Minimal Residual Disease Surveillance Research Articles

Tumor-informed deep sequencing of ctDNA detects minimal residual disease and predicts relapse in osteosarcoma

Current surveillance modalities of osteosarcoma relapse exhibit limited sensitivity and specificity. Although circulating tumor DNA (ctDNA) has been established as a biomarker of minimal residual disease (MRD) in many solid tumors, a sensitive ctDNA detection technique has not been thoroughly explored for longitudinal MRD detection in osteosarcoma. From August 2019 to June 2023, 59 patients diagnosed with osteosarcoma at the First Affiliated Hospital of Sun Yat-sen University were evaluated in this study. Tumor-informed MRD panels were developed through whole exome sequencing (WES) of tumor tissues. Longitudinal blood samples were collected during treatment and subjected to multiplex PCR-based next-generation sequencing (NGS). Kaplan-Meier curves and Log-rank tests were used to compare outcomes, and Cox regression analysis was performed to identify prognostic factors. WES analysis of 83 patients revealed substantial mutational heterogeneity, with non-recurrent mutated genes accounting for 58.1%. Tumor-informed MRD panels were successfully obtained for 85.5% of patients (71/83). Among 59 patients with successful MRD panel customization and available blood samples, 13 patients exhibited positive ctDNA detection after surgery. Patients with negative post-operative ctDNA had better event-free survival (EFS) compared to those with positive ctDNA, at 1-6 months after surgery, after adjuvant chemotherapy, and more than 6 months after surgery (p<0.05). In both univariate and multivariate Cox regression analysis, ctDNA results emerged as a significant predictor of EFS (p<0.05). ctDNA detection preceded positive imaging in 5 patients, with an average lead time of 92.6 days. Thirty-nine patients remained disease-free, with ctDNA results consistently negative or turning negative during follow-up. Our study underscores the applicability of tumor-informed deep sequencing of ctDNA in osteosarcoma MRD surveillance and, to our knowledge, represents the largest cohort to date. ctDNA detection is a significant prognostic factor, enabling the early identification of tumor relapse and progression compared to standard imaging, thus offering valuable insights in guiding osteosarcoma patient management. The Grants of National Natural Science Foundation of China (No. 82072964, 82072965, 82203798, 82203026), the Natural Science Foundation of Guangdong (No. 2023A1515012659, 2023A1515010302), and the Regional Combination Project of Basic and Applied Basic Research Foundation of Guangdong (No. 2020A1515110010).

Abstract 986: Mathematical modeling of ctDNA dynamics to predict utility of ultrasensitive MRD assays in early stage non-small cell lung cancer (NSCLC)

Abstract Background: ctDNA assay use for minimal residual disease (MRD) detection in early stage NSCLC has been limited by suboptimal sensitivity of first generation assays. It is unclear how improving assay limit of detection (LOD) would translate to improvements in clinical sensitivity. Here, we performed mathematical modeling of ctDNA dynamics after curative-intent treatment in early stage NSCLC to predict the clinical impact of MRD assays with improved analytical sensitivity. Methods: We analyzed a recently reported dataset of post-surgical ctDNA MRD surveillance in early stage NSCLC (Abbosh et al., Nature 2023) to investigate ctDNA growth dynamics. Of 70 relapsed patients in this cohort, 23 had 3 consecutive ctDNA-positive samples with no intervening therapy. We used these to generate patient-specific mathematical models for ctDNA variant allele fractions (VAF) over time, initially assuming exponential growth. We assessed this assumption by creating log-linear models for ctDNA increase in these cases. Using slopes of the log-linear models, we calculated patient-specific doubling times and assessed their relation to stage, histology, and outcome. We used the log-linear models to simulate a VAF distribution each day after surgery, enabling projection of clinical sensitivities and estimation of lead times for recurrence detection at varying LODs. Results: For the 23 cases with 3+ consecutive ctDNA positive samples, we developed log-linear models for ctDNA VAF growth, which were strongly correlated (r &amp;gt; 0.5) in 18/23 (78%) patients (median r = 0.89), supporting that ctDNA can be modeled as exponential growth. We calculated ctDNA doubling times for the 18 patients with exponential ctDNA kinetics and 10 patients with 2 consecutive ctDNA positive samples. Doubling times were remarkably consistent with median 51 days (IQR 40-79). Doubling times were longer for stage I tumors compared to stage II/III (p = 0.04) but were not different between NSCLC subtypes. Doubling time correlated with time to clinical relapse, with patients having more rapid ctDNA growth relapsing earlier (r = 0.52, p=0.03). Finally, we used patient-specific log-linear models to project ctDNA VAFs at a landmark 30 days after surgery. By improving the LOD for ctDNA detection from 10−4 to 10−6, the projected clinical sensitivity doubled (from 43% to 86%). Simulations predicted that this improvement in sensitivity would increase median lead time from 183 days for LOD=10−4 to 349 days for LOD=10−6 (p&amp;lt;0.0001). Conclusion: For most relapsed NSCLC patients, the natural history of ctDNA MRD growth can be reliably modeled with exponential growth kinetics. ctDNA doubling time is associated with stage and clinical outcome. Ultrasensitive MRD assays with LOD of 10−6 are predicted to meaningfully improve clinical sensitivity at the post-surgical landmark and to increase the lead time for MRD detection before relapse. Citation Format: Jordan S. Goldstein, Christopher Abbosh, Ash A. Alizadeh, Maximilian Diehn, David M. Kurtz. Mathematical modeling of ctDNA dynamics to predict utility of ultrasensitive MRD assays in early stage non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 986.

