Minimal Residual Disease Testing Research Articles

Individualized Antigen Expression in Precursor T-Cell Acute Lymphoblastic Leukemia

BACKGROUND: In T-acute lymphoblastic leukemia (T-ALL), multi-parametric flow cytometry can serve to detect minimal residual disease (MRD) by using immature or aberrant antigens expression as well as the altered expression of T-cell antigens. The latter approach has been specifically introduced to overcome the absence of leukemia-associated antigens. However, there is no agreed-upon method for the use of T-cell antigens in T-ALL MRD testing. AIMS AND OBJECTIVES: To compare the expression of classic T-cell antigens on T-lymphoblasts and T-lymphocytes to establish a protocol for their use in MRD analysis. MATERIALS AND METHODS: Flow cytometric data of PB or BM samples from 63 adults with T-ALL were collected. We assessed the frequency and degree of brightness or dimness of each T-cell marker, in addition to studying the uniformity of the events scatter of a total of 287 follow-up BM samples from 50 patients. RESULTS: Significant differences in expression intensity of T-cell markers were found between T-lymphoblasts and T-lymphocytes; they were reasonably stable on blasts in follow up samples. This detailed study has nominated the conjoint use sCD3neg/dim and CD5dim/neg in the identification of residual cells, to be supported by other T-cell markers. CONCLUSION: The suggested gating sequence showed an acceptable level of accuracy in detecting residual leukemia, supporting their use in T-ALL MRD especially when other distinguishing markers might be absent in the diagnosis sample, or susceptible to be lost with induction therapy.

Genomic Alterations in Acute Myeloid Leukemia in Routine Clinical Practice

Acute Myeloid Leukemia (AML), the most common acute leukemia in adults, is a genetically heterogeneous disease. Genomic alterations condition the pathophysiology of AML. Nowadays, these changes are considered to be important biomarkers for risk stratification, treatment decisions (including new targeted drugs) and Minimal Residual Disease (MRD) monitoring during followup of AML. Positive MRD in AML patients is associated with a higher risk of relapse and shorter overall survival compared to MRD negative individuals. Nevertheless, MRD-targets, diagnostic techniques, time points of MRD determination, and analyzed material are not standardized. Thus, integration of MRD testing in individual AML patients in routine practice is still a workin- progress. This review article comprehensively focuses on key molecular biomarkers in AML and their utilization in clinical practice, especially regarding risk assessment and MRD testing. Moreover, new AML molecular-targeted therapies are briefly summarized here.

Minimal residual disease detection by next-generation sequencing in multiple myeloma: Promise and challenges for response-adapted therapy.

Assessment of minimal residual disease (MRD) is becoming a standard diagnostic tool for curable hematological malignancies such as chronic and acute myeloid leukemia. Multiple myeloma (MM) remains an incurable disease, as a major portion of patients even in complete response eventually relapse, suggesting that residual disease remains. Over the past decade, the treatment landscape of MM has radically changed with the introduction of new effective drugs and the availability of immunotherapy, including targeted antibodies and adoptive cell therapy. Therefore, conventional serological and morphological techniques have become suboptimal for the evaluation of depth of response. Recently, the International Myeloma Working Group (IMWG) introduced the definition of MRD negativity as the absence of clonal Plasma cells (PC) with a minimum sensitivity of <10−5 either by next-generation sequencing (NGS) using the LymphoSIGHT platform (Sequenta/Adaptative) or by next-generation flow cytometry (NGF) using EuroFlow approaches as the reference methods. While the definition of the LymphoSIGHT platform (Sequenta/Adaptive) as the standard method derives from its large use and validation in clinical studies on the prognostic value of NGS-based MRD, other commercially available options exist. Recently, the LymphoTrack assay has been evaluated in MM, demonstrating a sensitivity level of 10−5, hence qualifying as an alternative effective tool for MRD monitoring in MM. Here, we will review state-of-the-art methods for MRD assessment by NGS. We will summarize how MRD testing supports clinical trials as a useful tool in dynamic risk-adapted therapy. Finally, we will also discuss future promise and challenges of NGS-based MRD determination for clinical decision-making. In addition, we will present our real-life single-center experience with the commercially available NGS strategy LymphoTrack-MiSeq. Even with the limitation of a limited number of patients, our results confirm the LymphoTrack-MiSeq platform as a cost-effective, readily available, and standardized workflow with a sensitivity of 10−5. Our real-life data also confirm that achieving MRD negativity is an important prognostic factor in MM.

