Perspective: sensitive detection of residual lymphoproliferative disease by NGS and clonal rearrangements—how low can you go?

•Ultradeep sequencing of immunoglobulin genes can identify residual disease.•Low-level detection clonally rearranged immunoglobulins is a stochastic process.•A sufficient amount DNA must be analyzed to detect one cell in million.•Standardization measurable disease by found imperative. Malignant lymphoproliferative disorders collectively constitute large fraction the hematological cancers, ranging from indolent highly aggressive neoplasms. Being diagnostically important hallmark, clonal gene rearrangements enable clinical course patients down minute malignant cells. The introduction next-generation (NGS) has provided unprecedented assay specificity, with sensitivity matching that polymerase chain reaction-based (MRD) 10–6 level. Although reaching 10–7 theoretically feasible, employing and coverage placed perspective practical challenges when relying on samples contrast controlled serial dilutions. As we discuss, randomness subsampling taken into account accommodate threshold—in terms both required number cells coverage. substantial part reviewed studies do not state depth or even some cases, call for increased transparency critical assessment MRD assays implementation feasibility. minimal/measurable concept its use hematology have been reinforced last couple years as become more accessible laboratories after continuous increase capacity lowered costs. NGS improved resolution nucleotide composition B T cells, making it effective longitudinal surveillance single but also multiple coexisting clones. Much work already focused superseding high quantitative reaction (qPCR) while increasing specificity circumventing need patient-specific PCR primers/probes. Because provide specific signatures B-cell receptor (BCR) T-cell (TCR) chains, these target sequences are immense importance diagnostic workup, sometimes prognostically, because they serve quantification setting disorders. A frequent notion relates capability detecting among normal counterparts out million healthy potentially beyond. Constituting key component adaptive immune system, lymphocytes specialized antigen presentation, BCR binding, mounting humoral response secretion antibodies. flexibility extensive diversity generated primarily light heavy loci. profound implication genomes differentiating static regions, introducing irreversible genomic changes. It this signature exploited diagnostics laboratory (Figure 1). After naive encounters an antigen, may undergo additional somatic hypermutations. proliferation represents counteraction inherent response, acute chronic excessive expansion result transformation, distinct clonotype constituting disorder. motivation performing analyses presence lymphoproliferation unique rearrangement at diagnosis during remission early sign relapse follow-up. In perspective, critically approach topic (IGH) 1) lymphoid We evaluate how sensitive being performed ultrahigh coverage, potential technical problems consider, biological pitfalls establish nuanced discourse sensitivities. Foremost, address theoretical reads needed confidently given level factors influence resolution. topics narrowed IGH rearrangements, general discussion pertains rearrangements. For reviews means instead refer previously published overviews [1Monter Nomdedeu JF. ClonoSEQ malignancies.Expert Rev Mol Diagn. 2019; 19: 571-578Crossref PubMed Scopus (14) Google Scholar, 2van Dongen JJ van der Velden VH Bruggemann M Orfao A. Minimal lymphoblastic leukemia: sensitive, fast, standardized technologies.Blood. 2015; 125: 3996-4009Crossref (278) 3Sanchez R Ayala Martinez-Lopez J. monitoring methodologies malignancies.Int J Sci. 20: 2832Crossref (17) 4Kotrova Trka Kneba M. 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Scholar] reported majority negative had level, noted recent developments comparable NGS. Patients using were superior progression-free survival Moreover, was determining factor stem transplantation [10Bader P Kreyenberg H Henze GH al.Prognostic value minimal before allogeneic stem-cell relapsed childhood ALL-REZ BFM Study Group.J Clin Oncol. 