Clinical validation and implementation of droplet digital PCR for the detection of BRAF mutations from cell-free DNA
Rainier ArnoldaKerryn HowlettTimmy ChanJeanette RaleighAthena HatzimihalisAnthony BellAndrew FellowesShahneen SandhuGrant A. McArthurStephen B. FoxSarah‐Jane DawsonChelsee HewittKate JonesStephen Q. Wong
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BackgroundA high percentage of patients diagnosed with localized colon cancer (CC) will relapse after curative treatment. Although pathological staging currently guides our treatment decisions, there are no biomarkers determining minimal residual disease (MRD) and patients are at risk of being undertreated or even overtreated with chemotherapy in this setting. Circulating-tumor DNA (ctDNA) can to be a useful tool to better detect risk of relapse.Patients and methodsOne hundred and fifty patients diagnosed with localized CC were prospectively enrolled in our study. Tumor tissue from those patients was sequenced by a custom-targeted next-generation sequencing (NGS) panel to characterize somatic mutations. A minimum variant allele frequency (VAF) of 5% was applied for variant filtering. Orthogonal droplet digital PCR (ddPCR) validation was carried out. We selected known variants with higher VAF to track ctDNA in the plasma samples by ddPCR.ResultsNGS found known pathological mutations in 132 (88%) primary tumors. ddPCR showed high concordance with NGS (r-=-0.77) for VAF in primary tumors. Detection of ctDNA after surgery and in serial plasma samples during follow-up were associated with poorer disease-free survival (DFS) [hazard ratio (HR), 17.56; log-rank P-=-0.0014 and HR, 11.33; log-rank P-=-0.0001, respectively]. Tracking at least two variants in plasma increased the ability to identify MRD to 87.5%. ctDNA was the only significantly independent predictor of DFS in multivariable analysis. In patients treated with adjuvant chemotherapy, presence of ctDNA after therapy was associated with early relapse (HR 10.02; log-rank P-<-0.0001). Detection of ctDNA at follow-up preceded radiological recurrence with a median lead time of 11.5-months.ConclusionsPlasma postoperative ctDNA detected MRD and identified patients at high risk of relapse in localized CC. Mutation tracking with more than one variant in serial plasma samples improved our accuracy in predicting MRD.
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Recent studies evaluated the diagnostic accuracy of circulating tumor DNA (ctDNA) analysis in the detection of epidermal growth factor receptor (EGFR) mutations from plasma of NSCLC patients, overall showing a high concordance as compared to standard tissue genotyping. However it is less clear if the location of metastatic site may influence the ability to identify EGFR mutations.This pooled analysis aims to evaluate the association between the metastatic site location and the sensitivity of ctDNA analysis in detecting EGFR mutations in NSCLC patients.Data from all published studies, evaluating the sensitivity of plasma-based EGFRmutation testing, stratified by metastatic site location (extrathoracic (M1b) vs intrathoracic (M1a)) were collected by searching in PubMed, Cochrane Library, American Society of Clinical Oncology, and World Conference of Lung Cancer, meeting proceedings. Pooled Odds ratio (OR) and 95% confidence intervals (95% CIs) were calculated for the ctDNA analysis sensitivity, according to metastatic site location.A total of ten studies, with 1425 patients, were eligible. Pooled analysis showed that the sensitivity of ctDNA-based EGFR-mutation testing is significantly higher in patients with M1b vs M1a disease (OR: 5.09; 95% CIs: 2.93 - 8.84). A significant association was observed for both EGFR-activating (OR: 4.30, 95% CI: 2.35-7.88) and resistant T790M mutations (OR: 11.89, 95% CI: 1.45-97.22), regardless of the use of digital-PCR (OR: 5.85, 95% CI: 3.56-9.60) or non-digital PCR technologies (OR: 2.96, 95% CI: 2.24-3.91).These data suggest that the location of metastatic sites significantly influences the diagnostic accuracy of ctDNA analysis in detecting EGFR mutations in NSCLC patients.
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In IgM monoclonal gammopathies MYD88L265P is a prognostic and predictive biomarker of therapy response. MYD88L265P detection is mainly performed by allele-specific quantitative PCR (ASqPCR), however recently, droplet digital PCR (ddPCR) has been proved to be suitable for MYD88L265P screening and minimal residual disease monitoring (MRD). This study compared ASqPCR and ddPCR to define the most sensitive method for MYD88L265P detection in bone marrow (BM), peripheral blood (PB) sorted or unsorted CD19+ cells, and in plasma cell-free DNA (cfDNA). Overall, the analysis showed a good concordance rate (74%) between the two methods, especially in BM samples, while discordances (26%) were mostly in favor of ddPCR (ddPCR+ vs. ASqPCR-) and were particularly evident in samples with low mutational burden, such as PB and cfDNA. This study highlights ddPCR as a feasible approach for MYD88L265P detection across different specimen types (including cfDNA). Interestingly, its high sensitivity makes CD19+ selection dispensable. On the other hand, our results showed that MYD88L265P detection on PB samples, especially with ASqPCR, is suboptimal for screening and MRD analysis. Finally, significantly different MYD88L265P mutational levels observed between Waldenström Macroglobulinemia and IgM monoclonal gammopathy of undetermined significance patients suggest the need for further studies in order to identify possible correlations between mutational levels and risk of progression to Waldenström.
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e22126 Background: Circulating tumor DNA (ctDNA) from cancer patient plasma is a promising biomarker with applications in screening, treatment selection, therapy response monitoring, minimal residual disease detection, and surveillance. We investigate the value of ctDNA as a biomarker in colorectal cancer to augment clinical decision-making. Methods: We present a ctDNA detection assay simultaneously capable of single molecule sensitivity, specificity to 0.01%– 0.001% mutation abundance versus wild-type, and multiplexing over 100 mutations from a single plasma sample. The employs a proprietary target selection process that removes wild-type DNA prior to library construction to greatly reduce the error rate of DNA sequencing. Using this assay, we analysed plasma from three cohorts of individuals including normal controls (n=13), age-matched normal controls (n=17), and colorectal cancer patients (n=17) across all stages of disease whose blood was drawn prior to treatment or surgery, and whose diagnosis confirms the presence of a tumor. Tissue from the primary tumor biopsy was also collected and analysed with the OnTarget assay. Results: We find a high correlation between the tumor mutational profile and the plasma mutation profile even for early stage patients demonstrating high sensitivity (92% plasma-tissue concordance over all patients) and exceptional specificity (100%: no false positives in normal cohorts). Additionally, mutations deemed from clinical evidence to originate from distant metastatic sites are detected in the plasma, highlighting the promise of the liquid biopsy approach for application in clinical care. We present concordance, sensitivity, and specificity results from the colorectal cancer patient cohort and the cohort of healthy individuals. Conclusions: Initial assay results from 30 healthy individuals and 17 CRC patients indicate that OnTarget detection of ctDNA provides an exceptionally specific assay for the presence of colorectal tumors and may have utility as a biomarker to guide colorectal cancer management across early-stage and metastatic disease.