Why is it Important to Identify Residual Cells in Leukaemia Patients after Treatment? A Review Article

Introduction: Most children diagnosed with acute lymphoblastic leukemia are cured and are not at risk of relapse. However, 20% of children are at a high risk of experiencing relapse later on in their lives. In order to detect risk and obtain prognostic information, the quantification of minimal residual disease (MRD) can be utilized. Detection of MRD can lead to efficient identification of relapse risk. However, there is limited understanding of the association between MRD and long-term outcomes after treatment in children. Therefore, this systematic review will examine existing literature to determine the strength of association between MRD negativity and relapse risk in children and its importance in the prediction of relapse. Methods: A systematic review of 5 articles centered around ALL in children was analyzed to examine the strength of association between MRD negativity and clinical outcomes of event-free survival (EFS) and overall survival (OS) following the PRISMA guideline. The literature search was done through databases such as NCBI, PubMed, and other childhood oncology databases. The inclusion criteria included peer-reviewed clinical studies that focus on ALL relapse risk and MRD detection. Additionally, reviews, abstracts, and studies with inadequate sample sizes or correlations were excluded. Data were extracted and organized based on criteria of MRD negativity, MRD detection type, and relapse risk level. The data collected from all studies were analyzed through a meta-analysis. The five publications discussed in this article were a total of 11,265 participants. Results: The results portion of your abstract should concisely describe a summary of the main findings. A positive correlation was determined between EFS and OS hazard ratios and MRD detection. Discussion: The analysis of the five publications demonstrated that MRD is an important marker and a strong predictor of relapse in children who are diagnosed with ALL. Conclusion: MRD detection can be proposed as a method of predicting a high risk of relapse in children with ALL. In essence, this literature review has the potential to identify the clinical and therapeutic significance of MRD testing which can be utilized to predict and prevent relapse of ALL in children.

Prognostic value of minimal residual disease among patients with classical Hodgkin lymphoma undergoing autologous stem cell transplantation

Improved biomarkers are needed to guide patient selection for autologous stem cell transplantation (ASCT) and post-ASCT maintenance therapies in relapsed/refractory classical Hodgkin lymphoma (cHL). To assess the prognostic value of minimal residual disease (MRD) using immunoglobulin-based high-throughput sequencing (Ig-HTS), we analyzed pre- and post-ASCT peripheral blood and pre-ASCT apheresis stem cell (ASC) samples in 36 cHL patients. A tumor clonotype was detected in only 12 patients (33%). Among these patients, MRD within plasma samples was closely associated with impending relapse. All patients (n = 3) with detectable MRD in any post-ASCT plasma sample relapsed (100% specificity), and MRD was not detected in any patients in remission. MRD testing from cellular specimens (peripheral blood mononuclear cell or ASC samples) was not associated with relapse. In this small cohort, plasma-based MRD testing appeared to be a promising biomarker in cHL, but given low clonotype detection rates with Ig-HTS, alternative MRD approaches should be investigated.

O-036 Safe and successful ovarian transplantations post leukemia after combined various MRD excluding tests

