pathogenesis and have demonstrated prognostic impact in MM. We tested the performance of digital multiplex ligation-dependent probe amplification (digitalMLPA), a novel technique combining MLPA and next-generation sequencing, to detect disease-related CNAs. Copy number status at 371 genomic loci was simultaneously analyzed in 56 diagnostic bone marrow samples, which were also examined by conventional MLPA and interphase fluorescence in situ hybridization (iFISH). On average, digitalMLPA identified 4.4 subchromosomal CNAs per patient. The increased number of probes compared with conventional MLPA allowed a detailed mapping of CNAs, especially on chromosome 1, where 24 different patterns were observed in 38 patients harboring loss(1p) and/or gain(1q). iFISH, MLPA, and digitalMLPA results at loci investigated by multiple methods showed a congruency of 95%. Besides precise characterization of hyperdiploid karyotypes not efficiently achievable by iFISH or MLPA, digitalMLPA unraveled 156 CNAs not detected by the other two methods in 45 patients (80%). In addition, we provide proof of principle that digitalMLPA can detect known point mutations, in this case the BRAFV600E. Our study demonstrates the robustness of digitalMLPA to profile CNAs and to screen point mutations in MM, which could efficiently be used in myeloma diagnostics. Multiple myeloma (MM) is currently an incurable malignant bone marrow disorder characterized by unleashed proliferation of plasma B cells.1Swerdlow S.H. Campo E. Pileri S.A. Harris N.L. Stein H. Siebert R. Advani R. Ghielmini M. Salles G.A. Zelenetz A.D. Jaffe E.S. The 2016 revision of the World Health Organization classification of lymphoid neoplasms.Blood. 2016; 127: 2375-2390Crossref PubMed Scopus (4524) Google Scholar, 2Rajkumar S.V. Multiple myeloma: 2016 update on diagnosis, risk-stratification, and management.Am J Hematol. 2016; 91: 719-734Crossref PubMed Scopus (303) Google Scholar The diverse genomic landscape of MM has extensively been profiled in large-scale studies using fluorescence in situ hybridization (FISH), single-nucleotide polymorphism–array, and next-generation sequencing (NGS).3Walker B.A. Leone P.E. Chiecchio L. Dickens N.J. Jenner M.W. Boyd K.D. Johnson D.C. Gonzalez D. Dagrada G.P. Protheroe R.K. Konn Z.J. Stockley D.M. Gregory W.M. Davies F.E. Ross F.M. Morgan G.J. A compendium of myeloma-associated chromosomal copy number abnormalities and their prognostic value.Blood. 2010; 116: e56-e65Crossref PubMed Scopus (270) Google Scholar, 4Jacobus S.J. Kumar S. Uno H. Van Wier S.A. Ahmann G.J. Henderson K.J. Callander N.S. Williams M.E. Siegel D.S. Greipp P.R. Rajkumar S.V. Fonseca R. Impact of high-risk classification by FISH: an Eastern Cooperative Oncology Group (ECOG) study E4A03.Br J Haematol. 2011; 155: 340-348Crossref PubMed Scopus (27) Google Scholar, 5Chapman M.A. Lawrence M.S. Keats J.J. Cibulskis K. Sougnez C. Schinzel A.C. et al.Initial genome sequencing and analysis of multiple myeloma.Nature. 2011; 471: 467-472Crossref PubMed Scopus (1162) Google Scholar, 6Boyd K.D. Ross F.M. Chiecchio L. Dagrada G.P. Konn Z.J. Tapper W.J. Walker B.A. Wardell C.P. Gregory W.M. Szubert A.J. Bell S.E. Child J.A. Jackson G.H. Davies F.E. Morgan G.J. NCRI Haematology Oncology Studies GroupA novel prognostic model in myeloma based on co-segregating adverse FISH lesions and the ISS: analysis of patients treated in the MRC Myeloma IX trial.Leukemia. 2012; 26: 349-355Crossref PubMed Scopus (250) Google Scholar, 7Kumar S. Fonseca R. Ketterling R.P. Dispenzieri A. Lacy M.Q. Gertz M.A. Hayman S.R. Buadi F.K. Dingli D. Knudson R.A. Greenberg A. Russell S.J. Zeldenrust S.R. Lust J.A. Kyle R.A. Bergsagel L. Rajkumar S.V. Trisomies in multiple myeloma: impact on survival in patients with high-risk cytogenetics.Blood. 2012; 119: 2100-2105Crossref PubMed Scopus (190) Google Scholar, 8Morgan G.J. Walker B.A. Davies F.E. The genetic architecture of multiple myeloma.Nat Rev Cancer. 