Phased Variants Allow Robust Profiling of Circulating Tumor DNA in Untreated Follicular Lymphomas

Background: In the absence of tumor tissue, several studies have shown the utility of plasma cell-free DNA (cfDNA) for blood based noninvasive genotyping of diverse human tumors, including aggressive lymphomas. However, it remains unclear whether these same cfDNA advantages also apply to follicular lymphoma (FL), and if they could inform noninvasive minimal residual disease (MRD) monitoring in the context of modern regimens inducing deep FL remissions. Here, we address these questions in previously untreated patients with FL receiving standard first-line (1L) therapy, using Phased Variant Enrichment and Detection Sequencing (PhasED-Seq, Kurtz et al 2021, Nat Biotech) to monitor ctDNA and MRD. Methods: We profiled 298 samples from 61 patients with FL, including archival formalin fixed paraffin embedded (FFPE) tumor tissues, and serial blood samples before, during, and following 1L therapy. All patients were treatment-naive, with most (75%) presenting with advanced stage FL. Induction regimens primarily comprised Bendamustine/Rituximab (66%) and R-CHOP (26%). At a median follow-up of 23 months from start of 1L therapy, 80% of patients were alive and progression free, while 11% experienced progression within 24 months (POD24). MRD was analyzed by PhasED-Seq (Foresight Diagnostics). Phased variants (PVs) were genotyped either using baseline blood plasma, or alternatively from FFPE tumor tissue; matched leukocytes were used as a source of constitutional DNA to censor germline variants and clonal hematopoiesis of indeterminate potential (CHIP). Baseline PV genotypes from each baseline source were then used to longitudinally assess MRD serially during 1L therapy. Blood timepoints included baseline (pre-treatment), Cycle 2 Day 1 (C2D1), C3D1, C4D1, C5D1, C6D1, at End of Induction (EOI), and serially thereafter. Levels of ctDNA were compared to known FL prognostic factors, including radiographic responses, POD24, and progression-free survival (PFS). Results: PVs were successfully identified from tumor FFPE samples in all patients (61/61), enabling ctDNA MRD assessment by PhasED-Seq. The median number of PVs identified was 530 (IQR 285-1152). Prior to therapy, ctDNA was detectable in 94% of cases (57/61). The median tumor fraction in baseline plasma was 0.28%, significantly lower than pretreatment ctDNA levels in diffuse large B-cell lymphoma (DLBCL) (DLBCL median = 6.1%, Roschewski, ASH 2022, P&amp;lt;0.0001 for FL vs DLBCL). Median pre-treatment ctDNA levels were significantly higher in advanced stage disease (stage I/II, 0.04%, stage III/IV, 0.53%, P=0.006), and correlated with FLIPI risk scores (P=0.04). We next tested whether adequate PVs can be directly identified from plasma without need for tumor tissue to allow MRD surveillance. Despite lower baseline ctDNA levels in FL, a sufficient number of PVs were identified directly in plasma to enable disease monitoring in 75% of cases (45/61). Moreover, ctDNA levels were highly correlated, regardless of the baseline sample used to identify PVs (i.e., tumor versus pretreatment plasma, Spearman rho=0.92, P&amp;lt;0.0001, Fig 1a). Despite high genotypic concordance of tumor vs plasma, we observed significant tumor heterogeneity across compartments with variable rates of concordance between specific mutated genes (Fig 1b). We next assessed the prognostic performance of ctDNA MRD to predict clinical outcomes. ctDNA levels prior to treatment were not significantly prognostic for time to progression. In contrast, in patients with ctDNA MRD assessed after two cycles of therapy (i.e., at C3D1), detection of residual ctDNA was significantly associated with inferior time to progression (log-rank P=0.03 HR = 8.9). Finally, we assessed the feasibility of serial ctDNA surveillance during radiographic remission. In patients with subsequent clinical relapse of disease, we detected reemergence of detectable MRD over two years prior to clinical disease progression. Conclusions: Circulating tumor DNA analysis with PhasED-Seq is feasible in FL, with baseline ctDNA levels significantly lower than those observed in DLBCL. In cases without tissue samples available, plasma DNA can frequently be used as a surrogate for tissue samples. ctDNA detection during treatment is strongly associated with eventual treatment responses, and serial disease surveillance with ctDNA can anticipate clinical relapse.

