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Ultrasensitive Detection of NOTCH1 c.7544_7545delCT Mutations in Chronic Lymphocytic Leukemia by Droplet Digital PCR Reveals High Frequency of Subclonal Mutations and Predicts Clinical Outcome in Cases with Trisomy 12

2020; Elsevier BV; Volume: 22; Issue: 4 Linguagem: Inglês

10.1016/j.jmoldx.2020.01.008

1943-7811

Catherine Hoofd, Steven J.T. Huang, Samuel Gusscott, Sonya H.L. Lam, Rachel Wong, A.J.B. Johnston, Susana Ben‐Neriah, Christian Steidl, David W. Scott, Hélène Bruyère, Tanya L. Gillan, Cynthia L. Toze, Alina S. Gerrie, Andrew P. Weng,

Lymphoma Diagnosis and Treatment

NOTCH1 is recurrently mutated in chronic lymphocytic leukemia (CLL), most commonly as a 2-bp frameshift deletion (c.7541_7542delCT). This mutated allele encodes a truncated form of the receptor (p.P2514Rfs∗4) lacking the C-terminal proline, glutamic acid, serine, and threonine (PEST) degradation domain that increases NOTCH1 signaling duration. NOTCH1 mutation has been associated with poor clinical outcomes in CLL. We validated a highly sensitive and quantitative droplet digital PCR assay for the NOTCH1 delCT mutation, which was anticipated to perform well compared with Sanger sequencing and allele-specific PCR. Performance characteristics of this assay were tested on 126 samples from an unselected CLL cohort and a separate cohort of 85 samples from patients with trisomy 12 CLL. The delCT mutation was detected at allele frequencies as low as 0.024%; 25% of unselected cases and 55% of trisomy 12 cases were positive at the 0.024% detection threshold. Mutational burdens ≥1% were significantly associated with shorter overall survival (OS) in patients with trisomy 12+ disease in multivariate analysis (median OS, 9.1 versus 13 years, with hazard ratio of 2.34; P = 0.031). Mutational burdens <1% correlated with shorter OS in univariate, but not multivariate, analyses. These results suggest that droplet digital PCR testing for NOTCH1 delCT mutation may aid in risk stratification and/or disease monitoring in certain subsets of patients with CLL. NOTCH1 is recurrently mutated in chronic lymphocytic leukemia (CLL), most commonly as a 2-bp frameshift deletion (c.7541_7542delCT). This mutated allele encodes a truncated form of the receptor (p.P2514Rfs∗4) lacking the C-terminal proline, glutamic acid, serine, and threonine (PEST) degradation domain that increases NOTCH1 signaling duration. NOTCH1 mutation has been associated with poor clinical outcomes in CLL. We validated a highly sensitive and quantitative droplet digital PCR assay for the NOTCH1 delCT mutation, which was anticipated to perform well compared with Sanger sequencing and allele-specific PCR. Performance characteristics of this assay were tested on 126 samples from an unselected CLL cohort and a separate cohort of 85 samples from patients with trisomy 12 CLL. The delCT mutation was detected at allele frequencies as low as 0.024%; 25% of unselected cases and 55% of trisomy 12 cases were positive at the 0.024% detection threshold. Mutational burdens ≥1% were significantly associated with shorter overall survival (OS) in patients with trisomy 12+ disease in multivariate analysis (median OS, 9.1 versus 13 years, with hazard ratio of 2.34; P = 0.031). Mutational burdens 30% of cases that had undergone Richter transformation to a higher histologic grade, most commonly diffuse large B-cell lymphoma.17Fabbri G. Khiabanian H. Holmes A.B. Wang J. Messina M. Mullighan C.G. Pasqualucci L. Rabadan R. Dalla-Favera R. Genetic lesions associated with chronic lymphocytic leukemia transformation to Richter syndrome.J Exp Med. 2013; 210: 2273-2288Crossref PubMed Scopus (216) Google Scholar Additional studies have demonstrated enrichment for NOTCH1 mutations in CLL patients harboring trisomy 12 (25% to 40%).18Balatti V. Bottoni A. Palamarchuk A. Alder H. Rassenti L.Z. Kipps T.J. Pekarsky Y. Croce C.M. NOTCH1 mutations in CLL associated with trisomy 12.Blood. 