2012; 12: 335-348Crossref PubMed Scopus (661) Google Scholar, 9Bolli N. Avet-Loiseau H. Wedge D.C. Van Loo P. Alexandrov L.B. Martincorena I. Dawson K.J. Iorio F. Nik-Zainal S. Bignell G.R. Hinton J.W. Li Y. Tubio J.M. McLaren S. O' Meara S. Butler A.P. Teague J.W. Mudie L. Anderson E. Rashid N. Tai Y.T. Shammas M.A. Sperling A.S. Fulciniti M. Richardson P.G. Parmigiani G. Magrangeas F. Minvielle S. Moreau P. Attal M. Facon T. Futreal P.A. Anderson K.C. Campbell P.J. Munshi N.C. Heterogeneity of genomic evolution and mutational profiles in multiple myeloma.Nat Commun. 2014; 5: 2997Crossref PubMed Scopus (633) Google Scholar, 10Walker B.A. Boyle E.M. Wardell C.P. Murison A. Begum D.B. Dahir N.M. Proszek P.Z. Johnson D.C. Kaiser M.F. Melchor L. Aronson L.I. Scales M. Pawlyn C. Mirabella F. Jones J.R. Brioli A. Mikulasova A. Cairns D.A. Gregory W.M. Quartilho A. Drayson M.T. Russell N. Cook G. Jackson G.H. Leleu X. Davies F.E. Morgan G.J. Mutational spectrum, copy number changes, and outcome: results of a sequencing study of patients with newly diagnosed myeloma.J Clin Oncol. 2015; 33: 3911-3920Crossref PubMed Scopus (388) Google Scholar, 11Chretien M.L. Corre J. Lauwers-Cances V. Magrangeas F. Cleynen A. Yon E. Hulin C. Leleu X. Orsini-Piocelle F. Blade J.S. Sohn C. Karlin L. Delbrel X. Hebraud B. Roussel M. Marit G. Garderet L. Mohty M. Rodon P. Voillat L. Royer B. Jaccard A. Belhadj K. Fontan J. Caillot D. Stoppa A.M. Attal M. Facon T. Moreau P. Minvielle S. Avet-Loiseau H. Understanding the role of hyperdiploidy in myeloma prognosis: which trisomies really matter?.Blood. 2015; 126: 2713-2719Crossref PubMed Scopus (78) Google Scholar The presence and combinations of various copy number alterations (CNAs) were reported to have prognostic significance in MM6Boyd K.D. Ross F.M. Chiecchio L. Dagrada G.P. Konn Z.J. Tapper W.J. Walker B.A. Wardell C.P. Gregory W.M. Szubert A.J. Bell S.E. Child J.A. Jackson G.H. Davies F.E. Morgan G.J. NCRI Haematology Oncology Studies GroupA novel prognostic model in myeloma based on co-segregating adverse FISH lesions and the ISS: analysis of patients treated in the MRC Myeloma IX trial.Leukemia. 2012; 26: 349-355Crossref PubMed Scopus (250) Google Scholar, 12Avet-Loiseau H. Li C. Magrangeas F. Gouraud W. Charbonnel C. Harousseau J.L. Attal M. Marit G. Mathiot C. Facon T. Moreau P. Anderson K.C. Campion L. Munshi N.C. Minvielle S. Prognostic significance of copy-number alterations in multiple myeloma.J Clin Oncol. 2009; 27: 4585-4590Crossref PubMed Scopus (240) Google Scholar, 13Shah V. Sherborne A.L. Walker B.A. Johnson D.C. Boyle E.M. Ellis S. Begum D.B. Proszek P.Z. Jones J.R. Pawlyn C. Savola S. Jenner M.W. Drayson M.T. Owen R.G. Houlston R.S. Cairns D.A. Gregory W.M. Cook G. Davies F.E. Jackson G.H. Morgan G.J. Kaiser M.F. Prediction of outcome in newly diagnosed myeloma: a meta-analysis of the molecular profiles of 1,905 trial patients.Leukemia. 2018; 32: 102-110Crossref PubMed Scopus (135) Google Scholar; therefore, development of efficient and rapid methods allowing the comprehensive screening of disease-relevant CNAs in clinical diagnostics is of high priority. Multiplex ligation-dependent probe amplification (MLPA) is an established multiplex PCR-based technique primarily developed for the detection of CNAs.14Schouten J.P. McElgunn C.J. Waaijer R. Zwijnenburg D. Diepvens F. Pals G. Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification.Nucleic Acids Res. 2002; 30: e57Crossref PubMed Scopus (2096) Google Scholar The relatively straightforward protocol i) needs only 50 ng of DNA input, ii) is able to analyze CNAs at single-exon resolution, iii) provides results within 24 hours, iv) is applicable even to the analysis of formalin-fixed, paraffin-embedded specimens, and v) has low reagent costs.15Hömig-Hölzel C. Savola S. Multiplex ligation-dependent probe amplification (MLPA) in tumor diagnostics and prognostics.Diagn Mol Pathol. 2012; 21: 189-206Crossref PubMed Scopus (110) Google Scholar, 16Atanesyan L. Steenkamer M.J. Horstman A. Moelans C.B. Schouten J.P. Savola S.P. Optimal fixation conditions and DNA extraction methods for MLPA analysis on FFPE tissue-derived DNA.Am J Clin Pathol. 