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Digital droplet PCR (ddPCR) is an implementation of conventional PCR, with the potential of overcoming some limitations of real-time quantitative PCR (RQ-PCR). To evaluate if ddPCR may improve the quantification of disease levels and refine patients' risk stratification, 116 samples at four time points from 44 (35 B-lineage and 9 T-lineage) adult Philadelphia-negative acute lymphoblastic leukemia patients enrolled in the GIMEMA LAL1913 protocol were analyzed by RQ-PCR and ddPCR. A concordance rate between RQ-PCR and ddPCR of 79% (P < 0.0001) was observed; discordances were identified in 21% of samples, with the majority being RQ-PCR-negative (NEG) or positive not quantifiable (PNQ). ddPCR significantly reduced the proportion of PNQ samples—2.6% versus 14% (P = 0.003)—and allowed disease quantifiability in 6.6% of RQ-PCR-NEG, increasing minimal residual disease quantification in 14% of samples. Forty-seven samples were also investigated by next-generation sequencing, which confirmed the ddPCR results in samples classified as RQ-PCR-PNQ or NEG. By reclassifying samples on the basis of the ddPCR results, a better event-free survival stratification of patients was observed compared to RQ-PCR; indeed, ddPCR captured more true-quantifiable samples, with five relapses occurring in three patients who resulted RQ-PCR-PNQ/NEG but proved ddPCR positive quantifiable. At variance, no relapses were recorded in patients whose follow-up samples were RQ-PCR-PNQ but reclassified as ddPCR-NEG. A broader application of ddPCR in acute lymphoblastic leukemia clinical trials will help to improve patients' stratification. Digital droplet PCR (ddPCR) is an implementation of conventional PCR, with the potential of overcoming some limitations of real-time quantitative PCR (RQ-PCR). To evaluate if ddPCR may improve the quantification of disease levels and refine patients' risk stratification, 116 samples at four time points from 44 (35 B-lineage and 9 T-lineage) adult Philadelphia-negative acute lymphoblastic leukemia patients enrolled in the GIMEMA LAL1913 protocol were analyzed by RQ-PCR and ddPCR. A concordance rate between RQ-PCR and ddPCR of 79% (P < 0.0001) was observed; discordances were identified in 21% of samples, with the majority being RQ-PCR-negative (NEG) or positive not quantifiable (PNQ). ddPCR significantly reduced the proportion of PNQ samples—2.6% versus 14% (P = 0.003)—and allowed disease quantifiability in 6.6% of RQ-PCR-NEG, increasing minimal residual disease quantification in 14% of samples. Forty-seven samples were also investigated by next-generation sequencing, which confirmed the ddPCR results in samples classified as RQ-PCR-PNQ or NEG. By reclassifying samples on the basis of the ddPCR results, a better event-free survival stratification of patients was observed compared to RQ-PCR; indeed, ddPCR captured more true-quantifiable samples, with five relapses occurring in three patients who resulted RQ-PCR-PNQ/NEG but proved ddPCR positive quantifiable. At variance, no relapses were recorded in patients whose follow-up samples were RQ-PCR-PNQ but reclassified as ddPCR-NEG. A broader application of ddPCR in acute lymphoblastic leukemia clinical trials will help to improve patients' stratification. Philadelphia-negative acute lymphoblastic leukemia (Ph− ALL) represents ≥70% of all adult ALL cases. Although the use of pediatric-inspired ALL treatment strategies has led to substantial improvements, about 50% of adults still relapse1Fielding A.K. Richards S.M. Chopra R. Lazarus H.M. Litzow M.R. Buck G. Durrant I.J. Luger S.M. Marks D.I. Franklin I.M. McMillan A.K. Tallman M.S. Rowe J.M. Goldstone A.H. Medical Research Council of the United Kingdom Adult ALL Working Party; Eastern Cooperative Oncology GroupOutcome of 609 adults after relapse of acute lymphoblastic leukemia (ALL); an MRC UKALL12/ECOG 2993 study.Blood. 2007; 109: 944-950Crossref PubMed Scopus (581) Google Scholar,2Gökbuget N. Stanze D. Beck J. Diedrich H. Horst H.A. Hüttmann A. Kobbe G. Kreuzer K.A. Leimer L. Reichle A. Schaich M. Schwartz S. Serve H. Starck M. Stelljes M. Stuhlmann R. Viardot A. Wendelin K. Freund M. Hoelzer D. German Multicenter Study Group for Adult Acute Lymphoblastic LeukemiaOutcome of relapsed adult lymphoblastic leukemia depends on response to salvage chemotherapy, prognostic factors, and performance of stem cell transplantation.Blood. 2012; 120: 2032-2041Crossref PubMed Scopus (294) Google Scholar. There is broad evidence that minimal residual disease (MRD) represents a major prognostic indicator; clinical protocols for childhood and adult ALL are tailored according to MRD assessments at different time points (TPs). Currently, real-time quantitative PCR (RQ-PCR) of clonotypic immunoglobulin and T-cell receptor (IG/TR) gene rearrangements is the most widely used molecular method for MRD assessment, standardized according to the EuroMRD consortium guidelines.3van der Velden V.H. Cazzaniga G. Schrauder A. Hancock J. Bader P. Panzer-Grumayer E.R. Flohr T. Sutton R. Cave H. Madsen H.O. Cayuela J.M. Trka J. Eckert C. Foroni L. Zur Stadt U. Beldjord K. Raff T. van der Schoot C.E. van Dongen J.J.M. European Study Group on MRD detection in ALL (ESG-MRD-ALL)Analysis of minimal residual disease by Ig/TCR gene rearrangements: guidelines for interpretation of real-time quantitative PCR data.Leukemia. 