2012; 119: 329-331Crossref PubMed Scopus (176) Google Scholar,19Del Giudice I. Rossi D. Chiaretti S. Marinelli M. Tavolaro S. Gabrielli S. Laurenti L. Marasca R. Rasi S. Fangazio M. Guarini A. Gaidano G. Foà R. NOTCH1 mutations in +12 chronic lymphocytic leukemia (CLL) confer an unfavorable prognosis, induce a distinctive transcriptional profiling and refine the intermediate prognosis of +12 CLL.Haematologica. 2012; 97: 437-441Crossref PubMed Scopus (163) Google Scholar Several groups have shown that NOTCH1 mutations are associated with poor clinical outcomes in CLL,12Fabbri G. Rasi S. Rossi D. Trifonov V. Khiabanian H. Ma J. Grunn A. Fangazio M. Capello D. Monti S. Cresta S. Gargiulo E. Forconi F. Guarini A. Arcaini L. Paulli M. Laurenti L. Larocca L.M. Marasca R. Gattei V. Oscier D. Bertoni F. Mullighan C.G. Foá R. Pasqualucci L. Rabadan R. Dalla-Favera R. Gaidano G. Analysis of the chronic lymphocytic leukemia coding genome: role of NOTCH1 mutational activation.J Exp Med. 2011; 208: 1389-1401Crossref PubMed Scopus (507) Google Scholar,13Puente X.S. Pinyol M. Quesada V. Conde L. Ordonez G.R. Villamor N. et al.Whole-genome sequencing identifies recurrent mutations in chronic lymphocytic leukaemia.Nature. 2011; 475: 101-105Crossref PubMed Scopus (1224) Google Scholar,19Del Giudice I. Rossi D. Chiaretti S. Marinelli M. Tavolaro S. Gabrielli S. Laurenti L. Marasca R. Rasi S. Fangazio M. Guarini A. Gaidano G. Foà R. NOTCH1 mutations in +12 chronic lymphocytic leukemia (CLL) confer an unfavorable prognosis, induce a distinctive transcriptional profiling and refine the intermediate prognosis of +12 CLL.Haematologica. 2012; 97: 437-441Crossref PubMed Scopus (163) Google Scholar, 20Rossi D. Rasi S. Fabbri G. Spina V. Fangazio M. Forconi F. Marasca R. Laurenti L. Bruscaggin A. Cerri M. Monti S. Cresta S. Famà R. De Paoli L. Bulian P. Gattei V. Guarini A. Deaglio S. Capello D. Rabadan R. Pasqualucci L. Dalla-Favera R. Foà R. Gaidano G. Mutations of NOTCH1 are an independent predictor of survival in chronic lymphocytic leukemia.Blood. 2012; 119: 521-529Crossref PubMed Scopus (369) Google Scholar, 21Sportoletti P. Baldoni S. Cavalli L. Del Papa B. Bonifacio E. Ciurnelli R. Bell A.S. Di Tommaso A. Rosati E. Crescenzi B. Mecucci C. Screpanti I. Marconi P. Martelli M.F. Di Ianni M. Falzetti F. NOTCH1 PEST domain mutation is an adverse prognostic factor in B-CLL.Br J Haematol. 2010; 151: 404-406Crossref PubMed Scopus (95) Google Scholar whereas others have found such associations to be less clear.22Baliakas P. Hadzidimitriou A. Sutton L.A. Rossi D. Minga E. Villamor N. Larrayoz M. Kminkova J. Agathangelidis A. Davis Z. Tausch E. Stalika E. Kantorova B. Mansouri L. Scarfo L. Cortese D. Navrkalova V. Rose-Zerilli M.J. Smedby K.E. Juliusson G. Anagnostopoulos A. Makris A.M. Navarro A. Delgado J. Oscier D. Belessi C. Stilgenbauer S. Ghia P. Pospisilova S. Gaidano G. Campo E. Strefford J.C. Stamatopoulos K. Rosenquist R. European Research Initiative on CLLRecurrent mutations refine prognosis in chronic lymphocytic leukemia.Leukemia. 