Abstract Study question Leukemias are considered high risk for minimal residual disease (MRD) contamination in cryopreserved ovarian tissue. How improving the safety of ovarian transplantation in leukemia patients? Summary answer Combination of various methods; immunohistochemistry, molecular biology and long period xenotransplantation to immune-deficient mice is obligatory for safety of ovarian transplantation (OT) in high-risk patients What is known already With high percentage of cancer survivors and high treatment induced ovarian failure in leukemia patients, more cured patients request OT to restore fertility. Studies using different methods have identified MRD contamination in cryopreserved ovarian tissue, which could potentially result in recurrence of the primary disease after reimplantation. This risk is high in leukemia patients thus many caregivers avoid OT in these patients. Today, different methods for identifying MRD like immunohistochemistry (IHC), FISH molecular methods, xenotransplantation into immune deficient mice are used to evaluate MRD in ovary before OT Study design, size, duration Between 2005 and 2022 ovarian tissue cryopreservation was performed in 74 leukemia patients. Twenty one leukemic patients were evaluated for MRD in the ovarian tissue. sixteen women were diagnosed with AML, 3 ALL and 2 suffered from CML. To evaluate MRD, one stored ovarian piece was thawed prior to transplantation, and different, personally determined methods, were used according with multidisciplinary team instructions. Only when all tests were negative OT was performed Participants/materials, setting, methods One ovarian piece was thawed and divided into 5 for evaluation. One used for H&amp;E and immunohistochemistry (IHC) to check for follicles presence and macroscopic malignancy evidence. One was used to check molecular markers by extracting DNA/RNA and performing molecular PCR, RT-PCR and next generation sequencing (NGS). In patients without molecular markers at diagnosis, leukemia cells were evaluated using NGS to identify new markers. Pieces were xenotransplanted into 3 SCID mice for 6 months. Main results and the role of chance For MRD assessment, H&amp;E and individualized IHC were performed in all ovarian grafts with no evidence of malignancy in 20 cases. However, in one AML patient IHC identified cluster of leukemic cells and further evaluation was not performed. In all patients presence of follicles was documented. Patient specific molecular markers were checked in ovarian tissue of 13/15 AML patients. One patient presented positive marker t(15;17) via RT-PCR while other methods were negative. Ten patients underwent ovarian tissue xenotransplantation in mice. In one patient, all 3 mice developed macroscopic leukemic masses after 5 months while other methods were negative. Eight OT were performed in 4 AML patients presenting with negative MRD tests resulting in 7 pregnancies and 4 live births. Long term follow up of up to 7 years showed no evidence of disease recurrence. Ovaries of 3 ALL patients were evaluated. In one patient, RT-PCR identified positive marker (IGH rearrangement). No evidence of malignancy was identified in all xenotransplanted mice. One patient underwent OT and achieved a twin pregnancy. Ovaries of 2 CML patients were checked. 1st patient showed BCR-ABL gene in ovary via RT-PCR. The ovary of second patient was free of disease, underwent OT. In this patient, no pregnancy achieved. Limitations, reasons for caution Safe transplantation requires checking molecular markers of diagnosis in ovary. The tumor cells aren't always available. In addition, not all leukemia patients carry molecular markers. Furthermore, since MRD is checked in one piece, malignant cells might be missed. Additionally, this piece doesn't represent the other pieces which would be transplanted Wider implications of the findings OT can restore fertility. High risk of ovary contamination in leukemia patients prevents centers to OT, leaving survivors infertile. Here we show safe and successful OT following meticulous MRD assessment by multiple methods. furthermore, MRD detection protocol might be practiced on testicular frozen tissue to restore fertility in young boys Trial registration number 7222-09-SMC

P973: REAL-WORLD STUDY ON ADOPTION OF STANDARD OF CARE (SOC) FOR FRONTLINE TRANSPLANT-ELIGIBLE MULTIPLE MYELOMA (FLTEMM) PATIENTS BETWEEN 2017 AND 2020/2021 ACROSS FRANCE, GERMANY, SPAIN, AND ITALY