2007; 21: 604-611Crossref PubMed Scopus (535) Google Scholar However, non-specific amplifications of spurious IG/TR gene rearrangements are hardly distinguishable from positive cases at a low level [positive not quantifiable (PNQ)] by RQ-PCR, with an intrinsic risk of false-positive/negative MRD detections. These cases are thus troublesome to interpret, representing a major challenge in the monitoring of patients at a time when MRD is incorporated in clinical trials and is guiding treatment decisions. Moreover, the use of RQ-PCR can be limited by the lack of sufficient diagnostic material because the method is based on the comparison, for each experiment, with a standard curve based on diagnostic neoplastic DNA, thus limiting the possibility of monitoring patients over time. Digital droplet PCR (ddPCR) and next-generation sequencing (NGS) are advanced molecular methods, investigated within the European Scientific Foundation for Laboratory Hemato-Oncology (ESLHO), in the EuroClonality and EuroMRD Consortium groups, that could help to overcome the limits of RQ-PCR and potentially provide a more precise definition of the MRD status.4Whale A.S. Huggett J.F. Cowen S. Speirs V. Shaw J. Ellison S. Foy C.A. Scott D.J. Comparison of microfluidic digital PCR and conventional quantitative PCR for measuring copy number variation.Nucleic Acids Res. 2012; 40: e82Crossref PubMed Scopus (312) Google Scholar, 5Hindson C.M. Chevillet J.R. Briggs H.A. Gallichotte E.N. Ruf I.K. Hindson B.J. Vessella R.L. Tewari M. Absolute quantification by droplet digital PCR versus analog real-time PCR.Nat Methods. 2013; 10: 1003-1005Crossref PubMed Scopus (876) Google Scholar, 6Kotrova M. van der Velden V.H. van Dongen J.J. Formankova R. Sedlacek P. Brüggemann M. Zuna J. Stary J. Trka J. Fronkova E. Next-generation sequencing indicates false-positive MRD results and better predicts prognosis after SCT in patients with childhood ALL.Bone Marrow Transplant. 2017; 52: 962-968Crossref PubMed Scopus (57) Google Scholar The ddPCR technology is an implementation of conventional PCR that allows the quantitation of nucleic acid targets without the need of the calibration curves.7Sanders R. Huggett J.F. Bushell C.A. Cowen S. Scott D.J. Foy C.A. Evaluation of digital PCR for absolute DNA quantification.Anal Chem. 2011; 83: 6474-6484Crossref PubMed Scopus (247) Google Scholar It has a sensitivity, accuracy, and reproducibility at least comparable to that of RQ-PCR and shows a good analytical performance to quantify low positive samples defined as PNQ by RQ-PCR,4Whale A.S. Huggett J.F. Cowen S. Speirs V. Shaw J. Ellison S. Foy C.A. Scott D.J. Comparison of microfluidic digital PCR and conventional quantitative PCR for measuring copy number variation.Nucleic Acids Res. 2012; 40: e82Crossref PubMed Scopus (312) Google Scholar,5Hindson C.M. Chevillet J.R. Briggs H.A. Gallichotte E.N. Ruf I.K. Hindson B.J. Vessella R.L. Tewari M. Absolute quantification by droplet digital PCR versus analog real-time PCR.Nat Methods. 2013; 10: 1003-1005Crossref PubMed Scopus (876) Google Scholar,8Vincent M.E. Liu W. Haney E.B. Ismagilov R.F. Microfluidic stochastic confinement enhances analysis of rare cells by isolating cells and creating high density environments for control of diffusible signals.Chem Soc Rev. 2010; 39: 974-984Crossref PubMed Scopus (82) Google Scholar resulting in a reliable quantification of MRD in about 20% to 30% of these samples.9Drandi D. Kubiczkova-Besse L. Ferrero S. Dani N. Passera R. Mantoan B. Gambella M. Monitillo L. Saraci E. Ghione P. Genuardi E. Barbero D. Omedè P. Barberio D. Hajek R. Vitolo U. Palumbo A. Cortelazzo S. Boccadoro M. Inghirami G. Ladetto M. Minimal residual disease detection by droplet digital PCR in multiple myeloma, mantle cell lymphoma, and follicular lymphoma: a comparison with real-time PCR.J Mol Diagn. 2015; 17: 652-660Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 10Della Starza I. Nunes V. Cavalli M. De Novi L.A. Ilari C. Apicella V. Vitale A. Testi A.M. Del Giudice I. Chiaretti S. Foà R. Guarini A. Comparative analysis between RQ-PCR and digital-droplet-PCR of immunoglobulin/T-cell receptor gene rearrangements to monitor minimal residual disease in acute lymphoblastic leukaemia.Br J Haematol. 2016; 174: 541-549Crossref PubMed Scopus (45) Google Scholar, 11Cavalli M. De Novi L.A. Della Starza I. Cappelli L.V. Nunes V. Pulsoni A. Del Giudice I. Guarini A. Foà R. Comparative analysis between RQ-PCR and digital droplet PCR of BCL2/IGH gene rearrangement in the peripheral blood and bone marrow of early stage follicular lymphoma.Br J Haematol. 2017; 177: 588-596Crossref PubMed Scopus (30) Google Scholar Likewise, several groups have documented the value of NGS technologies for MRD detection in precursor and mature B-cell tumors.6Kotrova M. van der Velden V.H. van Dongen J.J. Formankova R. Sedlacek P. Brüggemann M. Zuna J. Stary J. Trka J. Fronkova E. Next-generation sequencing indicates false-positive MRD results and better predicts prognosis after SCT in patients with childhood ALL.Bone Marrow Transplant. 2017; 52: 962-968Crossref PubMed Scopus (57) Google Scholar,12Logan A.C. Zhang B. Narasimhan B. Carlton V. Zheng J. Moorhead M. Krampf M.R. Jones C.D. Waqar A.N. Faham M. Zehnder J.L. Miklos D.B. Minimal residual disease quantification using consensus primers and high-throughput IGH sequencing predicts post-transplant relapse in chronic lymphocytic leukemia.Leukemia. 2013; 27: 1659-1665Crossref PubMed Scopus (116) Google Scholar,13Ladetto M. Brüggemann M. Monitillo L. Ferrero S. Pepin F. Drandi D. Barbero D. Palumbo A. Passera R. Boccadoro M. Ritgen M. Gökbuget N. Zheng J. Carlton V. Trautmann H. Faham M. Pott C. Next-generation sequencing and real-time quantitative PCR for minimal residual disease detection in B-cell disorders.Leukemia. 2014; 28: 1299-1307Crossref PubMed Scopus (227) Google Scholar Studies using the NGS platform in ALL have demonstrated that a sensitivity level of 10–612Logan A.C. Zhang B. Narasimhan B. Carlton V. Zheng J. Moorhead M. Krampf M.R. Jones C.D. Waqar A.N. Faham M. Zehnder J.L. Miklos D.B. Minimal residual disease quantification using consensus primers and high-throughput IGH sequencing predicts post-transplant relapse in chronic lymphocytic leukemia.Leukemia. 2013; 27: 1659-1665Crossref PubMed Scopus (116) Google Scholar,13Ladetto M. Brüggemann M. Monitillo L. Ferrero S. Pepin F. Drandi D. Barbero D. Palumbo A. Passera R. Boccadoro M. Ritgen M. Gökbuget N. Zheng J. Carlton V. Trautmann H. Faham M. Pott C. Next-generation sequencing and real-time quantitative PCR for minimal residual disease detection in B-cell disorders.Leukemia. 