Minimal residual disease response-adapted therapy in newly diagnosed multiple myeloma (MASTER): final report of the multicentre, single-arm, phase 2 trial

For patients with newly diagnosed multiple myeloma, reaching minimal residual disease (MRD) negativity after treatment is associated with improved outcomes; however, the use of MRD to modulate therapy remains elusive. We present the final analysis of the MASTER trial of daratumumab, carfilzomib, lenalidomide, and dexamethasone (Dara-KRd) therapy in patients with newly diagnosed multiple myeloma, in which MRD status is used to modulate treatment duration and cessation. MASTER was a multicentre, single-arm, phase 2 trial conducted in five academic medical centres in the USA. Eligible participants were 18 years or older with newly diagnosed multiple myeloma (measurable by serum or urine protein electrophoresis or serum free light chains), a life expectancy of at least 12 months, and an Eastern Cooperative Oncology Group performance status of 0-2, and had received no previous treatment for multiple myeloma except up to one cycle of therapy containing bortezomib, cyclophosphamide, and dexamethasone. The study was enriched for participants with high-risk chromosome abnormalities (HRCAs). During the induction phase, participants received four 28-day cycles of Dara-KRd, each comprising daratumumab (16 mg/kg intravenously on days 1, 8, 15, and 22), carfilzomib (56 mg/m2 intravenously on days 1, 8, and 15), lenalidomide (25 mg orally on days 1-21), and dexamethasone (40 mg orally or intravenously on days 1, 8, 15, and 22); induction was followed by autologous haematopoietic stem-cell transplantation and up to two phases of consolidation with Dara-KRd. We assessed MRD by next-generation sequencing after or during each phase. The primary endpoint was reaching MRD negativity (<10-5). Participants who reached MRD negativity after or during two consecutive phases stopped treatment and began observation with MRD surveillance (MRD-SURE); participants who did not reach two consecutive MRD-negative results received maintenance lenalidomide. Secondary endpoints included progression-free survival and cumulative incidence of progression. All analyses were conducted in the intention-to-treat population. This trial is registered with ClinicalTrials.gov, NCT03224507, and is complete. Between Mar 21, 2018, and Oct 23, 2020, 123 participants were recruited to the study, of whom 70 (57%) were men, 53 (43%) were women, 94 (76%) were non-Hispanic White, 25 (20%) were non-Hispanic Black, and four (3%) were of another race or ethnicity. The median age of participants was 61 years (IQR 55-68), and 24 (20%) were aged 70 years or older. The median duration of follow up was 42·2 months (IQR 34·5-46·0). Of the 123 participants, 53 (43%) had no HRCAs, 46 (37%) had one HRCA, and 24 (20%) had two or more HRCAs. For 118 (96%) of 123 participants, MRD was evaluable by next-generation sequencing; the remaining five had an absence of sufficiently unique clonogenic sequences to enable tracking by the assay. Of these 118 participants, 96 (81%, 95% CI 73-88) reached MRD of less than 10-5 (comprising 39 [78%, 64-88] of 50 participants with no HRCAs, 38 [86%, 73-95] of 44 participants with one HRCA, and 19 [79%, 58-93] of 24 participants with two or more HRCAs) and 84 (71%, 62-79) reached MRD-SURE and treatment cessation. 36-month progression-free survival among all 123 participants was 88% (95% CI 78-95) for participants with no HRCAs, 79% (67-88) for those with one HRCA, and 50% (30-70) for those with two or more HRCAs. For the 84 participants reaching MRD-SURE, the 24-month cumulative incidence of progression from cessation of therapy was 9% (95% CI 1-19) for participants with no HRCAs, 9% (1-18) for those with one HRCA, and 47% (23-72) for those with two or more HRCAs. 61 participants (comprising 52% of 118 MRD-evaluable participants and 73% of 84 participants who reached MRD-SURE) remain free of therapy and MRD-negative as of Feb 7, 2023. The most common grade 3-4 adverse events were neutropenia (43 patients, 35%), lymphopenia (28 patients, 23%), and hypertension (13 patients, 11%). Three treatment-emergent deaths were recorded: two sudden deaths and one due to viral infection, none of which were judged to be treatment-related. This approach provided positive outcomes and a pathway for treatment cessation in most patients with newly diagnosed multiple myeloma. Outcomes for patients with ultra-high-risk multiple myeloma, defined as those with two or more HRCAs, remain unsatisfactory, and these patients should be prioritised for trials with early introduction of therapies with novel mechanisms of action. Amgen and Janssen Pharmaceuticals.

Utility of circulating tumor DNA as a tool to detect minimal residual disease in patients with metastatic cancer and durable response following treatment with immune checkpoint inhibitors.

e21563 Background: Circulating tumor DNA (ctDNA) is tumor-derived fragment of DNA circulating in plasma. ctDNA has been used to detect minimal residual disease (MRD) in early-stage cancers after curative intent therapy. However, ctDNA based MRD concept is not investigated in metastatic disease. There is a lack of clarity around optimal timing to stop immune checkpoint inhibitors (ICI) in melanoma and lung cancer patients who have achieved durable response (DR) and MRD may have a role here to select right patients to stop therapy at an optimal time. Methods: This is prospective study on metastatic lung cancer and melanoma patients with DR treated with ICI. DR is identified as objective response (CR/PR) by modified WHO criteria lasting ≥6 months continuously. We utilized Signatera assay by Natera. Signatera uses tissue from original biopsy of the patients and develop multiplex PCR based personalized assay to detect MRD. The level of ctDNA is reported in mean tumor molecules per mL. We obtained ctDNA assay at baseline, month 3 and month 6 at their routine follow up visits. If applicable, ctDNA assay was done at the time of progression, and 3 months after resuming treatment. Results: We identified 29 melanoma and 17 Lung cancer (both small cell and non-small cell) patients with metastases and DR. Median age was 69 years in both cohorts. Median duration of ICI was 14 months in melanoma and 24 months in lung cancer cohort. ctDNA assay was obtained in 26 melanoma and 13 lung cancer patients. One small cell lung cancer patient had detectable ctDNA at baseline, although radiographically, the patient had DR. The rest of the patients in both cohorts had undetectable ctDNA. 2 melanoma patients developed detectable ctDNA on subsequent assays and their therapies were changed. One small cell lung cancer patient has persistently detectable ctDNA on subsequent assays but remained in DR. A total of 10 patients in both cohorts decided to stop their therapy following several -ve ctDNA assay. Conclusions: The role of ctDNA needs to be investigated in advanced/metastatic settings as MRD may have a role to identify patients who may benefit from treatment break. MRD surveillance may also support radiographic assessment. [Table: see text]