2015; 29: 329-336Crossref PubMed Scopus (236) Google Scholar, 23Chiaretti S. Marinelli M. Del Giudice I. Bonina S. Piciocchi A. Messina M. Vignetti M. Rossi D. Di Maio V. Mauro F.R. Guarini A. Gaidano G. Foà R. NOTCH1, SF3B1, BIRC3 and TP53 mutations in patients with chronic lymphocytic leukemia undergoing first-line treatment: correlation with biological parameters and response to treatment.Leuk Lymphoma. 2014; 55: 2785-2792Crossref PubMed Scopus (38) Google Scholar, 24Lionetti M. Fabris S. Cutrona G. Agnelli L. Ciardullo C. Matis S. Ciceri G. Colombo M. Maura F. Mosca L. Gentile M. Recchia Anna G. Ilariucci F. Musolino C. Molica S. Di Raimondo F. Cortelezzi A. Rossi D. Gaidano G. Morabito F. Ferrarini M. Neri A. High-throughput sequencing for the identification of NOTCH1 mutations in early stage chronic lymphocytic leukaemia: biological and clinical implications.Br J Haematol. 2014; 165: 629-639Crossref PubMed Scopus (48) Google Scholar Furthermore, it was reported that NOTCH1 mutations in CLL associate with poorer response to rituximab therapy when given in the context of fludarabine plus cyclophosphamide,25Stilgenbauer S. Schnaiter A. Paschka P. Zenz T. Rossi M. Döhner K. Bühler A. Böttcher S. Ritgen M. Kneba M. Winkler D. Tausch E. Hoth P. Edelmann J. Mertens D. Bullinger L. Bergmann M. Kless S. Mack S. Jäger U. Patten N. Wu L. Wenger M.K. Fingerle-Rowson G. Lichter P. Cazzola M. Wendtner C.M. Fink A.M. Fischer K. Busch R. Hallek M. Döhner H. Gene mutations and treatment outcome in chronic lymphocytic leukemia: results from the CLL8 trial.Blood. 2014; 123: 3247-3254Crossref PubMed Scopus (386) Google Scholar an unexpected finding that may be related to lower levels of CD20 expression on the cell surface.26Pozzo F. Bittolo T. Arruga F. Bulian P. Macor P. Tissino E. Gizdic B. Rossi F.M. Bomben R. Zucchetto A. Benedetti D. Degan M. D'Arena G. Chiarenza A. Zaja F. Pozzato G. Rossi D. Gaidano G. Del Poeta G. Deaglio S. Gattei V. Dal Bo M. NOTCH1 mutations associate with low CD20 level in chronic lymphocytic leukemia: evidence for a NOTCH1 mutation-driven epigenetic dysregulation.Leukemia. 2016; 30: 182-189Crossref PubMed Scopus (71) Google Scholar One striking, and as yet unexplained, feature of NOTCH1 mutations in CLL is that >80% are represented by a single dinucleotide deletion, c.7541_7542delCT (COSM12774, COSV53024776) or delCT, that results in frameshift (p.P2514Rfs∗4) and deletion of the PEST domain.12Fabbri G. Rasi S. Rossi D. Trifonov V. Khiabanian H. Ma J. Grunn A. Fangazio M. Capello D. Monti S. Cresta S. Gargiulo E. Forconi F. Guarini A. Arcaini L. Paulli M. Laurenti L. Larocca L.M. Marasca R. Gattei V. Oscier D. Bertoni F. Mullighan C.G. Foá R. Pasqualucci L. Rabadan R. Dalla-Favera R. Gaidano G. Analysis of the chronic lymphocytic leukemia coding genome: role of NOTCH1 mutational activation.J Exp Med. 2011; 208: 1389-1401Crossref PubMed Scopus (507) Google Scholar,13Puente X.S. Pinyol M. Quesada V. Conde L. Ordonez G.R. Villamor N. et al.Whole-genome sequencing identifies recurrent mutations in chronic lymphocytic leukaemia.Nature. 2011; 475: 101-105Crossref PubMed Scopus (1224) Google Scholar The striking preponderance of the delCT mutation in CLL has prompted the development of dedicated assays, such as allele-specific PCR, which have detected this particular mutation in up to 20% of CLL patients at diagnosis.21Sportoletti P. Baldoni S. Cavalli L. Del Papa B. Bonifacio E. Ciurnelli R. Bell A.S. Di Tommaso A. Rosati E. Crescenzi B. Mecucci C. Screpanti I. Marconi P. Martelli M.F. Di Ianni M. Falzetti F. NOTCH1 PEST domain mutation is an adverse prognostic factor in B-CLL.Br J Haematol. 