2017; 147: 60-68PubMed Google Scholar In addition, it does not require highly specialized equipment, only a thermocycler and a capillary electrophoresis instrument. MLPA has extensively been tested on clinical patient samples in various hematological malignancies, such as acute lymphoblastic leukemia, chronic lymphocytic leukemia, follicular lymphoma, chronic myeloid leukemia, myelodysplastic syndrome, and acute myeloid leukemia.17Buijs A. Krijtenburg P.J. Meijer E. Detection of risk-identifying chromosomal abnormalities and genomic profiling by multiplex ligation-dependent probe amplification in chronic lymphocytic leukemia.Haematologica. 2006; 91: 1434-1435PubMed Google Scholar, 18Coll-Mulet L. Santidrián A.F. Cosialls A.M. Iglesias-Serret D. de Frias M. Grau J. Menoyo A. González-Barca E. Pons G. Domingo A. Gil J. Multiplex ligation-dependent probe amplification for detection of genomic alterations in chronic lymphocytic leukaemia.Br J Haematol. 2008; 142: 793-801Crossref PubMed Scopus (39) Google Scholar, 19Fabris S. Scarciolla O. Morabito F. Cifarelli R.A. Dininno C. Cutrona G. Matis S. Recchia A.G. Gentile M. Ciceri G. Ferrarini M. Ciancio A. Mannarella C. Neri A. Fragasso A. Multiplex ligation-dependent probe amplification and fluorescence in situ hybridization to detect chromosomal abnormalities in chronic lymphocytic leukemia: a comparative study.Genes Chromosomes Cancer. 2011; 50: 726-734Crossref PubMed Scopus (21) Google Scholar, 20Donahue A.C. Abdool A.K. Gaur R. Wohlgemuth J.G. Yeh C.H. Multiplex ligation-dependent probe amplification for detection of chromosomal abnormalities in myelodysplastic syndrome and acute myeloid leukemia.Leuk Res. 2011; 35: 1477-1483Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 21Schwab C.J. Jones L.R. Morrison H. Ryan S.L. Yigittop H. Schouten J.P. Harrison C.J. Evaluation of multiplex ligation-dependent probe amplification as a method for the detection of copy number abnormalities in B-cell precursor acute lymphoblastic leukemia.Genes Chromosomes Cancer. 2010; 49: 1104-1113Crossref PubMed Scopus (95) Google Scholar, 22Alpar D. de Jong D. Savola S. Yigittop H. Kajtar B. Kereskai L. Pajor L. Szuhai K. MLPA is a powerful tool for detecting lymphoblastic transformation in chronic myeloid leukemia and revealing the clonal origin of relapse in pediatric acute lymphoblastic leukemia.Cancer Genet. 2012; 205: 465-469Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar We reported the first MLPA study in MM, and the robustness of the approach was later confirmed by other groups.23Alpar D. de Jong D. Holczer-Nagy Z. Kajtar B. Savola S. Jakso P. David M. Kosztolanyi S. Kereskai L. Pajor L. Szuhai K. Multiplex ligation-dependent probe amplification and fluorescence in situ hybridization are complementary techniques to detect cytogenetic abnormalities in multiple myeloma.Genes Chromosomes Cancer. 2013; 52: 785-793Crossref PubMed Scopus (15) Google Scholar, 24Boyle E.M. Proszek P.Z. Kaiser M.F. Begum D. Dahir N. Savola S. Wardell C.P. Leleu X. Ross F.M. Chiecchio L. Cook G. Drayson M.T. Owen R.G. Ashcroft J.M. Jackson G.H. Anthony Child J. Davies F.E. Walker B.A. Morgan G.J. A molecular diagnostic approach able to detect the recurrent genetic prognostic factors typical of presenting myeloma.Genes Chromosomes Cancer. 2015; 54: 91-98Crossref PubMed Scopus (28) Google Scholar, 25Zang M. Zou D. Yu Z. Li F. Yi S. Ai X. Qin X. Feng X. Zhou W. Xu Y. Li Z. Hao M. Sui W. Deng S. Acharya C. Zhao Y. Ru K. Qiu L. An G. Detection of recurrent cytogenetic aberrations in multiple myeloma: a comparison between MLPA and iFISH.Oncotarget. 2015; 6: 34276-34287Crossref PubMed Scopus (10) Google Scholar The number of genomic loci that can simultaneously be analyzed in one conventional MLPA reaction is, however, limited to 55 to 60, typically including at least eight silent reference regions that are needed for data normalization and interpretation of the results.15Hömig-Hölzel C. Savola S. Multiplex ligation-dependent probe amplification (MLPA) in tumor diagnostics and prognostics.Diagn Mol Pathol. 