Abstract PR007: A liposomal priming agent increases the sensitivity of liquid biopsies

Abstract Liquid biopsy measurements, such as analysis of circulating tumor DNA (ctDNA) shed by cancer cells, have garnered significant attention for their potential to empower the field of precision oncology. Given that ctDNA tests could enable minimally invasive monitoring and molecular profiling of disease, they are being investigated for use in earlier detection via pan-cancer screening tests, for tracking tumor evolution to inform therapy selection, and for making treatment decisions during minimal residual disease surveillance. However, a typical blood draw carries ultra-low levels of ctDNA (as low as 1.7 copies of the tumor genome in 15mL of blood for a 1cm lung tumor), and this fundamentally limits the sensitivity and the clinical utility of ctDNA-testing in many settings. To push beyond current ctDNA detection limits, we present a first-in-class liquid biopsy priming agent that is given prior to a blood draw to increase the abundance of ctDNA in circulation. Our priming agent consists of liposomes that transiently block the uptake of cell free DNA (cfDNA) by macrophages in the liver, resulting in increased cfDNA available for diagnostic analysis in blood. Using an in vitro 2D assay, we first identified a DSPE-based liposomal formulation that inhibits the uptake of cfDNA by two independent murine macrophage cell lines. Next, we injected our liposomal agent into healthy or tumor-bearing mice and collected blood for further analysis. We found that in healthy mice priming increases the half-life and the recovery of cfDNA from a blood draw, assayed via qPCR. In tumor-bearing mice, the priming agent increases the recovery of ctDNA by up to 60-fold (P = 0.0103) and improves the sensitivity of a ctDNA diagnostic assay from 0% to 75% at low tumor burden. Importantly, cfDNA levels in mice return to baseline within five hours of agent administration and repeated dosing shows no evidence of toxicity. Our priming strategy should be of interest for precision oncology applications, including early detection, longitudinal monitoring of therapeutic response, and surveillance for minimal residual disease. This tumor-agnostic priming agent should also improve the performance of ctDNA analytical techniques other than mutational profiling and may even increase the recovery of cfDNA from other body fluids beyond plasma. Moreover, we believe that this work sets a precedent for the potential development of priming agents for liquid biopsy at large across other analytes. In summary, here we present a first-in-class liquid biopsy priming agent capable of improving the sensitivity and the robustness of ctDNA testing in tumor-bearing mice by modulating liver cfDNA clearance. We envision that further development of liquid biopsy priming agents will signify a major step forward for the successful deployment of precision oncology tools across all stages of cancer management. Citation Format: Carmen Martin-Alonso, Shervin Tabrizi, Kan Xiong, Ahmet Bekdemir Bekdemir, Sahil Patel, Zhenyi An, Timothy Blewett, Sainetra Sridhar, Douglas Shea, Ava Amini, Jesse D. Kirkpatrick, Jin Bae, Eugenia Roberts, Ruolin Liu, Justin Rhoades, Todd Golub, J. Christopher Love, Viktor A. Adalsteinsson, Sangeeta N. Bhatia. A liposomal priming agent increases the sensitivity of liquid biopsies. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr PR007.

Analytical validation of OriMIRACLE S, a personalized and tumor-informed approach to minimal residual disease surveillance.

e15013 Background: The presence of minimal residual disease (MRD) after curative-intent therapies in solid tumors is recognized as a risk factor for relapse. Glycoprotein-based tumor markers lack sensitivity and specificity for clinical application. Circulating tumor DNA (ctDNA) based MRD detection which can identify disease relapse ahead of radiological imaging has shown promising performance. The objective of this study was to develop and validate OriMIRACLE S (Minimal Residual Circulating Nucleic Acid Longitudinal Detection in Solid Tumor), a highly sensitive and specific tumor-informed assay for MRD detection via plasma cell-free DNA. Methods: Tumor-specific somatic single nucleotide variants (SNVs) were identified by whole exome sequencing of tumor tissue and matched germline DNA. Clonal SNVs were selected by OriSelector algorithm for patient-specific multiplex PCR-based NGS assay for MRD tracking. The limit of detection (LOD), specificity, sensitivity, repeatability, and reproducibility of this assay were determined experimentally with a series of standard references and in silico. Standard references were developed as following for assessing assay performance. The somatic variant references were two commercialized standards each containing five positive sites at variant allele frequency [VAF] of 0.1% and 1%, and a wild type sample. The single nucleotide polymorphism (SNP) references were made by blending three gDNA samples (160 sites) with gDNA samples without these SNPs to VAFs of 0%, 0.005%, 0.01%, 0.02%, 0.05% and 0.1%. And cfDNA references were made by titrating the clinical cfDNA mixture from five patients containing 31 positive sites with mononucleosomal HEK293 DNA to VAFs of 0.01-0.3% and 0.1-3%. Results: Site level sensitivity of ctDNA samples were 68.8% to 100% with 10 to 50ng input ctDNA (3,000 to 15,000 haploid genome equivalents). Site level reproducibility and repeatability were 98.0% and 97.0% using somatic variant references, respectively. The detection of three positive sites was sufficient to achieve a nearly 100% overall sample level sensitivity and specificity (6,000 haploid genome equivalents of gDNA samples) determined either by calculating a binomial probability or in silico with customized panels have 21 to 30 variants. The LOD of this assay was as low as 0.01% with the algorithm of OriCleverClover which could filter process and sequencing-related artifacts. Conclusions: OriMIRACLE S has been analytically validated as a highly sensitive and specific method to detect low quantities of ctDNA in plasma. The low limit of detection at a VAF of only 0.01% with low DNA input. OriMIRACLE S has wide-ranging potential applications in the clinical setting and drug development in a real-world scenario.