2010; 151: 404-406Crossref PubMed Scopus (95) Google Scholar Even further, a study of 384 cases assayed by next-generation sequencing identified the delCT mutation in 38% of cases,24Lionetti M. Fabris S. Cutrona G. Agnelli L. Ciardullo C. Matis S. Ciceri G. Colombo M. Maura F. Mosca L. Gentile M. Recchia Anna G. Ilariucci F. Musolino C. Molica S. Di Raimondo F. Cortelezzi A. Rossi D. Gaidano G. Morabito F. Ferrarini M. Neri A. High-throughput sequencing for the identification of NOTCH1 mutations in early stage chronic lymphocytic leukaemia: biological and clinical implications.Br J Haematol. 2014; 165: 629-639Crossref PubMed Scopus (48) Google Scholar presumably because of greater sensitivity of next-generation sequencing over allele-specific PCR. This article reports the validation of an ultrasensitive and highly quantitative droplet digital PCR (ddPCR) assay for detection of the NOTCH1 delCT mutation in clinical patient samples and demonstrates its use for stratifying risk in patients with trisomy 12 CLL, where it is highly prevalent. All samples were obtained under approved research ethics board protocols at the British Columbia Cancer Agency (Vancouver, BC, Canada) and University of British Columbia (Vancouver, BC, Canada). MOLT4, P12 Ichikawa, PEER, RPMI 8402, and TALL-1 cell lines were gifted from Dr. Jon Aster (Brigham & Women's Hospital, Boston, MA) and verified by short tandem repeat profiling (Promega PowerPlex 16HS; Genetica DNA Laboratories, Cincinnati, OH). Dual-color FISH analysis was performed using bacterial artificial chromosome probes covering the NOTCH1 locus within 9q34.3 (labeled in spectrum orange) and a control region within 9p21.3 (labeled in spectrum green). Two bacterial artificial chromosome probes were used for each locus: RP11-707O3 (chromosome 9: 138370668 to 138573626) and RP11-370H5 (chromosome 9: 138418844 to 138587998) for the 9q34.3 region and RP11-149I2 (chromosome 9: 021899259 to 022000413) and RP11-478M20 (chromosome 9: 021937304 to 022100179) for the 9p21.3 region. The NOTCH1 locus spans the region chromosome 9: 138508717 to 138560059. A total of 200 interphase nuclei were scored for each cell line. DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, Toronto, ON, Canada) from either live, intact nucleated cells from peripheral blood after ammonium chloride red blood cell lysis (StemCell Technologies, Vancouver, BC, Canada) or methanol/acetic acid (MAA)–fixed nucleated cell pellets remaining after clinical FISH testing.10Gerrie A.S. Huang S.J.T. Bruyere H. Dalal C. Hrynchak M. Karsan A. Ramadan K.M. Smith A.C. Tyson C. Toze C.L. Gillan T.L. Population-based characterization of the genetic landscape of chronic lymphocytic leukemia patients referred for cytogenetic testing in British Columbia, Canada: the role of provincial laboratory standardization.Cancer Genet. 2014; 207: 316-325Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar Commercial predesigned primer sets were selected to amplify both the wild-type (WT) and delCT (COSM12774) alleles of the human NOTCH1 locus, which each produce 65-bp amplicons and include HEX (WT) and FAM (delCT) TaqMan probes for multiplex detection (PrimePCR ddPCR Mutation Assays dHsaCP2500501 and dHsaCP2500500; Bio-Rad, Mississauga, ON, Canada). High-molecular-weight genomic DNA (gDNA) was added to a mixture containing HaeIII restriction enzyme, WT(HEX) and delCT(FAM) primer sets/TaqMan probes, and ddPCR Supermix for Probes (catalog number 1863010; Bio-Rad). Samples were then processed according to the manufacturer's protocol. Briefly, 20-μL reactions were partitioned into at