Background: Newer therapies for frontline transplant-eligible multiple myeloma (FLTEMM) are underway, but an accurate and updated overview of standard MM treatment management is lacking. Repeated analyses and real-world data are warranted to describe current MM standard of care (SoC), treatment lines, and patient clinical outcomes in daily practice. Aims: This non-interventional, cross-sectional, retrospective observational database study described the current SoC for FLTEMM patients in France, Germany, Spain, and Italy, and recorded the evolution in regimen adoption in distinct elements of frontline treatment (induction, consolidation, and maintenance) during 2017-2020/2021. Methods: Clinical information on ongoing (population I) or previous (population II) FLTEMM patients was extracted from the Cancerology database. The primary objective was to describe the treatment regimen considered current SoC for FLTEMM management in all four countries using study population I, and to study the evolution in the use of different regimens in induction phase using study population II. The secondary objective was to describe clinical outcomes, including best tumor response and minimal residual disease (MRD) status, associated with each induction regimen in study population II. Results: In study populations I and II, the median (min, max) age of the patients at induction initiation was 59.0 (35.0, 86.0) and 60.0 (23.0, 80.0) years and 62.9% (227/361) and 64.8% (287/443) patients were male, respectively. In study population I, the most common induction regimens were bortezomib/lenalidomide/dexamethasone (VRd) in France (75.3%, 33/44) and Spain (44.1%, 53/120), bortezomib/thalidomide/dexamethasone (VTd) in Italy (65.2%, 76/116), and others in Germany (58.9%, 48/81) (including daratumumab-based [19.8%, 16/81] or not). Maintenance was ongoing or planned for 78.3% (34/44), 62.3% (51/81), 65.2% (78/120), and 61.4% (71/116) patients in France, Germany, Spain, and Italy, respectively. Among study population I patients on an ongoing maintenance regimen, lenalidomide was received by 99.6% (20/20) patients in France, 86.8% (34/40) in Germany, 80.2% (45/56) in Spain, and 90.7% (30/33) in Italy. In study population II, VRd use as 1L induction increased from 27.0% (10/36) in 2017 to 65.7% (12/18) in 2019 in France and was relatively low in Germany, Spain, and Italy. The most common 1L induction regimen in Germany was VCd, but its use decreased from 85.2% (31/37) in 2017 to 64.1% (24/38) in 2019. VTd was the most common 1L induction in Spain and Italy, but its use declined from 58.3% (16/27) and 72.4% (34/47) in 2017 to 17.3% (4/22) and 52.8% (19/36) in 2019, respectively. In total, 37.5% (166/443) study population II patients achieved at least a complete response, which was the highest for those on VRd (59.2% versus 34.3% for VTd, 33.4% for VCd, and 33.7% for other non-daratumumab-based regimens, respectively). In total, 42.5% (188/443) study population II patients were tested for MRD status and 18.2% (81/443) were found negative. MRD testing rate varied from 26.2% in France to 14.8% in Germany, 84.4% in Spain, and 47.7% in Italy. Summary/Conclusion: The use of bortezomib triplets in induction varied markedly over time and between selected countries. Despite not being approved by the European Medicines Agency specifically for FLTEMM patients, VRd use tended to increase with time in France and to a lesser extent in Spain and Italy in this population.

Abstract 3815: Investigating the role of bone marrow microenvironment dysregulation at remission in disease outlook of multiple myeloma

Abstract Multiple myeloma (MM) is a cancer characterized by the unchecked proliferation of antibody-secreting plasma cells (PCs), which to date remains incurable despite therapeutic advancements. The idea that deeper tumor clearance leads to better outcomes is widely accepted in MM treatment: minimal residual disease (MRD) testing, whereby ultra-sensitive sequencing or fluorescence-based cytometric detection of the clonal immunoglobulin secreted by tumor PCs, has been found to be the strongest independent prognostic factor when predicting survival. However, MRD status remains an imperfect predictor. As PCs have a well-defined physiological niche within the bone marrow (BM), and patients often exhibit immunosuppressive BM dysregulation, we investigate the presence of BM microenvironment signatures present at remission that may influence clinical outcomes insufficiently explained by tumor risk strata or MRD status. We conducted 3’ single-cell RNA sequencing of 36 remission, 8 primary diagnosis, and 3 relapse whole BM aspirates taken from 35 patients enrolled in an ongoing clinical trial evaluating a treatment regimen consisting of ixazomib, lenalidomide, and dexamethasone (IRD). 19 of 35 patients were MRD+ at remission; among these, 6 have been reported as having poor outcome (PO; progression-free survival &amp;lt;2.5yrs post sample collection), 12 have had average outcomes (AO; PFS &amp;gt;2.5yrs), and 1 is awaiting assessment. The remaining 16 patients were MRD- at remission, 6 of whom had poor outcome, 7 average, and 3 awaiting assessment. Preliminary analysis of 43 samples have yielded an average capture of 8,620 cells per sample with 1,247 median genes per cell. In comparing 3 PO to 8 AO MRD- remission samples, we see clear polarity between PO and AO cells in every major immune lineage, with a majority of clusters being predominantly (&amp;gt;80%) made of either PO or AO cells. Surprisingly, T, NK, and B cells from AO samples exhibit FOS/JUN upregulation relative to those from PO samples, indicative of AP-1 transcription factor activity that is typically associated with cell proliferation. PO samples also exhibited signs of active cytotoxicity, including upregulated granzymes H and B and IL7R in T and NK cells. Sizeable PC populations were detected in several MRD- remission samples with subpopulation heterogeneity in the expression of MM marker genes; comparison against PCs isolated from healthy donor BM suggests possible persistence of disease phenotypes not captured by MRD testing. We further investigate BM microenvironment evolution between primary diagnosis, remission, and relapse timepoints to elucidate changes in response to tumor clearance. Overall, this study enhances our understanding of how immune surveillance and interaction may influence treatment response in MM. Citation Format: Julia T. Wang, Mark Fiala, Julie Fortier, Ruiyang Liu, Reyka Jayasinghe, Ravi Vij, Li Ding. Investigating the role of bone marrow microenvironment dysregulation at remission in disease outlook of multiple myeloma [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 3815.