Liquid Biopsy-Based Biosensors for MRD Detection and Treatment Monitoring in Non-Small Cell Lung Cancer (NSCLC).

Globally, non-small cell lung cancer (NSCLC) is the leading cause of cancer deaths. Despite advancements in chemotherapy and targeted therapies, the 5-year survival rate has remained at 16% for the past forty years. Minimal residual disease (MRD) is described as the existence of either isolated tumour cells or circulating tumour cells in biological liquid of patients after removal of the primary tumour without any clinical signs of cancer. Recently, liquid biopsy has been promising as a non-invasive method of disease monitoring and treatment guidelines as an MRD marker. Liquid biopsy could be used to detect and assess earlier stages of NSCLC, post-treatment MRD, resistance to targeted therapies, immune checkpoint inhibitors (ICIs) and tumour mutational burden. MRD surveillance has been proposed as a potential marker for lung cancer relapse. Principally, biosensors provide the quantitative analysis of various materials by converting biological functions into quantifiable signals. Biosensors are usually operated to detect antibodies, enzymes, DNA, RNA, extracellular vesicles (EVs) and whole cells. Here, we present a category of biosensors based on the signal transduction method for identifying biosensor-based biomarkers in liquid biopsy specimens to monitor lung cancer treatment.

Minimal residual disease surveillance at day 90 predicts long-term survival in pediatric patients with T-cell acute lymphoblastic leukemia

To evaluate the optimal time to monitor minimal residual disease (MRD) for pediatric patients with T-cell acute lymphoblastic leukemia (T-ALL). Children newly diagnosed with T-ALL were treated per the CCLG-ALL2008 protocol in our hospital. MRD was monitored at days 15, 33 and 90, and the patients were stratified as low-, intermediate- or high-risk according to MRD at days 33 and 90. The 5-year event-free survival (EFS) and overall survival (OS) rates for all patients were 60.1 ± 5.6% and 63.1 ± 5.6%, respectively. The median follow-up time was 54 (0.3–120) months. Univariate analysis showed that the 5-year EFS rate correlated with MRD at days 33 and 90 (p < .01). Multivariate analysis demonstrated that only MRD at day 90 and involvement of the central nervous system (CNS) were independent prognostic factors. MRD at day 90 likely provides better prognostic value for pediatric T-ALL patients. Clinical Trial Registration: ClinicalTrials.gov identifier: NCT00707083

MiR-638 in circulating leukaemia cells as a non-invasive biomarker in diagnosis, treatment response and MRD surveillance of acute promyelocytic leukaemia.

MicroRNA (miRNA) expression has been implicated in leukaemia. In recent years, miRNAs have been under investigation for their potential as non-invasive biomarkers in acute promyelocytic leukaemia (APL). We investigated whether miR-638 in circulating leukaemia cells is a non-invasive biomarker in diagnosis, assessment of the treatment response and minimal residual disease (MRD) surveillance of APL. Sixty cases of acute myeloid leukaemia (AML), including 30 cases of APL and 30 cases of non-APL AML, were selected. Thirty healthy controls were also selected. Bone marrow (BM) and peripheral blood (PB) samples were collected from APL patients at diagnosis and post-induction. Microarray analysis and quantitative real-time PCR (qRT-PCR) were performed for miRNA profiling and miR-638 expression analysis, respectively. For statistical analysis, Mann-Whitney U test, Wilcoxon Signed Rank test, receiver operating characteristic (ROC) curve analysis and Spearman's rho correlation test were used. Both microarray and qRT-PCR data showed that miR-638 was significantly upregulated in BM after APL patients received induction therapy. Moreover, miR-638, which is specifically downregulated in APL cell lines, was upregulated after all-trans retinoic acid (ATRA)-induced myeloid differentiation. Receiver operating characteristic (ROC) curve analyses revealed that miR-638 could serve as a valuable biomarker for differentiating APL from controls or non-APL AML. Furthermore, miR-638 expression was sharply increased after induction therapy and complete remission (CR). An inverse correlation was observed between miR-638 and PML-RARα transcripts levels in BM samples, while a positive correlation was revealed between PB miR-638 and BM miR-638 levels in APL patients after induction therapy. Our study suggested that miR-638 may serve as a potential APL biomarker for diagnosis and assessment of the response to targeted therapy, and PB miR-638 could be used for non-invasive MRD surveillance in APL.