2014; 28: 1299-1307Crossref PubMed Scopus (227) Google Scholar is achievable when higher amounts of DNA are used. Many authors have reported that NGS may be more specific than RQ-PCR in predicting relapse in ALL patients after induction as well as after allogenic stem cell transplantation.6Kotrova M. van der Velden V.H. van Dongen J.J. Formankova R. Sedlacek P. Brüggemann M. Zuna J. Stary J. Trka J. Fronkova E. Next-generation sequencing indicates false-positive MRD results and better predicts prognosis after SCT in patients with childhood ALL.Bone Marrow Transplant. 2017; 52: 962-968Crossref PubMed Scopus (57) Google Scholar,14Kotrova M. Muzikova K. Mejstrikova E. Novakova M. Bakardjieva-Mihaylova V. Fiser K. Stuchly J. Giraud M. Salson M. Pott C. Bruggemann M. Fullgrabe M. Stary J. Trka J. Fronkova E. The predictive strength of next-generation sequencing MRD detection for relapse compared with current methods in childhood ALL.Blood. 2015; 126: 1045-1047Crossref PubMed Scopus (69) Google Scholar In this study, we analyzed by RQ-PCR and ddPCR 116 follow-up (FU) samples collected at four TPs from 44 adult ALL patients enrolled in the GIMEMA LAL1913 front-line protocol for Ph− ALL,15Bassan R. Chiaretti S. Paoloni F. Audisio E. Marbello L. Borlenghi E. Zappasodi P. Di Raimondo F. Martinelli G. Mattei D. Cortelezzi A. Bocchia M. De Fabritiis P. Bonifacio M. Candoni A. Cassibba V. Di Bartolomeo P. Latte G. Offidani M. Della Starza I. Spinelli O. Santoro A. Elia L. De Propris M.S. Guarini A. Vitale A. Fazi P. Vignetti M. Rambaldi A. Foa R. First results of new GIMEMA trial LAL1913 for adult patients with Philadelphia-negative acute lymphoblastic leukemia (PH- ALL). European Hematology Association 23rd Congress. EHA.2018Google Scholar to evaluate the potential of ddPCR to redefine the MRD status, to increase and/or recover the rate of quantification of low disease levels, and to ultimately improve patients' risk stratification. The analysis was also conducted by NGS and in a subgroup of Ph− ALL samples. A total of 116 bone marrow (BM) samples from 44 newly diagnosed adult Ph− ALL patients (35 B-lineage and 9 T-lineage), aged between 18 and 65 years and enrolled in the GIMEMA LAL1913 protocol, were studied by RQ-PCR and ddPCR using IG/TR gene rearrangements as molecular markers. The analysis was performed by both methods at four TPs (TP1, TP2, TP3, and TP4), and the sample distribution was 38 at TP1, 40 at TP2, 6 at TP3, and 32 at TP4. Forty-seven of the 116 samples from 18 patients, depending on material availability, were also studied by NGS. The median follow-up of the cohort hereby analyzed was 33 months (range, 6 to 74 months). An updated clinical risk classification based on diagnostic characteristics, immunophenotype, cytogenetics, and molecular biology was used at onset to identify standard-risk, high-risk, and very-high-risk patients at presentation.15Bassan R. Chiaretti S. Paoloni F. Audisio E. Marbello L. Borlenghi E. Zappasodi P. Di Raimondo F. Martinelli G. Mattei D. Cortelezzi A. Bocchia M. De Fabritiis P. Bonifacio M. Candoni A. Cassibba V. Di Bartolomeo P. Latte G. Offidani M. Della Starza I. Spinelli O. Santoro A. Elia L. De Propris M.S. Guarini A. Vitale A. Fazi P. Vignetti M. Rambaldi A. Foa R. First results of new GIMEMA trial LAL1913 for adult patients with Philadelphia-negative acute lymphoblastic leukemia (PH- ALL). European Hematology Association 23rd Congress. EHA.2018Google Scholar All patients received homogeneous induction/early consolidation chemotherapy, with concurrent MRD analysis at four TPs [weeks 4 (TP1), 10 (TP2), 16 (TP3), and 22 (TP4) of induction/consolidation], to optimize risk classification and support risk/MRD-oriented therapy: patients with MRD ≥10−4 at TP2 or TP3, or ≥10−4 at TP4, were considered MRD positive, and patients with MRD <10−4 at TP2 and TP3 and negative at TP4 were considered MRD negative. MRD results at TP2 and TP4 represented the decisional time points for allogeneic transplant (allogeneic stem cell transplantation) allocation. Diagnostic DNA samples were screened by PCR amplification to identify IGH, IGK, TRG, TRD, and TRB gene rearrangements,16Pongers-Willemse M.J. Seriu T. Stolz F. dAniello E. Gameiro P. Pisa P. Gonzalez M. Bartram C.R. Panzer-Grumayer E.R. Biondi A. San Miguel J.F. van Dongen J.J. Primers and protocols for standardized detection of minimal residual disease in acute lymphoblastic leukemia using immunoglobulin and T cell receptor gene rearrangements and TAL1 deletions as PCR targets: report of the BIOMED-1 CONCERTED ACTION: investigation of minimal residual disease in acute leukemia.Leukemia. 1999; 13: 110-118Crossref PubMed Scopus (321) Google Scholar, 17van Dongen J.J. Langerak A.W. Bruggemann M. Evans P.A.S. Hummel M. Lavender F.L. Delabesse E. Davi F. Schuuring E. Garcia-Sanz R. van Krieken J.H.J.M. Droese J. González D. Bastard C. White H.E. Spaargaren M. Gonzalez M. Parreira A. Smith J.L. Morgan G.J. Kneba M. Macintyre E.A. Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936.Leukemia. 2003; 17: 2257-2317Crossref PubMed Scopus (2444) Google Scholar, 18Szczepanski T. van der Velden V.H.J. Hoogeveen P.G. de Bie M. Jacobs D.C.H. van Wering E.R. van Dongen J.J.M. Vdelta2-Jalpha rearrangements are frequent in precursor-B-acute lymphoblastic leukemia but rare in normal lymphoid cells.Blood. 2004; 103: 3798-3804Crossref PubMed Scopus (54) Google Scholar used as markers for MRD evaluation. MRD RQ-PCR assessment was performed and interpreted according to the EuroMRD guidelines, as previously described.3van der Velden V.H. Cazzaniga G. Schrauder A. Hancock J. Bader P. Panzer-Grumayer E.R. Flohr T. Sutton R. Cave H. Madsen H.O. Cayuela J.M. Trka J. Eckert C. Foroni L. Zur Stadt U. Beldjord K. Raff T. van der Schoot C.E. van Dongen J.J.M. European Study Group on MRD detection in ALL (ESG-MRD-ALL)Analysis of minimal residual disease by Ig/TCR gene rearrangements: guidelines for interpretation of real-time quantitative PCR data.Leukemia. 