Abstract 2793: Minimal residual disease (MRD) as a predictive biomarker/endpoint for novel drug development

Abstract MRD is defined as the continual presence of cancer cells in patients post therapy that is below the threshold of detection by conventional morphologic or radiologic assessments. Monitoring patient status clinically as MRD negative or MRD positive has become the basis for predicting outcomes, remission or reoccurrence, as well as determining therapeutic options. The three primary methods to assess MRD status in patients include Flow Cytometry (FC), Polymerase Chain Reaction (PCR) and Next-Generation Sequencing (NGS). All three are highly quantitative measurements with a high degree of sensitivity to detect “new” circulating tumor cells or ctDNA that can potentially indicate disease recurrence before it happens. The choice of MRD methodology usually depends on the tumor type, liquid or solid. Flow Cytometry has been widely used to monitor MRD status in patients with hematologic malignancies. Specifically designed MRD panels have been used clinically in CLL, MM, CML and AML patients. In addition to its prognostic/diagnostic relevance, recent guidance by the FDA is now driving the use of MRD testing as a predictive biomarker/endpoint for novel cancer drug development. NeoGenomics Pharma Services has provided flow cytometry MRD expertise to multiple sponsors conducting clinical trials to evaluate the effectiveness of novel cancer therapy. In a CLL study, NeoGenomics employed the ERIC MRD assessment to monitor patient status during therapeutic intervention at various points throughout treatment. Representative patient data show progression from full, active disease prior to therapy to non-detectable disease levels below 0.01%MRD. These examples show patient progression through multiple cycles of treatment to full remission. Clearly, this is an example of how MRD can serve as the primary “biomarker” to evaluate a patient’s response to novel drug therapy in clinical trials. This presentation will further discuss the data and the relevance of MRD testing to support new drug development. Citation Format: Nicholas Jones, Brian Ngo, Floyd Davis, Benjamin Fancke. Minimal residual disease (MRD) as a predictive biomarker/endpoint for novel drug development [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 2793.

Circulating Tumor DNA and Late Recurrence in High-Risk Hormone Receptor-Positive, Human Epidermal Growth Factor Receptor 2-Negative Breast Cancer.