2012; 21: 189-206Crossref PubMed Scopus (110) Google Scholar Recently, a novel technique, called digitalMLPA, has been developed; this technique combines the advantages of NGS and MLPA.26Benard-Slagter A. Zondervan I. de Groot K. Ghazavi F. Sarhadi V. Van Vlierberghe P. De Moerloose B. Schwab C. Vettenranta K. Harrison C.J. Knuutila S. Schouten J. Lammens T. Savola S. Digital multiplex ligation-dependent probe amplification for detection of key copy number alterations in T- and B-cell lymphoblastic leukemia.J Mol Diagn. 2017; 19: 659-672Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar DigitalMLPA works on the basis of the same principles as conventional MLPA, with the difference that specific adapters allowing sequencing of the amplified probes on Illumina (San Diego, CA) instruments are attached to the PCR products during the protocol. After sequencing, the copy number status of the loci of interest is determined by relative read number quantification of the various amplicons. A major advantage of digitalMLPA is the greatly increased number of probes, allowing the investigation of hundreds of genomic loci in a single reaction. In this study, we explored the power of digitalMLPA to detect both subchromosomal and chromosomal CNAs in MM by comparing its performance with interphase FISH (iFISH) and conventional MLPA. Our results suggest that digitalMLPA is a robust and fast high-throughput technique to detect disease-relevant key CNAs and known point mutations; hence, it could efficiently be included in the routine workflow of myeloma diagnostics. Diagnostic bone marrow samples from 56 patients diagnosed with multiple myeloma on the basis of the International Myeloma Working Group's criteria were investigated in this study.27Rajkumar S.V. Dimopoulos M.A. Palumbo A. Blade J. Merlini G. Mateos M.V. Kumar S. Hillengass J. Kastritis E. Richardson P. Landgren O. Paiva B. Dispenzieri A. Weiss B. LeLeu X. Zweegman S. Lonial S. Rosinol L. Zamagni E. Jagannath S. Sezer O. Kristinsson S.Y. Caers J. Usmani S.Z. Lahuerta J.J. Johnsen H.E. Beksac M. Cavo M. Goldschmidt H. Terpos E. Kyle R.A. Anderson K.C. Durie B.G. Miguel J.F. International Myeloma Working Group updated criteria for the diagnosis of multiple myeloma.Lancet Oncol. 2014; 15: e538-e548Abstract Full Text Full Text PDF PubMed Scopus (2584) Google Scholar Immunophenotypic characterization, including CD45-PerCP-Cy5.5, CD19-phosphatidylethanolamine-Cy7, CD38–fluorescein isothiocyanate, CD138-allophycocyanin, and CD56-phosphatidylethanolamine markers, was performed by five-color flow cytometry (CyFlow space; Partec GmbH, Görlitz, Germany). In cases where plasma cell purity decreased below 20% to 30%, immunomagnetic cell enrichment (BD IMag; BD Biosciences, San Jose, CA) was applied, according to the manufacturer's instructions, using CD56 or CD138 antibody, depending on the CD56 expression of the myeloma cells. This was followed by a second immunophenotyping based on CD38 and CD138 expressions to assess the efficacy of the enrichment. Peripheral blood samples from healthy volunteers and bone marrow samples from patients without malignant disorder were used as negative controls for all genetic analyses. Ethical committee approval and written informed consent from the patients were obtained for the study, which was conducted in accordance with the Declaration of Helsinki. Genomic DNA was extracted from fixed cells stored in 70% ethanol using a Biorobot EZ1 system (Qiagen, Valencia, CA), also considering and avoiding potential pitfalls that could have had an adverse effect on the performance of digitalMLPA and MLPA protocols (http://www.mlpa.com, last accessed August 31, 2018). DNA (40 ng) was subjected to each digitalMLPA reaction using a test version of the D006 probemix (lot X1-0613), which has recently been