Accurate and Sensitive Analysis of Minimal Residual Disease in Acute Myeloid Leukemia Using Deep Sequencing of Single Nucleotide Variations

Minimal residual disease (MRD) in acute myeloid leukemia (AML) is of major prognostic importance. The genetic landscape of AML is characterized by numerous somatic mutations, which constitute potential MRD markers. Leukemia-specific mutations can be identified with exome sequencing at diagnosis and assessed during follow-up at low frequencies by using targeted deep sequencing. Our aim was to further validate this patient-tailored assay for substitution mutations. By applying a statistical model, which corrects for position-specific errors, a limit of detection for single nucleotide variations of variant allele frequency (VAF) of 0.02% was achieved. The assay was linear in MRD range (0.03% to 1%) with good precision [CV, 4.1% (2.2% to 5.7%) at VAF 1% and 13.3% (8.8% to 19.4%) at VAF 0.1%], and low relative bias [7.9% (2.5% to 15.3%) at VAF 1%]. When applied to six childhood AML cases and compared with multiparameter flow cytometry for MRD analysis, deep sequencing showed concordance and superior sensitivity. Further high concordance was found with expression of fusion transcripts RUNX1-RUNX1T1 and KMT2A-MLLT10. The deep sequencing assay also detected mutations in blood when VAF in bone marrow exceeded 0.1% (n=19). In conclusion, deep sequencing enables reliable detection of low levels of residual leukemic cells. Introduction of this method in patient care will allow for highly sensitive MRD surveillance in virtually every patient with AML.

Simple deep sequencing-based post-remission MRD surveillance predicts clinical relapse in B-ALL

BackgroundNext-generation sequencing (NGS) of the rearranged immunoglobulin heavy-chain gene has emerged as a highly sensitive method to detect minimal residual disease (MRD) in B acute lymphoblastic leukemia/lymphoma (B-ALL). However, a sensitive and easily implemented NGS methodology for routine clinical laboratories is lacking and clinical utility of NGS-MRD surveillance in a post-remission setting to predict clinical relapse has not been determined.MethodsHere we described a simple and quantitative NGS platform and assessed its performance characteristics, quantified NGS-MRD levels in 122 B-ALL samples from 30 B-ALL patients, and explored the clinical merit of NGS-based MRD surveillance.ResultsThe current NGS platform has an analytic sensitivity of 0.0001% with excellent specificity and reproducibility. Overall, it performs better than routine multi-color flow cytometry (MCF) in detecting MRD. Utilizing this assay in MRD surveillance in a post-remission setting showed that it detected conversion to positive MRD (CPMRD) in patients with NGS-based molecular remission much earlier than MCF, and that positive MRD conversion could be detected as early as 25.6 weeks prior to clinical relapse in closely surveilled patients. Post-remission CPMRD, but not NGS-based MRD positivity at end of induction, can accurately predict clinical relapse in our limited cohort of B-ALL patients.ConclusionsThis pilot proof-of-concept study illustrates the clinical utility of a simple, sensitive, and clinically feasible MRD detection platform in post-remission NGS-based MRD surveillance and early relapse detection in B-ALL patients.

Novel markers for determining risk and evaluation of minimal residual disease in diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease that is usually curable with frontline immunochemotherapy and whose prognosis is directly impacted by the usual prognostic factors grouped within the International Prognostic Index (IPI). Despite the overall improvement in the outcome of patients with DLBCL since the introduction of rituximab, 40% of patients are primarily refractory or experience short-term relapse and have an extremely poor prognosis. The prognostic impact of the response to treatment as assessed by intermediate PET could be increased by combining it with the results of the modern molecular biology techniques, which include measuring the VDJ rearrangement in blood, or Next generation sequencing (NGS) of a dedicated somatic mutation panel in circulating tumor DNA, according to the concept of liquid biopsy. Emerging data has also suggested that these methods would be a powerful tool in the assessment of minimal residual disease (MRD) in DLBCL. Important questions remain about the most appropriate timing and the most efficient technique for evaluating the MRD throughout the course of the disease. We conducted a review of the recent literature to present, summarize and explain the major pilot studies that have established novel markers for determining baseline risk in DLBCL, early assessment of disease response and post-therapeutic MRD surveillance. Indeed, in the coming years, the evaluation of the MRD will be a secondary objective of all major clinical trials in this pathology, in order to confirm its clinical relevance and reproducibility, before setting up clinical trials based on the evaluation of the MRD as the primary decision-making criterion for the adaptation of patient treatment.