To examine the prevalence and dynamics of circulating tumor DNA (ctDNA) and its association with metastatic recurrence in patients with high-risk early-stage hormone receptor-positive breast cancer (HR+ BC) more than 5 years from diagnosis. We enrolled 103 patients with high-risk stage II-III HR+ BC diagnosed more than 5 years prior without clinical evidence of recurrence. We performed whole-exome sequencing (WES) on primary tumor tissue to identify somatic mutations tracked via a personalized, tumor-informed ctDNA test to detect minimal residual disease (MRD). We collected plasma at the time of consent and at routine visits every 6-12 months. Patients were followed for clinical recurrence. In total, 85 of 103 patients had sufficient tumor tissue; of them, 83 of 85 (97.6%) patients had successful whole-exome sequencing. Personalized ctDNA assays were designed targeting a median of 36 variants to test 219 plasma samples. The median time from diagnosis to first sample was 8.4 years. The median follow-up was 10.4 years from diagnosis and 2.0 years from first sample. The median number of plasma samples per patient was two. Eight patients (10%) had positive MRD testing at any time point. Six patients (7.2%) developed distant metastatic recurrence, all of whom were MRD-positive before overt clinical recurrence, with median ctDNA lead time of 12.4 months. MRD was not identified in one patient (1.2%) with local recurrence. Two of eight MRD-positive patients had not had clinical recurrence at last follow-up. In this prospective study, in patients with high-risk HR+ BC in the late adjuvant setting, ctDNA was identified a median of 1 year before all cases of distant metastasis. Future studies will determine if ctDNA-guided intervention in patients with HR+ BC can alter clinical outcomes.

P-283 Performance characteristics of a tumor-informed circulating tumor DNA (ctDNA) minimal residual disease (MRD) assay in stage 1-3 colorectal cancer (CRC) in clinical practice

Detection of MRD has emerged as a tool for risk stratification in patients with early stage CRC with potential implication for adjuvant therapy. Recently, the sensitivity of MRD testing for early detection of tumor recurrence/ metastasis compared with conventional imaging has been questioned. We sought to evaluate the diagnostic performance characteristics of the Signatera™ ctDNA assay in patients with stage 1-3 CRC at West Cancer Center and Research Institute (WCCRI). Patients with CRC who received ctDNA MRD testing 3-8 weeks after curative surgical resection for stage 1-3 disease from 12/2019 to 02/2022 were evaluated. Both patients with single testing and serial ctDNA assays were included. Individual chart review was performed to collect demographic information, verify diagnosis, stage, pathology, ctDNA and imaging results, and treatment course. ctDNA positivity rates were calculated and analyzed by diagnosis, stage, and clinical course. MRD results from 104 patients were available, 64 of which had sequential results. Positivity rate at initial testing was associated with stage: stage 1 0/9 pts, stage 2 5/40 (12.5%), stage 3 15/55 (27%). In the 64 patients with sequential data, conversion from negative to positive occurred in 7/50 (14%), 8/14 patients initially positive results converted to negative after adjuvant therapy, 5 of which developed ctDNA positivity in further monitoring after 3-14 months (range) with to date 4 recurrences confirmed by imaging. 27/104 patients were found to be ctDNA positive at any time point of which 2 patients were initially ctDNA negative with presence of overt disease (1 small pulmonary metastases, 1 incidentally found peritoneal nodule). In sequential ctDNA monitoring, both patients eventually showed positive ctDNA results. Only 1 patient had a false positive result at low Mean Tumor Molecule level (0.06 MTM/mL). Tumor-informed ctDNA MRD testing has very high sensitivity and specificity, but a one-time negative test result does not preclude the presence of metastatic disease, in particular with regard to pulmonary and peritoneal tumor manifestations. Prospective randomized and non-randomized studies are underway to further assess the clinical utility of ctDNA MRD testing in early stage CRC.

Liquid biopsy (LB)-based comprehensive genomic profiling (CGP) of circulating tumor DNA (ctDNA) for the evaluation of patients with myeloid neoplasms.