developed by MRC-Holland (Amsterdam, the Netherlands), and provided to collaborating laboratories for testing and validation. The probemix included the following: i) 268 target probes for regions recurrently altered by CNAs in MM; ii) one probe for the specific detection of the BRAFV600E mutation; iii) 105 reference probes hybridizing to copy number stable regions; and iv) 128 internal control probes for sample identification, as well as quantity and quality assessment (Supplemental Table S1). Reference probes were used for data normalization and, together with a subset of the target probes, for the identification of whole chromosome gains and losses. This karyotyping set of probes covered all chromosomes at 194 different loci near the telomere, the centromere, or the middle of the arm. The digitalMLPA protocol started with mixing sample DNA with unique barcode solution, followed by sample denaturation and the addition of digitalMLPA probes with buffer to the mixture. Each probe comprised two or three oligonucleotides with a specific 25- to 50-bp hybridizing sequence. Probe oligonucleotides binding to a specific locus were designed to hybridize adjacently; therefore, if perfectly hybridized, they could be ligated into a single molecule using ligase-65 enzyme. Each ligated probe was then amplified by a universal primer pair compatible with all Illumina sequencing platforms. After amplification, sample-specific products from different reactions were pooled, diluted, denatured, and loaded into a MiSeq v3 standard flow cell (Illumina) for 115-bp single-read sequencing. After quality assessment of the exported FASTQ files, assignment of the reads to digitalMLPA probes and subsequent data analysis were performed in two main consecutive steps using in-house software (MRC-Holland).26Benard-Slagter A. Zondervan I. de Groot K. Ghazavi F. Sarhadi V. Van Vlierberghe P. De Moerloose B. Schwab C. Vettenranta K. Harrison C.J. Knuutila S. Schouten J. Lammens T. Savola S. Digital multiplex ligation-dependent probe amplification for detection of key copy number alterations in T- and B-cell lymphoblastic leukemia.J Mol Diagn. 2017; 19: 659-672Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar First, the read number for each probe was normalized by the median read number generated from reference probes hybridizing to usually conservative regions in the same genome (intrasample normalization). The relative read number generated for each probe was then compared with the matching values obtained in all reference samples (intersample normalization). The final probe ratio value, called dosage quotient, was around 1.0 if the region of interest was unaffected by CNA, whereas an increased or decreased value indicated the presence and level of gain or loss, respectively. Tumor cell purity, as measured by flow cytometry, was also considered at the interpretation of the results. For example, a dosage quotient of 0.6 was interpreted as monoallelic or biallelic loss if the plasma cell ratio in the sample was 80% or 40%, respectively. In addition, a normal dosage quotient range (average ± 3 SDs) was calculated for each digitalMLPA probe on the basis of values measured in the negative control samples. In patient samples, only dosage quotient values falling outside this normal range were considered as losses or gains. CNAs were interpreted as being subclonal if multiple consecutive probes had dosage quotients clearly outside the normal range but without reaching the expected level of monoallelic loss, as calculated on the basis of plasma cell purity and compared with other affected loci within the same specimen. Oligonucleotides of the digitalMLPA probe, designed to detect the V600E mutation of the BRAF kinase gene, were only ligated; thus, the corresponding sequencing reads were produced if the point mutation was present in the sample. Detailed general laboratory and bioinformatic protocols have recently been published.26Benard-Slagter A. Zondervan I. de Groot K. Ghazavi F. Sarhadi V. Van Vlierberghe P. De Moerloose B. Schwab C. Vettenranta K. Harrison C.J. Knuutila S. Schouten J. Lammens T. Savola S. Digital multiplex ligation-dependent probe amplification for detection of key copy number alterations in T- and B-cell lymphoblastic leukemia.J Mol Diagn. 