Noninvasive Genotyping and Assessment of Treatment Response in Diffuse Large B Cell Lymphoma

Background and MethodsDLBCL patients exhibit striking heterogeneity in therapeutic responses, yet current methods to assess residual disease have shown suboptimal performance. Circulating tumor DNA (ctDNA) is a promising noninvasive biomarker for disease burden. While High-Throughput Sequencing of immunoglobulin genes (Ig-HTS) has shown utility for ctDNA detection in DLBCL, only a small number of tumor markers are profiled, limiting its sensitivity and broad applicability (Kurtz DM, Blood 2015 and Roschewski M, Lancet Oncology 2015). Here, we studied DLBCL patients using CAPP-Seq, a recently developed approach for ultrasensitive assessment of ctDNA that profiles multiple genomic regions across hundreds of kilobases in a single HTS assay (Newman AM, Nat. Med. 2014).ResultsWe designed a 250kb DLBCL CAPP-Seq panel targeting 1053 regions from 268 genes, incorporating recurrent single nucleotide variants (SNVs), insertions/deletions (indels) and rearrangements, and evaluated its genotyping performance on diverse tumors from 63 patients (Table 1). We detected somatic mutations in 98% of patients (median: 143 variants/pt), including AID-related events in well-known hotspots. Moreover, we detected 100% of BCL2/BCL6 rearrangements and most MYC translocations previously identified by FISH (n = 30).Biopsy-confirmed tumor mutations were detectable with 99.3% specificity in 97% of pretreatment plasma samples (n = 37) with a range of 0.007% to 32% mean fractional abundance. Median recovery rates for both SNVs and fusions in plasma exceeded 85%, indicating robust tumor genotyping performance. Furthermore, in most patients, paired allelic fractions between tumor biopsies and pretreatment plasma samples were significantly correlated (P < 0.05), demonstrating that ctDNA can accurately mirror the mutational profiles found in tumor tissues.We next evaluated the utility of direct biopsy-free genotyping of pretreatment plasma using a novel ctDNA genotyping approach. We noninvasively identified variants in 97% of cases (n=37), and confirmed a median of 95% of SNVs per cfDNA sample in paired tumor biopsies, indicating that plasma DNA is an effective surrogate for direct tumor genotyping.Having established the technical performance in pretreatment cfDNA, we next assessed utility of CAPP-Seq for minimal residual disease (MRD) detection (Figure 1). Plasma samples at time points of complete response (n = 22) were collected from 10 patients, all of whom ultimately relapsed. Each plasma sample was then analyzed for evidence of biopsy-confirmed mutations from pretreated tumors. Strikingly, ctDNA was detected in 9 of 10 patients (90%), including 3 of 3 isolated CNS relapses, with fractional abundances as low as 0.004%. Among patients with CAPP-Seq detectable MRD, the median time between first detection and relapse was 162 days, and all patients whose MRD levels were uniformly below the detection limit had blood collections >8 months prior to relapse.ConclusionsTo summarize, we validated robust technical performance of a novel approach for noninvasive tumor genotyping in DLBCL using pretreatment plasma, and for MRD surveillance. Given the demonstrated advantages of CAPP-Seq in the setting of radiographic remission, we envision its utility for improved relapse prediction. Moreover, we anticipate that this biopsy-free genotyping approach will have applications for monitoring subclonal dynamics and emergent mutations in response to therapy.FS, DMK and AMN contributed equally. [Display omitted] [Display omitted] DisclosuresNewman:Roche: Consultancy. Klass:Roche: Employment. Diehn:Roche: Consultancy. Alizadeh:Genentech: Consultancy; Celgene: Consultancy; Roche: Consultancy.

Retrospective Analysis of Minimal Residual Disease-Guided Preemptive Treatment in Patients with AML and MDS - the SAL Study Group Experience

IntroductionDetection of minimal residual disease (MRD) in patients with acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) in complete hematological remission (CR) offers a potential therapeutic window allowing early interventions in order to prevent overt hematological relapse. Due to the absence of generic markers in AML/MDS, MRD assessment seems to be a complex procedure. As prospective clinical studies are sparse, we initiated a retrospective survey within the SAL (Study Alliance Leukemia) study group and members of the German Cancer Consortium to evaluate the current clinical use of the approach and related clinical outcome of treated patients.PatientsBased on questionnaires we analyzed 84 patients from five clinical centers (pts, m/f=46/38; median age 52 yrs (19-74)) with either AML (n=77) or MDS (n=7) in CR after conventional intensive chemotherapy (CTx; n=23) or allogeneic hematopoietic stem cell transplantation (allo-HSCT; n=61), undergoing preemptive therapy. Mutations, cytogenetic aberrations or donor chimerism analysis for transplanted pts were monitored as MRD markers. Thirty-three pts had a normal and 14 pts - a complex karyotype; 6 pts carried -7 and/or inv(3); other aberrations were detected in 29 pts; in 2 pts no data were available. Among molecular aberrations, NPM1 (n=23) and FLT3-ITD (n=13) were the most frequent, followed by CBFβ/MYH11 (n=8), RUNX1/RUNX1T1 (n=8) and MLL/AF6 (n=3). In 20 pts no mutations were found. For 5 pts data on molecular diagnostics were not available and MRD was assessed by chimerism.ResultsThe median time to MRD positivity after completion of intensive therapy was 12 months (range, 1-97). Subsequent MRD-guided therapy in pts treated only with CTx included hypomethylating agents (HMA, 34%), clofarabine (17%), additional intensive CTx (9%), targeted therapy (gemtuzumab ozogamycin, 9%), low-dose cytarabine (9%), and allo-HSCT (22%). Other treatment strategies included interferon alfa and sorafenib. In transplanted patients the most preferred treatment for the molecular relapse was donor lymphocyte infusion (26 pts, 43%; median number of DLI=3, range, 1-6), alone or in combination (54% with HMA). 32 MRD-based treated pts (43%) did not experience a hematological relapse and were either alive (23 pts, median observation time 953 days, range, 30-3660), or died due to another cause (9 pts) with median survival of 359 days (range, 125-954). In 9 pts without hematological relapse more than one MRD recurrence was observed. In the remaining 57% relapsing patients, median time to hematological relapse was 252 days (range, 24-1161) after MRD positivity. Interestingly, in some pts the hematological relapse was observed in the absence of the known mutation, indicating that the disease probably progressed due to another subclone. Thus, an accurate initial diagnostic is essential, ideally a multiple gene approach and comprehensive follow-up.ConclusionsThe retrospective analysis demonstrates that MRD-guided therapies have already entered routine practice in patients with AML and MDS. The heterogeneous nature of both cancer entities is reflected by the variety of MRD surveillance protocols among clinical centers. Therefore, clinical trials are needed to better define molecular markers and subsets of patients who might benefit from this approach. DisclosuresMayer:Janssen: Research Funding. Thiede:AgenDix GmbH: Equity Ownership. Platzbecker:Novartis: Honoraria; GlaxoSmithKline: Honoraria, Research Funding; Celgene: Honoraria; Amgen, Inc.: Honoraria.