e19064 Background: LB-based CGP of ctDNA can facilitate the diagnosis, molecular profiling, and monitoring of patients with myeloid neoplasia, particularly when standard CGP is not feasible due to a lack of circulating disease or insufficient tumor in the marrow or tissue. Methods: Retrospective study of LB samples from patients reported to have histiocytic neoplasms (HNs), myelodysplastic syndrome (MDS), myeloproliferative neoplasms (MPN), or acute myeloid leukemia (AML) tested using hybrid-capture next-generation sequencing using the FoundationOne Liquid CDx (targeting 324 genes), FoundationOne Liquid (70 genes), or FoundationACT (62 genes) assays during routine clinical care between 5/2016 and 9/2021. Results: Among 48,459 unique patient samples submitted for CGP, 83 were from myeloid neoplasia: 34 HN, 23 MDS, 15 MPN, and 11 AML. The median cell-free DNA yield was 74.9 ng (interquartile range [IQR]: 52.0-167.4), well above the minimum needed for analysis. Reportable pathogenic alterations were detected in 62.7% (52/83) of cases. The median maximum somatic allele frequency (MSAF) was 17.0% (IQR: 1.7-49.0%), with AML (45.7%), MDS (25.9%), and MPN (43.8%) having higher median MSAFs than HNs (3.2%), not unexpected given that the former naturally circulate. In all, 147 pathogenic short variants and 6 pathogenic rearrangements were detected (median variant allele frequency [VAF]: 2.1%, IQR: 0.3-20.5%, range: 0.11-99.8%). Sixty-one had VAFs &lt; 1.0%, indicating excellent sensitivity. HNs exhibited activating RAS-RAF-MEK pathway alterations in BRAF (31.3% of cases in which pathogenic alterations were identified), NF1 (12.5%), MAP2K1 (6.3%), and NRAS (6.3%), detected at VAFs as low as 0.21%, 0.13%, 0.11%, and 4.0%, respectively. AML cases exhibited pathogenic alterations in TP53 (57.1%), FLT3 (28.6%), IDH2 (28.6%), NPM1 (28.6%), KRAS (14.3%), and NRAS (14.3%), among others. Subclonal heterogeneity was identified, including an AML case with KRAS G12A and G13R and NRAS G12A mutations detected at VAFs of 0.24%, 0.19%, and 0.2%, respectively. Altered genes in patients undergoing workup for MDS or MPN included TP53 (41.4%), JAK2 (37.9%), DNMT3A (13.8%), CHEK2 (10.3%), PTPN11 (10.3%), SF3B1 (10.3%), TET2 (10.3%), ASXL1 (6.9%), MPL (3.5%), and U2AF1 (3.5%), among others. Last, among 48,243 patients with a LB for a solid tumor diagnosis, 3,487 JAK2 V617F, 366 CALR C-terminal truncation, and 343 MPL W515X mutations were detected, potentially indicating a concurrent or occult MPN. Conclusions: LB identified clinically relevant genomic alterations in myeloid neoplasia, offering a powerful tool that may be used pre- or post-treatment when CGP of the buffy coat, marrow, or tissue is infeasible. Given its low limit of detection, LB can also identify low-level emerging or persistent subclones, which may facilitate monitoring and minimal residual disease testing.

Lab validation of an ultrasensitive ctDNA pan-cancer MRD assay using whole-genome sequencing.

e13582 Background: Minimal residual disease (MRD) monitoring using liquid biopsy for solid tumors requires a highly sensitive and specific assay that can overcome the limitation of low abundance cfDNA in a standard blood draw. We developed a whole-genome sequencing (WGS)-based assay to detect the presence of circulating tumor DNA (ctDNA) in plasma. The C2i assay is a tumor-informed assay that uses personalized tumor signature, advanced noise models, and artificial intelligence (AI) modalities to interrogate plasma for the presence of ctDNA longitudinally. Methods: The C2i test was developed in accordance with CAP/CLIA and New York state validation principles. We used contrived samples to establish analytical validation of the assay performance, which was then validated with a large clinical cohort of early-stage patients across various cancer types. Briefly, aggregated tumor signatures derived from cancer cell lines were fragmented and spiked into a contrived healthy plasma pool; the mixed samples were used to assess the presence of tumor DNA signature down to tumor fractions of 10e-4. Positive samples are identified by tumor-derived variants detected above the noise levels. Noise modeling was established using a panel of normal (PON) approach. We assessed the analytical sensitivity, specificity, and accuracy using 348 contrived samples derived from five different cancer cell lines. Reproducibility and precision were assessed with multiple replicates, and statistical concordance was reported. This validation was complemented by a cohort of 200 patients and ̃1000 plasma samples across a variety of cancer types including, NSCLC, MIBC, CRC, GBM, Breast Cancer, and a mixture of other cancer types. Results: Cancer cell lines, representing the five most prevalent disease indications, used for determining analytical sensitivity are as follows: CRC HT-29, Breast SK-BR3, Bladder HT-1376, Lung HCI-H526, and Prostate LNCaP. The cell line DNA was enzymatically fragmented and size-selected to mimic ctDNA. This ctDNA was spiked into cfDNA extracted from healthy volunteers at various dilution levels, varying from 10e0 to 10e-4. The 95% probability of detecting ctDNA was established at 10e-4. The reproducibility of tumor signature between replicates was assessed to be greater than 90%. The assay was performed using both normal and maximum input amounts. These performance estimates were then validated on a cohort of plasma collected from early-stage (stage I-III) patients across various cancer types. Conclusions: C2i MRD test is an ultrasensitive pan-cancer MRD monitoring assay used in several clinical trials across the world. We present an extensive analytical and clinical validation of the assay supporting its high performance.