2009; 27: 377-384Crossref (263) Scholar], TCR targets ALL. less 1 10,000 higher probability event-free lower cumulative incidence relapse. One interesting finding results related “dose-dependent” relation between Before NGS, leap rearrangement, normally within range 10–4 [11Leisch Jansko Zaborsky Greil Pleyer L. Next generation AML—on becoming new standard treatment initiation and/or modulation?.Cancers (Basel). 11: 252Crossref (16) compared fragment analyses. qPCR, Southern blots gold standard. thorough recombinations concerning standardization protocols (BIOMED-2), [12van Langerak al.Design primers suspect lymphoproliferations: report BIOMED-2 Concerted Action BMH4-CT98-3936.Leukemia. 2003; 17: 2257-2317Crossref (2372) assessed 5%–10% cumbersome. That article recommendations still relevant age (IGH, IGK, IGL) (TCRB, TCRD, TCRG; TCRA included PCR) addressed study, fact all expanded arose underlying malignancy. This argument central strategy mapping clonotypes reliable measurements achieved, regardless type. Gene directly lineage specific, aptly exemplified immature B- leukemias [13Beishuizen Verhoeven Wering ER Hahlen K Hooijkaas JJ. Analysis Ig 40 subsequent relapse: implications analysis.Blood. 1994; 83: 2238-2247Crossref 14Meleshko AN Belevtsev MV Savitskaja TV Potapnev MP. B-lineage immunophenotype fusion oncogene expression.Leuk Res. 2006; 30: 795-800Crossref (12) 15Nosaka Kita Miwa al.Cross-lineage human leukemic B-precursor occur frequently V-DJ IgH genes.Blood. 1989; 74: 361-368Crossref neoplasms necessarily detectable applied technologies. approximately 80%–90% referred here, identified [16Li Z Jiang Lim EH al.Identifying clones RNA-Seq.Leukemia. 34: 2418-2429Crossref 17Cavagna Guinea Montalvo ML Tosi al.Capture-based identification immunoglobulin/T-cell markers mutations adult lacking molecular probes.Cancers 12: 1505Crossref (5) 18Gawad Pepin Carlton VE al.Massive evolution locus children precursor leukemia.Blood. 2012; 120: 4407-4417Crossref (90) such, primary is, clonotype, implement follow-up quantification. Failure any clone arise primer issues, biologically incomplete [19Gonzalez Gonzalez Alonso ME al.Incomplete DJH novel tumor quantitation real-time PCR.Leukemia. 1051-1057Crossref (24) possibly malignancies devoid immunoglobulin. existed used several foundation, methods, calculations changed lack modalities. Currently, assumed [6Kruse although achievements fully demonstrated practice. eventually empirically, without extrapolating curves dilutions, which statistically feasible larger cohorts analyzed. However, applicability remains somewhat speculative, especially cost sensitivity. Recently, Yao [20Yao Q Bai Y Chim CS. Standardized myeloma.Front 9: 449Crossref concordance allele-specific oligonucleotide (ASO-qPCR) similar described earlier [21Ladetto Monitillo L al.Next-generation disorders.Leukemia. 2014; 28: 1299-1307Crossref (219) negativity positivity dynamic ranges. authors information rationale obtaining 10–5 triplicates µg each commented articles orders magnitude Considering “sequencing limited only input thus levels well below leukocytes” [22Faham Zheng Moorhead al.Deep-sequencing 5173-5180Crossref (294) question account. particular stated 106, thus, Poisson sampling detrimental effect assay. cannot will converted one-to-one manner. if based total of, example, nucleated then sequence background suffice aforementioned threshold. On hand, ask why currently reported, threshold sensitivities literature (Table 1), stable another issue. Neither does sporadic correspond established nor dilution assays, demonstrating linearity million, applicable robust laboratory. Another issue aspects. briefly note, values indicate cure impossible, logically, long-term guaranteed.Table 1Reported across selected studiesStudyDiseasePlatformAmount material/readsEstimated sensitivityCohortaCohort defined estimated included.23van Seriu Panzer-Grumayer leukaemia childhood.Lancet. 