2007; 21: 604-611Crossref PubMed Scopus (535) Google Scholar The MRD ddPCR and NGS analyses were performed as published.6Kotrova M. van der Velden V.H. van Dongen J.J. Formankova R. Sedlacek P. Brüggemann M. Zuna J. Stary J. Trka J. Fronkova E. Next-generation sequencing indicates false-positive MRD results and better predicts prognosis after SCT in patients with childhood ALL.Bone Marrow Transplant. 2017; 52: 962-968Crossref PubMed Scopus (57) Google Scholar,19Della Starza I. De Novi L.A. Santoro A. Salemi D. Tam W. Cavalli M. Menale L. Soscia R. Apicella V. Ilari C. Vitale A. Testi A.M. Inghirami G. Chiaretti S. Foà R. Guarini A. Digital droplet PCR and next-generation sequencing refine minimal residual disease monitoring in acute lymphoblastic leukemia.Leuk Lymphoma. 2019; 60: 2838-2840Crossref PubMed Scopus (12) Google Scholar, 20Faham M. Zheng J. Moorhead M. Carlton V.E. Stow P. Coustan-Smith E. Pui C.H. Campana D. Deep-sequencing approach for minimal residual disease detection in acute lymphoblastic leukemia.Blood. 2012; 120: 5173-5180Crossref PubMed Scopus (303) Google Scholar, 21Della Starza I. Nunes V. Lovisa F. Silvestri D. Cavalli M. Garofalo A. Campeggio M. De Novi L.A. Soscia R. Oggioni C. Mussolin L. Biondi A. Guarini A. Valsecchi M.G. Conter V. Biffi A. Basso G. Foà R. Cazzaniga G. Droplet digital PCR improves IG-/TR-based MRD risk definition in childhood B-cell precursor acute lymphoblastic leukemia.Hemasphere. 2021; 5: e543Crossref PubMed Scopus (12) Google Scholar P values for differences in categorical variables were calculated using the χ2 test or the Fisher exact test. Disease levels by RQ-PCR or ddPCR were expressed in natural logarithmic scale. Event-free survival was defined as the time from the date of diagnosis to relapse/treatment failure or last follow-up and calculated using the Kaplan-Meier method; the log-rank test was used to evaluate differences between factors. All data were analyzed using the R software version 3.6.2 (R Core Team 2021; R Foundation for Statistical Computing, Vienna Austria, httsp://www.R-project.org, last accessed March 1, 2022). The activities described in the article, regarding the MRD monitoring within the GIMEMA MRD network, were conducted on samples of adults affected by Ph− ALL enrolled in a GIMEMA trial. The GIMEMA trial is LAL1913 (EudraCT number 2014-000383-18), approved by the Ethical Committee of the Coordinating Center on May 26, 2014, and by all other participating centers (NCT02067143, https://www.clinicaltrials.gov, last accessed May 11, 2022). In this trial, MRD monitoring was part of the protocol; all patients entering the trial signed the corresponding informed consent. Overall, the comparison of MRD results obtained by RQ-PCR and ddPCR showed a concordance rate of 79% [92/116 (P < 0.0001)] for samples classified as positive quantifiable (Q) or negative (NEG) by both techniques, whereas discordant results were detected in 21% (24/116) of samples. Sixteen were RQ-PCR-PNQ, 5 of which were ddPCR-Q and 11 of which were ddPCR-NEG; in the remaining 8 discordant FU samples, 5 were RQ-PCR-NEG/ddPCR-Q and 3 were RQ-PCR-NEG/ddPCR-PNQ. Overall, the use of ddPCR significantly reduced the proportion of samples defined as PNQ—3 of 116 (2.6%) versus 16 of 116 (14%) (P = 0.003)—because quantified MRD in 5 of 16 RQ-PCR-PNQ samples and proved NEG in 11 of 16. Moreover, ddPCR also allowed to quantify the disease in 5 of 75 (6.6%) samples that were RQ-PCR-NEG, modifying MRD quantification by 14% (16/116) in samples defined RQ-PCR-PNQ or NEG (Figure 1 and Table 1).Table 1Overall Comparison of RQ-PCR and ddPCR Plus NGS MRD ResultsRQ-PCRNEGPNQQTotalddPCR NEG67(25 NGS-NEG)(2 NGS-Q)11(1 NGS-Q)—78 PNQ3(1 NGS-NEG)——3 Q5(4 NGS-Q)5(4 NGS-Q)25(10 NGS-Q)35 Total751625116Analyses were performed on 116 samples at four time points (1, 2, 3, and 4) by RQ-PCR and ddPCR. Of 116 samples, 47 were also analyzed by NGS. NGS data are reported in italics.ddPCR, droplet digital PCR; MRD, minimal residual disease; NEG, negative; NGS, next-generation sequencing; PNQ, positive not quantifiable; Q, positive quantifiable; RQ-PCR, real-time quantitative PCR. Open table in a new tab Analyses were performed on 116 samples at four time points (1, 2, 3, and 4) by RQ-PCR and ddPCR. Of 116 samples, 47 were also analyzed by NGS. NGS data are reported in italics. ddPCR, droplet digital PCR; MRD, minimal residual disease; NEG, negative; NGS, next-generation sequencing; PNQ, positive not quantifiable; Q, positive quantifiable; RQ-PCR, real-time quantitative PCR. In 47 of the 116 samples analyzed by RQ-PCR and ddPCR, MRD was also analyzed by NGS: 21 of 47 samples resulted Q, and 26 of 47 samples resulted NEG. The concordance rate between NGS and RQ-PCR and ddPCR was 75% (35/47 samples) and 92% (43/47 samples), respectively. Thus, NGS quantified the disease in 11 samples classified as PNQ or NEG by RQ-PCR, confirming the ddPCR results in 8 of the 11 samples; in 3 samples that were ddPCR-NEG, NGS was able to quantify the disease with an MRD level of 10−5. Moreover, in one RQ-PCR-PNQ sample, NGS was NEG and in one sample, ddPCR-PNQ but RQ-PCR-NEG, NGS was NEG (Table 2).Table 2Comparison of RQ-PCR versus NGS and ddPCR versus NGS MRD ResultsNGSRQ-PCRQPNQNEGTotalQ100010PNQ5016NEG602531Total2102647ddPCRQPNQNEGTotalQ180018PNQ0011NEG302528Total2102647Analyses were performed on 47 samples at four time points (1, 2, 3, and 4). Concordant values between RQ-PCR and NGS and ddPCR and NGS are indicated in bold.ddPCR, droplet digital PCR; MRD, minimal residual disease; NEG, negative; NGS, next-generation sequencing; PNQ, positive not quantifiable; Q, positive quantifiable; RQ-PCR, real-time quantitative PCR. Open table in a new tab Analyses were performed on 47 samples at four time points (1, 2, 3, and 4). Concordant values between RQ-PCR and NGS and ddPCR and NGS are indicated in bold. ddPCR, droplet digital PCR; MRD, minimal residual disease; NEG, negative; NGS, next-generation sequencing; PNQ, positive not quantifiable; Q, positive quantifiable; RQ-PCR, real-time quantitative PCR. The RQ-PCR and ddPCR MRD analysis was performed at TP1, TP2, TP3, and TP4 for a