2017; 19: 659-672Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar MLPA reactions were performed as previously described.23Alpar D. de Jong D. Holczer-Nagy Z. Kajtar B. Savola S. Jakso P. David M. Kosztolanyi S. Kereskai L. Pajor L. Szuhai K. Multiplex ligation-dependent probe amplification and fluorescence in situ hybridization are complementary techniques to detect cytogenetic abnormalities in multiple myeloma.Genes Chromosomes Cancer. 2013; 52: 785-793Crossref PubMed Scopus (15) Google Scholar Briefly, 150 ng input genomic DNA was denatured and hybridized using the SALSA P425 version A1 probemix (MRC-Holland) containing 42 probes targeting regions affected by recurrent CNAs of prognostic significance in MM: 1p32 (FAF1, CDKN2C, PLPP3, and DAB1), 1p21, 1q21.3 (CKS1B), 1q23.3, 5q31.3, 12p13.31, 13q14 (RB1 and DLEU1/DLEU2), 16q12 (CYLD), 16q23 (WWOX), and 17p13 (TP53) (Supplemental Table S2). The reactions, including negative control samples, were performed according to the vendor's instructions. The amplified probes were analyzed using an ABI 3730 DNA analyzer (Life Technologies, Bleiswijk, the Netherlands) and GeneMarker software version 1.95 (SoftGenetics, LLC, State College, PA). After intrasample and intersample normalizations, copy number status at each locus was estimated, as described previously,21Schwab C.J. Jones L.R. Morrison H. Ryan S.L. Yigittop H. Schouten J.P. Harrison C.J. Evaluation of multiplex ligation-dependent probe amplification as a method for the detection of copy number abnormalities in B-cell precursor acute lymphoblastic leukemia.Genes Chromosomes Cancer. 2010; 49: 1104-1113Crossref PubMed Scopus (95) Google Scholar also considering the tumor cell ratio measured by flow cytometry and the variability of probe performance in the negative control samples, as mentioned above. iFISH was performed on archived cells fixed in Carnoy solution. Selected DNA loci were in situ stained for visualizing Δ13 (LSI D13S319 Spectrum Orange/13q34 Spectrum Green), deletion of the TP53 gene (LSI TP53 SO/CEP17 SG), abnormalities of chromosome 5 (LSI D5S23 SG/EGR1 5q31 SO), disruption of the immunoglobulin heavy chain gene (IGH) locus (LSI IGH DC BA), and, in case of positivity for the latter aberration, for the specific identification of IGH translocations most frequently occurring in MM: LSI IGH/FGFR3 DC DF, LSI IGH/CCND1 XT DC DF, and LSI IGH/MAF DC DF (Vysis, Downers Grove, IL). FISH procedures were performed following the vendor's recommendations, and the signal pattern of 200 nuclei was evaluated using Zeiss Axioplan2 (ie, MOT; Metasystems, Altlussheim, Germany) and Zeiss AxioImager A1 microscopes (Carl Zeiss Technika Kft, Budapest, Hungary). For the cell-based detection of loss(1p) and gain(1q), bacterial arteficial chromosome clones specific for regions 1p32, 1p21, and 1q21 were selected, labeled, and validated, as described previously.23Alpar D. de Jong D. Holczer-Nagy Z. Kajtar B. Savola S. Jakso P. David M. Kosztolanyi S. Kereskai L. Pajor L. Szuhai K. Multiplex ligation-dependent probe amplification and fluorescence in situ hybridization are complementary techniques to detect cytogenetic abnormalities in multiple myeloma.Genes Chromosomes Cancer. 2013; 52: 785-793Crossref PubMed Scopus (15) Google Scholar FISH signal patterns were evaluated by following the European Myeloma Network's recommendations.28Ross F.M. Avet-Loiseau H. Ameye G. Gutiérrez N.C. Liebisch P. O'Connor S. Dalva K. Fabris S. Testi A.M. Jarosova M. Hodkinson C. Collin A. Kerndrup G. Kuglik P. Ladon D. Bernasconi P. Maes B. Zemanova Z. Michalova K. Michau L. Neben K. Hermansen N.E. Rack K. Rocci A. Protheroe R. Chiecchio L. Poirel H.A. Sonneveld P. Nyegaard M. Johnsen H.E. European Myeloma NetworkReport from the European Myeloma Network on interphase FISH in multiple myeloma and related disorders.Haematologica. 