1998; 352: 1731-1738Abstract Full Text PDF (790) ScholarALLPCR1 µg, ∼6.5 × 105 cells10–4–10–6251 samples/168 patients10Bader ScholarALLPCR (BIOMED-1)—10–3–10–491 patients22Faham ScholarB-ALLNGS (LymphoSIGHT), ASO-qPCR, FC6.0 105(0.9–17 105) cells10–6 (10–7)106 patients24Thol Kolking Damm myeloid FLT3-ITD NPM1 mutations.Genes Chromosomes Cancer. 51: 689-695Crossref ScholarAMLNGS (FLT3/NPM1)7,758 15,278(393–24,997) reads5 10–480 samples21Ladetto ScholarALL/MCL/MMNGS, (Multiplex)1.5 μg (3 500 ng)10–5378 samples/55 patients25Logan AC Vashi Faham al.Immunoglobulin quantifies post-transplantation survival.Biol Blood Marrow Transplant. 1307-1313Abstract (94) ScholarALLNGS (LymphoSIGHT)10 mL PB/5 BM (MNCs)10–4–10–6237 samples/29 patients26Martinez-Lopez Lahuerta deep method myeloma.Blood. 123: 3073-3079Crossref (305) ScholarMMNGS (LymphoSIGHT)<300,000 cells10–3–10–5133 patients27Borowitz MJ Wood BL Devidas significance B-ALL: Children's Oncology Group study AALL0232.Blood. 126: 964-971Crossref (189) ScholarB-ALLFC>750,000 cells10–42,479 patients7Pulsipher (ImmunoSeq IgH)3 (sequences)10–741 patients28Attal Lauwers-Cances V Hulin al.Lenalidomide, bortezomib, dexamethasone myeloma.N Engl Med. 376: 1311-1320Crossref (573) ScholarMMFC—10–4700 patients29Flores-Montero Sanoja-Flores Paiva al.Next myeloma.Leukemia. 31: 2094-2103Crossref (289) ScholarMMFC∼107 cells10–5–10–6385 samples/patients30Kotrova VHJ JJM indicates false-positive better SCT ALL.Bone 52: 962-968Crossref (48) (BIOMED-2)500 ng10–4 (–10–5)228 samples/30 patients31Takamatsu Takezako sequencing-based who underwent autologous transplantation.Ann 2503-2510Abstract (27) (LymphoSIGHT)103 (59–288) μg/19 (6–36) µg10–6–10–7125 patients32Cheng S Inghirami G Cheng Tam W. Simple post-remission B-ALL.J Hematol 105Crossref (LymphoTrack)0.5–5 (105 reads)10–6122 patients33Levis Perl AE Altman JK al.A AML mutations.Blood Adv. 2: 825-831Crossref (60) (FLT3-ITD)0.7 ∼100,000 cells10–580 patients34Perrot Corre major 132: 2456-2464Crossref (173) (LymphoSIGHT)—10–6700 overall study8Wood (ImmunoSEQ), FC0.4–8 µg10–6619 patients20Yao (Lymphotrack)3 µg10–54 patients9Thompson ScholarCLLNGS (ClonoSEQ)N/A (>1.9 106 cells?)10–662 patients17Cavagna ScholarT-ALLNGS (capture-based panel)0.6–1 µg10–4–10–523 patients35Drandi Alcantara Benmaad I al.Droplet Digital Quantification mantle lymphoma four prospective trials European MCL Network.Hemasphere. 4: e347Crossref ScholarMCLPCR (digital droplet)—10–4–10–5416 samples/166 patients16Li (RNA)0.4–1 μg10–4–10–5258 patientsB-ALL=B-cell leukemia; FC=flow cytometry; MCL=mantle lymphoma; T-ALL=T-cell leukemia.a Cohort included. Open table tab B-ALL=B-cell leukemia. Several criteria achieving short variants error rate platform [36Levine RL Valk PJM. Next-generation leukemia.Haematologica. 104: 868-871Crossref (22) poses problem rely fingerprint. relate base mismatches accept including frequency. known constant length sequence. no consensus exclude detected clonotype. LymphoTrack allows up (Instructions Use, Nos. 280364 280473, Invivoscribe, San Diego, CA, USA). process drawing liquid tissue biopsy, through extraction, PCR, final computational output comprises discrete steps contribute effort exerted understanding kinetics reactions, amplification simple exponential growth rate, depending starting amount, efficiency, thermocycles 2A). oversimplification model stretches sample, involves targeting leader region FR1, FR2, FR3, FR4 regions Such multiplex reactions templates entail different concentrations varying efficiencies. Thus, end product considered sum identical, nonidentical, amplified Figure 2A (Eq. ratio expected 2A, Eq. 2), consequence analyzing patient sample replicate contain zero copies. Also, uneven efficiency differs background. With step makes follow distribution 3), failing demonstrate increases dramatically small 2B). Basically, importantly, helps predict mean targets. actually known, fixed intended implemented t