total of 116 BM samples analyzed. At weeks 4 and 10 (TP1 and TP2, considered together to increase the sample size), the RQ-PCR and ddPCR analyses were performed on 78 BM samples. By RQ-PCR, 22 of 78 were Q, 9 of 78 were PNQ, and 47 of 78 were NEG, whereas by ddPCR, 29 of 78 were Q, 1 of 78 was PNQ, and 48 of 78 were NEG. Overall, at these TPs, the methods provided discordant results in 14 of 78 samples (18%): 6 were RQ-PCR-PNQ/ddPCR-NEG, 3 were RQ-PCR-PNQ/ddPCR-Q, 4 were RQ-PCR-NEG/ddPCR-Q, and 1 was RQ-PCR-NEG/ddPCR-PNQ. A subgroup of 31 of the 78 BM samples was analyzed also by NGS: 16 samples proved Q, and 15 were NEG. In the NGS group, 8 of 31 samples were RQ-PCR/ddPCR discordant (25.8%): 4 samples were RQ-PCR-NEG/ddPCR-Q and NGS resulted Q, 2 were RQ-PCR-PNQ/ddPCR-Q and NGS was Q, and 2 were RQ-PCR-PNQ/ddPCR-NEG while NGS was NEG and Q, showing that ddPCR and NGS provided reproducible results at the early TP. At weeks 16 and 22 (TP3 and TP4), 38 BM samples were analyzed by RQ-PCR and ddPCR. By RQ-PCR, 3 resulted Q, 7 resulted PNQ, and 28 resulted NEG, whereas by ddPCR, 7 proved Q, 1 proved PNQ, and 30 proved NEG. The methods were discordant in 10 of 38 samples (26.3%): 2 RQ-PCR-PNQ/ddPCR-Q, 5 RQ-PCR-PNQ/ddPCR-NEG, 2 RQ-PCR-NEG/ddPCR-Q, and 1 RQ-PCR-NEG/ddPCR-PNQ. Of the 38 BM samples, 16 were also analyzed by NGS—11 NEG and 5 samples proved Q—with 3 being RQ-PCR/ddPCR discordant: 2 samples that were RQ-PCR-PNQ/ddPCR-Q resulted Q by NGS, and 1 that was RQ-PCR-NEG/ddPCR-PNQ was NEG by NGS. Overall, 5 of 44 patients relapsed (11%), after a median follow-up of 33 months (range, 6 to 74 months). Three relapses occurred in patients who were PNQ/NEG by RQ-PCR but proved Q by ddPCR and/or NGS (60%), and in 2 patients defined Q by all methods (40%). Event-free survival was evaluated on the basis of the patients' disease levels at a single TP (TP1, TP2, and TP4). At TP1, by means of RQ-PCR, there were no statistically significant differences between Q (n = 15), NEG (n = 21), and PNQ (n = 5). At variance, evaluation by ddPCR resulted in the abrogation of PNQ cases, leading to an increase in both Q (n = 18) and NEG (n = 20) cases. As such, event-free survival was statistically different between the two subsets (P = 0.016) (Figure 2). At TP2 and TP4, representing decisional time points for patients' allocation to allogeneic transplant, patients with Q levels by both RQ-PCR and ddPCR showed a significantly inferior outcome compared to those with PNQ or no detectable disease. In line with the results observed at TP1, ddPCR successfully quantified more patients who presented a relapse (four of five at TP2 and three of five at TP4) compared to RQ-PCR (two of five at TP2 and three of five at TP4). Finally, patients were classified based on the possibility to successfully quantify disease levels by their specific marker(s) longitudinally in more than one TP (marker tracing) (Figure 3). Multiple FUs allow us to measure the dynamics of patients' specific markers over time and to identify those with quantifiable disease in more than one TP. Of the 37 of 41 patients with more than one FU available, 5 were traceable by RQ-PCR (13%) and 10 were traceable by ddPCR (27%). Patients traceable by ddPCR had a significantly inferior event-free survival than those traceable by RQ-PCR (Figure 3). Marker tracing along greater than one TP was possible in four of five relapsed patients by ddPCR versus one of five by RQ-PCR (Figure 3). During RQ-PCR MRD monitoring in ALL, a consistent fraction of samples with low MRD levels cannot be properly quantified despite the use of the specifically developed EuroMRD guidelines.3van der Velden V.H. Cazzaniga G. Schrauder A. Hancock J. Bader P. Panzer-Grumayer E.R. Flohr T. Sutton R. Cave H. Madsen H.O. Cayuela J.M. Trka J. Eckert C. Foroni L. Zur Stadt U. Beldjord K. Raff T. van der Schoot C.E. van Dongen J.J.M. European Study Group on MRD detection in ALL (ESG-MRD-ALL)Analysis of minimal residual disease by Ig/TCR gene rearrangements: guidelines for interpretation of real-time quantitative PCR data.Leukemia. 2007; 21: 604-611Crossref PubMed Scopus (535) Google Scholar Since low disease levels are close to the sensitivity limit of the current analytical methods, it is difficult to obtain reproducible results, and this inevitably hampers a precise MRD definition in these cases. In the present study, 116 samples from 44 adult ALL patients enrolled in the front-line GIMEMA LAL1913 protocol for Ph− ALL at four TPs were analyzed by RQ-PCR and ddPCR to evaluate the potential of ddPCR to redefine the MRD status, improving quantification of low disease levels and patients' risk stratification. The comparison of MRD results obtained by RQ-PCR and ddPCR showed a concordance rate of 79% at the tested TPs. The greater accuracy of ddPCR allowed us to discriminate low/not quantifiable positive samples and to quantify the disease in 6.6% of samples defined as negative by RQ-PCR, modifying MRD quantification of 14%. These data underline the greater accuracy and sensitivity of ddPCR that enabled us to identify an MRD signal also in low positive samples and, in particular, that ddPCR can provide a more robust and precise stratification for cases with a positivity <10−4, allowing us to distinguish true-positive cases from those defined as negative or PNQ by RQ-PCR. Moreover, the value of 10−4 represents the most challenging cutoff both clinically and methodologically. At weeks 4 and 10 (TP1 and TP2), RQ-PCR and ddPCR were discordant in 14 samples, with 7 becoming Q by ddPCR. Similarly, at weeks 16 and 22 (TP3 and TP4), RQ-PCR and ddPCR were discordant in 10 samples, and in 4 ddPCR evaluation changed the MRD status compared with RQ-PCR (Q versus PNQ/NEG). By survival analysis, both ddPCR and RQ-PCR were able to predict patients' relapse at TP2 and TP4 individually, with an advantage of ddPCR in capturing more events than RQ-PCR. Despite the small number of cases, this difference has important implications because the detection of Q levels of disease could lead to further therapeutic interventions in clinical trials and potentially rescue patients from relapse. On the other hand, it is likewise relevant to underline that no relapses were recorded in patients whose FU samples were defined as RQ-PCR-PNQ but proved NEG by ddPCR. These findings complement in part those previously reported by Raff et al,22Raff T. Gökbuget N. Lüschen S. Reutzel R. Ritgen M. Irmer S. Böttcher S. Horst H.A. Kneba M. Hoelzer D. Brüggemann M. Study Group G.M.A.L.L. Molecular relapse in adult standard-risk ALL patients detected by prospective MRD monitoring during and after maintenance treatment: data from the GMALL 06/99 and 07/03 trials.Blood. 