least 12,000-nL droplets with a QX200 Droplet Generator (Bio-Rad), then amplified in a thermal cycler with the following program: 95°C enzyme activation for 10 minutes × 1 cycle, 94°C denaturation for 30 seconds, followed by 55°C annealing/extension for 1 minute × 40 cycles (2.5°C/second ramp rate), and 98°C hold for 10 minutes × 1 cycle. Two-color fluorescence (FAM and HEX) of individual droplets was measured using a QX200 Droplet Reader (Bio-Rad), and absolute DNA concentrations were calculated using QuantaSoft software version 1.7.4.0917 (Bio-Rad). Discrimination between positive and negative droplets was determined by manual gating for droplet events that exhibited fluorescence values at least approximately 10 SDs greater than the mean of the negative population, and not greater than approximately 4 SDs greater than the mean of the positive population. This latter criterion was applied to exclude outlier droplets showing presumed non-specific fluorescence signal. For each reaction, 300 ng gDNA extracted from live, intact cells, or 600 ng gDNA extracted from MAA-fixed cell pellets was inputed. These input DNA amounts were selected empirically on the basis of titration data to target approximately 80% positive droplets in each assay. Statistical analyses were performed using QuantaSoft (Bio-Rad), Prism version 8.0.1 (GraphPad Software Inc., San Diego, CA), and STATA version 12.0 (StataCorp, College Station, TX) software packages. The assay linearity was first assessed using various admixtures of gDNA from two reference T-cell acute lymphoblastic leukemia cell lines: MOLT4, which is heterozygous for the delCT mutation; and P12 Ichikawa, which is homozygous WT at this site.16Weng A.P. Ferrando A.A. Lee W. Morris IV, J.P. Silverman L.B. Sanchez-Irizarry C. Blacklow S.C. Look A.T. Aster J.C. Activating mutations of NOTCH1 in human T cell acute lymphoblastic leukemia.Science. 2004; 306: 269-271Crossref PubMed Scopus (2243) Google Scholar The assay was observed to be highly log-linear over the full range of allele admixtures tested, ranging from 100% down to approximately 1% MOLT4 gDNA (Supplemental Figures S1 and S2). After counting droplets positive for the delCT and/or WT probes, the instrument software outputs a fractional abundance (FA) value with 95% CI based on a Poisson distribution statistical model. To convert this output FA value to a mutant allele frequency (MAF) value, a set of standard curves was generated using mixtures of gDNA from our reference MOLT4 and P12 Ichikawa cell lines (Supplemental Figure S3). Of note, MOLT4 is hypertetraploid (modal n = 98; range, 94 to 101), whereas P12 Ichikawa is hypotetraploid (modal n = 82 to 84)27Drexler H.G. The Leukemia-Lymphoma Cell Line Facts Book. Academic Press, San Diego, CA2000Google Scholar,28Drexler H.G. Guide to Leukemia-Lymphoma Cell Lines.ed 2. DSMZ, Braunschweig, Germany2010Google Scholar and thus a correction factor is necessary to adjust for the difference in ploidy between the two cell lines. FISH was used to quantify the average copies/cell of chromosome 9 (where the NOTCH1 locus resides) in each of the cell lines. An average of 3.74 copies/cell was found in MOLT4, and an average of 2.80 copies/cell was found in P12 Ichikawa, which results in a correction factor of 1.1277 to convert the input mass ratio to copies of chromosome 9/cell ratio (Supplemental Table S1). Human T-cell acute lymphoblastic leukemia cell lines lacking the delCT mutation were used as negative controls to determine the assay false-positive/background rate. Genomic DNA from four different