Minimal residual disease surveillance in chronic lymphocytic leukemia by fluorescence-activated cell sorting.

Achievement of complete response (CR) to therapy in chronic lymphocytic leukemia (CLL) has become a feasible goal, directly correlating with prolonged survival. It has been established that the classic definition of CR actually encompasses a variety of disease loads, and more sensitive multiparameter flow cytometry and polymerase chain reaction methods can detect the disease burden with a much higher sensitivity. Detection of malignant cells with a sensitivity of 1 tumor cell in 10,000 cells (10–4), using the abovementioned sophisticated techniques, is the current cutoff for minimal residual disease (MRD). Tumor burdens lower than 10–4 are defined as MRD-negative. Several studies in CLL have determined the achievement of MRD negativity as an independent favorable prognostic factor, leading to prolonged disease-free and overall survival, regardless of the treatment protocol or the presence of other pre-existing prognostic indicators. Minimal residual disease evaluation using flow cytometry is a sensitive and applicable approach which is expected to become an integral part of future prospective trials in CLL designed to assess the role of MRD surveillance in treatment tailoring.

Combination Imatinib with Interferon-α For Philadelphia Positive Acute Lymphocytic Leukemia: A Mutiple Centers Study In China

BackgroundImatinib combined with chemotherapy for induction and intensification therapy has become the standard strategy for Philadelphia-positive acute lymphocytic leukemia (Ph+ALL). Anyhow, intensified chemotherapy lead to about 5% of early death. Because we believed that imatinib plays the most important role in Ph+ALL treatment, we started Ph+ALL-HX-200803 trial (WHO ICTRP Registry No. ChiCTR-TNRC-00000309 ) to test the effect of combining imatinib and low dose chemotherapy and added interferon-¦Á in maintenance to prevent relapse. MethodAccording to our protocol, all patients received imatinib 400mg daily, vindesine 4 mg weekly and dexamethasone 10mg/m2/day for 4 days per week for 4 weeks as induction therapy. For patients under 55 years old, we give them three sequential courses of intensified chemotherapy (CAM, high dose MTX+L-Asp, and MA, CALLG2008 protocol). Patients in CR1 would receive allogenic HSCT if they had suitable donors. Those who were reluctant to receive or unsuitable for allo-HSCT received maintenance therapy with imatinib 400mg daily, interferon-¦Á 3 million unit 2-3 doses per week and chemotherapy. Maintenance chemotherapy including vindesine and dexamethasone was given monthly in the first year, once every two months in the second year, and once every three months in the third year. For patients over 55 years old, we skipped intensified chemotherapy and gave them maintenance therapy directly. Minimal residual disease surveillance was conducted by BCR/ABL fusion gene quantification. The main endpoints were 3-year overall survival and disease free survival. ResultBetween 2008 and 2012, 50 patients with newly diagnosed Ph+ALL were enrolled. The median age of this group of patients was 35.5 years old. All but one patients achieved complete remission (98%) after 4 weeks induction therapy. No patient died during induction therapy. The median follow-up time was 27 months. The estimated 3-year DFS and OS were 38.8 ±9.2% and 52.7±10.4%. Five patients received allo-HSCT in CR1. For patients who did not receive allo-HSCT in CR1, 1 patient survived for more than 5 years, 7 patients survived for more than 3 years. In post-hoc analysis, patients achieved MRD negativity at six month showed better median DFS (not reached vs. 11 moths, p=0.001) and OS (not reached vs. 22 months, p=0.015) compared to those who did not. ConclusionThe outcomes of our study suggest that imatinib combing with low dose chemotherapy showed a high and safe induction remission rate, combination of interferon-¦Á with imatinib and maintenance chemotherapy might improve the outcomes of patients with Ph+All who were not eligible for Allo-HSCT. MRD status at six month is an important prognostic indicator. The long term disease-free survivors may signal the possibility of a cure even without allo-HSCT. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.