2007; 109: 910-915Crossref PubMed Scopus (176) Google Scholar where relapses were observed also in cases with MRD within the quantitative range. As a matter of fact, in the present cohort, three relapses were documented in RQ-PCR-PNQ/NEG patients; noteworthy, these three cases were Q by ddPCR, thus showing the reliability of ddPCR over Q-PCR. Finally, when observing the disease level trend along multiple TPs (marker tracing), ddPCR was able to predict the occurrence of relapse. We believe this composite outcome measure is more precise than individual TPs (ie, TP2 or TP4) as it keeps into account the dynamic fluctuations of ALL over time. Taken together, all these findings highlight, with the limit of the small sample size, the clinical advantage of ddPCR over RQ-PCR in patients' stratification. In a smaller number of samples (n = 47), MRD was also analyzed by NGS, which showed a concordance rate of 75% with RQ-PCR and of 92% with ddPCR. It confirmed the ddPCR quantification in samples classified as RQ-PCR-PNQ or NEG and allowed us to increase the rate of quantification, defining as positive and quantifiable three samples that resulted ddPCR-NEG (RQ-PCR-Q 21% versus ddPCR-Q 38% versus NGS-Q 45%). We are working on increasing the number of cases to analyze also by NGS. However, although NGS might in principle be more sensitive in quantifying MRD levels, it must be recalled that it requires higher amounts of DNA, not always available at follow-up TPs, and requires bioinformatics skill. An earlier publication described preliminary analysis by all three methods in 23 samples from 11 patients enrolled in two GIMEMA trials (6 cases from LAL1308 and 5 from LAL1913).19Della Starza I. De Novi L.A. Santoro A. Salemi D. Tam W. Cavalli M. Menale L. Soscia R. Apicella V. Ilari C. Vitale A. Testi A.M. Inghirami G. Chiaretti S. Foà R. Guarini A. Digital droplet PCR and next-generation sequencing refine minimal residual disease monitoring in acute lymphoblastic leukemia.Leuk Lymphoma. 2019; 60: 2838-2840Crossref PubMed Scopus (12) Google Scholar By increasing the number of patients and samples from a single protocol, we can confirm the strength of ddPCR in improving the rate of quantification in critically low positive samples, with a greater concordance compared with NGS (92% versus 87%). In keeping with these observations, in a recent comparative analysis between RQ-PCR and ddPCR performed in a large pediatric cohort in collaboration with the AIEOP cooperative study group,21Della Starza I. Nunes V. Lovisa F. Silvestri D. Cavalli M. Garofalo A. Campeggio M. De Novi L.A. Soscia R. Oggioni C. Mussolin L. Biondi A. Guarini A. Valsecchi M.G. Conter V. Biffi A. Basso G. Foà R. Cazzaniga G. Droplet digital PCR improves IG-/TR-based MRD risk definition in childhood B-cell precursor acute lymphoblastic leukemia.Hemasphere. 2021; 5: e543Crossref PubMed Scopus (12) Google Scholar we showed that within a selected subset of childhood ALL patients defined as slow early responders (ie, having a high disease burden at day 33 and resulting RQ-PCR PNQ at day 78), most relapses occurred in cases that proved MRD quantifiable by ddPCR at day 78 (P < 0.001). On the contrary, patients with a negative or PNQ ddPCR MRD at day 78 had a better outcome than patients with a high MRD (≥5.0 × 10−4) at day 33 and negative at day 78, and similar to that of medium-risk patients enrolled in the same protocol. Overall, these data indicate that ddPCR is a technique as sensitive as RQ-PCR in detecting and quantifying MRD at all analyzed TPs and more accurate when the RQ-PCR quantitative range is inferior to 10−4, given its greater amplification efficiency and reproducibility. Moreover, ddPCR allows the quantitation of nucleic acid targets without the need of the calibration, thus not limiting the possibility of monitoring patients over time. Several studies have suggested that ddPCR is an attractive tool to monitor MRD in different hematologic malignancies, underlining its capability to predict relapse by quantifying low-positive samples and to lead to a potential refinement in patients' risk stratification.11Cavalli M. De Novi L.A. Della Starza I. Cappelli L.V. Nunes V. Pulsoni A. Del Giudice I. Guarini A. Foà R. Comparative analysis between RQ-PCR and digital droplet PCR of BCL2/IGH gene rearrangement in the peripheral blood and bone marrow of early stage follicular lymphoma.Br J Haematol. 2017; 177: 588-596Crossref PubMed Scopus (30) Google Scholar,21Della Starza I. Nunes V. Lovisa F. Silvestri D. Cavalli M. Garofalo A. Campeggio M. De Novi L.A. Soscia R. Oggioni C. Mussolin L. Biondi A. Guarini A. Valsecchi M.G. Conter V. Biffi A. Basso G. Foà R. Cazzaniga G. Droplet digital PCR improves IG-/TR-based MRD risk definition in childhood B-cell precursor acute lymphoblastic leukemia.Hemasphere. 2021; 5: e543Crossref PubMed Scopus (12) Google Scholar,23Drandi D. Alcantara M. Benmaad I. Söhlbrandt A. Lhermitte L. Zaccaria G. Ferrante M. Genuardi E. Mantoan B. Villarese P. Cheminant M. Starza I.D. Ciabatti E. Bomben R. Jimenez C. Callanan M. Abdo C. Eckert C. Ribrag V. Cortelazzo S. Dreyling M. Hermine O. Delfau-Larue M.H. Pott C. Ladetto M. Ferrero S. Macintyre E. Droplet digital PCR quantification of mantle cell lymphoma follow-up samples from four prospective trials of the European MCL network.Hemasphere. 