2012; 97: 1272-1277Crossref PubMed Scopus (219) Google Scholar All patient samples were screened by two independent investigators (B.K. and D.A.). Detection and quantification of V600E substitution in the BRAF kinase gene was performed with the PyroMark Q24 system (Qiagen GmbH, Hilden, Germany) using 100 ng input DNA and following the vendor's protocol. Codon 600 was amplified, followed by immobilization of the biotinylated PCR products on streptavidin-coated Sepharose High Performance beads. After bead capture on a vacuum workstation and multiple washing steps, single-stranded DNA suitable for pyrosequencing was generated by denaturation. Sequencing was performed in the reverse direction, producing a CAC wild-type sequence in normal samples and a CTC genotype in case of V600E mutation. Results were analyzed using the PyroMark Q24 software version 2.0.8. Validation of the BRAFV600E mutation with an increased sensitivity was achieved by using droplet digital PCR. The reaction was performed with 50 ng input DNA and with commercially available BRAF assays specific for the wild-type (dHsaCP2000028) and mutant (dHsaCP2000027) targets, following the manufacturer's protocol. Droplets were generated by the QX200 Droplet Generator, and reading was completed with the QX200 Droplet Digital PCR system (Bio-Rad Laboratories, Hercules, CA). Results were analyzed using the QuantaSoft software version 1.7.4.0917 (Bio-Rad Laboratories). The BRAFmut allelic burden was determined as fractional abundance (FA) on the basis of the percentage ratio between the number of mutant DNA molecules (a) and the number of mutant (a) plus wild-type (b) molecules detected: FA=a/(a+b). Congruency between digitalMLPA results and iFISH as well as MLPA data was evaluated with Fisher's exact test using the SPSS 15.0 software (SPSS Inc., Chicago, IL). DigitalMLPA provided informative results at 372 genomic loci for each patient sample. Two target probes showed an SD of >0.09 across the negative control samples; therefore, they have been excluded from the downstream analysis. The laboratory protocol was completed within 24 hours, including overnight hybridization of the digitalMLPA probes as well as loading of the pooled libraries into the MiSeq instrument. Using a standard flow cell with v3 chemistry, the 115-bp single-read run took approximately 9.5 hours and produced on average 1169 ± 184 sequencing reads per probe. In total, 210 whole chromosome aberrations were identified by digitalMLPA, including 65 losses and 145 gains in 47 patients (84%). Monosomy 13 proved to be the most abundant alteration, with most defects occurring among the nonhyperdiploid cases. Most (94%) of trisomies were observed in the 20 patients displaying a hyperdiploid karyotype (Figure 1). In these cases, additional copies of odd-number chromosomes were predominantly detected (Supplemental Figure S1), with chromosomes 3 and 9 displaying trisomy the most commonly, followed by chromosomes 11, 19, 15, 5, 7, and 21 (Supplemental Figure S2). DigitalMLPA unveiled 246 subchromosomal CNAs (Supplemental Figure S3 and Supplemental Table S3). Gain(1q) was the most frequently occurring lesion among those detected in more than three patients, followed by loss(1p), loss(8p), loss(16q), loss(12p), loss(14q), gain(8q), gain(Xq), loss(13q), loss(6q), gain(14q), loss(17p), loss(20p), loss(22q), loss(5q), gain(6p), and gain(9q) (Table 1). The average number of CNAs per case was 4.4 (range, 0 to 13), with 3.7 in the hyperdiploid subgroup and 4.8 among the nonhyperdiploid patients. DigitalMLPA detected at least one subchromosomal CNA in 53 of 56 cases (95%), with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, and 13 aberrations identified in 9, 7, 7, 4, 8, 6, 4, 1, 1, 4, 1, and 1 case, respectively (Supplemental Table S3). In 10 patients, biallelic del" @default.
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