cell lines (P12 Ichikawa, PEER, RPMI 8402, and TALL-1) was assayed a total of 16 times, which yielded one to three positive delCT droplets in four instances and zero in the remaining 12 instances, with an average of 12,841 ± 3019 positive WT droplets (Supplemental Figures S4 and S5). The four instances with positive delCT droplets imputed FA values ranging from 0.003% to 0.006% (95% CI, 0%–0.021%). It was specified that for a sample to be deemed positive for delCT mutation, it must yield an FA value of >0.021%, thus setting the diagnostic threshold above the 95% CI for the highest observed assay background level. An FA value of 0.021% corresponds to an MAF value of 0.024%, and we defined the assay limit of detection at this level. Of note, the lowest assayed concentration of MOLT4/P12 Ichikawa gDNA (ploidy-corrected MAF = 0.0149%) yielding a deemed positive result detected 2 delCT and 7026 WT positive droplets among 17,288 total droplets, with an observed FA of 0.022% (Supplemental Figure S6). Samples with zero delCT positive droplets were reasonably deemed negative; however, they cannot be assigned a nominal MAF value of 0 because one cannot exclude the possibility that the n + 1 droplet could have scored as positive. Accordingly, in cases with zero delCT positive droplets, the assay result was scored as negative, but a nominal MAF value of 1/(number of positive WT droplets) was assigned. For example, a sample with zero delCT positive droplets and 2000 or 200 positive WT droplets was deemed negative, but assigned an MAF value of 0.05% or 0.5%, respectively (Supplemental Figure S7). Similarly, assays yielding FA values below our positivity threshold (FAs ≤0.021%) were scored as negative, and also assigned nominal MAF values of 1/(number of positive WT droplets). In essence, the nominal MAF values for negative assays indicate the sensitivity down to which the negative result is informative. As this study included analysis of DNA extracted from MAA-fixed cell pellets remaining after clinical FISH testing, it was determined whether MAA fixation might have any detrimental effect on the assay background. Accordingly, DNA extracted from MAA-fixed P12 Ichikawa cells were tested, and false-positive droplet events were observed at frequencies in keeping with nonfixed gDNA samples (data not shown). The presumed false-positive rate among actual MAA-fixed patient peripheral blood and bone marrow samples, which were processed per routine handling conditions in the clinical FISH laboratory over the same approximate time interval as those used for the trisomy 12+ cohort (see DelCT Mutation Frequency and Clinical Significance in a Cohort of CLL Patients with Trisomy 12), was also examined. Twenty-five randomly chosen non-CLL patient samples submitted for assessment of hematologic malignancy were identified, and a single delCT positive droplet was detected in six of the 25 samples tested (24%), and none in the rest. In all six cases, the corresponding FA values fell below the previously established 0.021% FA cutoff, and thus are not reliably distinguished from known true-negative cell line controls. The first cohort of cases investigated consisted of 126 patient samples, including 107 samples that were referred to the British Columbia Cancer Agency Clinical Flow Cytometry Laboratory for primary investigation of lymphocytosis/lymphadenopathy or follow-up of known small lymphocytic lymphoma/CLL/monoclonal B-lymphocytosis, and 19 samples that were obtained from the Hematology Cell Bank of British Columbia. Samples originating from the Brit