2020; 4: e347Crossref PubMed Scopus (22) Google Scholar,24Coccaro N. Tota G. Anelli L. Zagaria A. Specchia G. Albano F. Digital PCR: a reliable tool for analyzing and monitoring hematologic malignancies.Int J Mol Sci. 2020; 21: 3141Crossref Scopus (18) Google Scholar The new-generation approaches have the remarkable advantage of a greater applicability (≥95% of cases) and sensitivity, and the possibility of proving additional information about the whole clonal immune gene rearrangement status of each patient. At the present time, their use to monitor MRD in clinical protocols is prevented by the lack of published international guidelines, a prerequisite requirement to compare MRD data in different clinical protocols. The EuroMRD Consortium (http://www.euromrd.org, last accessed February 16, 2022) is actively working to rapidly achieve this goal. The next step will be a parallel prospective analysis by ddPCR, for which more data are available, of samples classified as PNQ and/or NEG by RQ-PCR at clinically critical TPs to conclusively clarify its contribution to a further improvement of ALL patients' stratification and outcome. I.D.S. and L.A.D.N. analyzed the data and wrote the manuscript; A.S., D.S., O.S., M.T., R.S., M.C., V.A., V.B., and E.D.L. performed experiments; F.P. and L.V.C. performed statistical analysis; A.V., M.V., F.F., A.R., and R.B. provided clinical care, collected patient data, and revised the manuscript; and A.G., S.C., and R.F. supervised the experimental work and wrote the manuscript.
Minimal Residual Disease
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Abstract Introduction: Non-invasive monitoring of variants in a cancer patient for minimal residual disease, recurrence and/or resistance has tremendous clinical utility and warrants the development of a cell-free circulating tumor DNA panel, which involves the use of a simple blood draw. Towards this end, JAX has validated a new liquid biopsy assay called the Plasma MonitorTM that focuses on 84 clinically significant hotspots across 14 genes to complement our current clinical test menu, with a focus on comprehensive profiling of cancer. Methods: Post development and optimization of a custom amplicon panel, which included running multiple batches of samples through the assay to determine appropriate extraction methods, input DNA QC metrics, sequencing batch size, and sequencing loading concentrations, clinical validation of The Plasma MonitorTM was initiated. Validation included the evaluation of 20 uncharacterized plasma samples and 14 known controls and was executed in 5 phases: (1) Sample Processing for Validation Parameter determination; (2) LOD, sensitivity, specificity and accuracy (3) inter-assay concordance; (4) intra-assay concordance; (5) clinical validity in terms of interpretation and reporting of variants identified. Results: The final clinical protocol was developed using an input of 10ng of ctcfDNA quantified and qualified by a custom qPCR assay. Wet lab results of the first validation batch can be seen in Table 1. Using samples containing variants with known allele frequencies and a droplet digital PCR (ddPCR) based confirmation of novel variants identified by our assay, we established the assay's limit of detection (LOD) to be 0.9%. At this LOD, sensitivity, specificity, and accuracy were found to be 96.6%, 100%, and 98% respectively. Conclusion: Based on the results of this validation, the Plasma Monitor assay will be incorporated into the JAX clinical test menu. This new assay allows for comprehensive profiling and monitoring of cancer progression, response to therapy and minimal residual disease, and a significant benefit to both patients and clinicians. Citation Format: Kevin J. Kelly, Jasmina Uvalic, Daniel Bergeron, Shelbi Burns, Melissa Soucy, Guruprasad Ananda, Andrew Hesse, Pavalan Panneer Selvam, Honey V. Reddi. Development and validation of the plasma monitor test system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3428.
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Abstract Oropharyngeal squamous cell carcinoma (OPSCC) is increasing in global prevalence and is divided into two types dependent on association with human papillomavirus (HPV), with a more favorable prognosis in HPV+ve tumors. Assay of HPV copy number in plasma cell-free DNA (cfDNA) provides a minimally invasive method for detecting and monitoring tumor-derived HPV, with potential for enhancing clinical care. We have evaluated the utility of cfDNA droplet digital PCR (ddPCR) as a method for characterisation and longitudinal monitoring of patients with OPSCC in a prospectively recruited cohort of 104 OPSCC patients. ddPCR assay of cfDNA for five HPV types showed overall 95% concordance with p16 immunohistochemistry (IHC) and PCR analysis of solid tumor tissue. Longitudinal sampling in 48 HPV+ve patients, with median follow-up of 20 months, strongly predicted patient outcomes. Progression-free survival, stratified respectively by the presence or absence of detectable HPV cfDNA at a median of 13 weeks post-treatment, was 50% and 88% (p=0.001, hazard ratio 10.0 (95% CI 2.1-47.1)). In two patients, greater reliance on sequential HPV measurement would have avoided surgical intervention which ultimately did not confirm disease recurrence. The high concordance of pre-treatment plasma cfDNA-HPV analysis with p16 IHC and HPV-PCR of solid tissue, together with the predictive value and utility of sequentially measured post-treatment cfDNA-HPV copy number, provide a compelling case for the routine use of cfDNA-HPV ddPCR in management of OPSCC and for clinical trials to assess its impact on treatment outcomes. Citation Format: Martyna Joanna Adamowicz, Sophie J. Warlow, John P. Thomson, Lara M. Carey, Helen Thain, Robert Wescott, Kate Cuschieri, Lucy Q. Li, Brendan Conn, Ashley Hay, Iain J. Nixon, Timothy J. Aitman. Longitudinal measurement of HPV copy number in cell free DNA predicts progression free survival in HPV positive oropharyngeal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3408.
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