Minimal residual disease comparison between Ig/TCR PCR versus NGS assays in children with Philadelphia chromosome-positive acute lymphoblastic leukemia: A report from the COG AALL1631 study.

10023 Background: Minimal residual disease (MRD) assessment by immunoglobulin/T-cell receptor (Ig/TCR) polymerase chain reaction (PCR) is currently being used in the international pediatric Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) trial EsPhALL2017/AALL1631 for risk stratification. MRD concordance has previously been demonstrated between Ig/TCR PCR and flow cytometry in Ph+ALL. We sought to assess concordance of MRD assessment between conventional Ig/TCR PCR and next-generation sequencing (NGS) assays. Methods: MRD was assessed in all pts on AALL1631 by Ig/TCR PCR at end-induction IB; those with MRD &lt;5x10-4 were classified as standard-risk (SR) and randomized to treatment with imatinib and one of two chemotherapy regimens without hematopoietic stem cell transplant (HSCT), whereas pts with end-induction 1B MRD ≥ 5x10-4 were considered high-risk (HR) and assigned to HSCT after consolidation chemotherapy. Residual diagnostic and end-induction IB samples from consenting pts were assessed for NGS MRD by the clonoSEQ assay (Adaptive Biotechnologies) in blinded fashion and subsequently compared to Ig/TCR MRD to determine concordance as related to MRD-based HSCT recommendations ( ie, MRD ≥ 5x10-4 consistent with HR group assignment). MRD values were calculated using the kappa statistic for agreement above chance. Results: Sixty-seven pts had matched samples available for MRD assessment at end-induction 1B by both Ig/TCR PCR and NGS (Table). NGS MRD was evaluable for all 67 pts and stratified as 62 SR (&lt;5x10-4) and 5 HR (≥5x10-4). In contrast, Ig/TCR PCR results were inevaluable for 3 pts (unsatisfactory sample quality) and indeterminate (positive, but not quantifiable) in 4 pts. Of the remaining 60 pts, 55 met SR and 5 HR criteria using Ig/TCR PCR. There was only 1 discordant case between the two methods for MRD-based HSCT recommendation among these 60 pts with a kappa statistic for agreement above chance of 0.88. Conclusions: NGS and Ig/TCR PCR assays were highly concordant in MRD assessment for risk stratification at a threshold of 5x10-4 in pediatric pts with Ph+ALL enrolled on AALL1631. Of note, the NGS assay yielded MRD results amenable for risk stratification in 100% pts compared to 89.6% for the Ig/TCR PCR methodology. These data support the use of NGS MRD testing for risk stratification in pediatric Ph+ALL.[Table: see text]

Clinical Significance of Circulating Clonal Plasma Cells Detected by a Novel Microfluidic Chip in Multiple Myeloma

Circulating Clonal Plasma cells (cCPCs) in peripheral blood is emerged as an important biomarker in evaluating the Minimal Residual Disease (MRD) status in Multiple myeloma (MM). The elevation of cCPCs is closely related to the recurrence of disease. Comparing to the conventional bone marrow aspirate, it can be accessed regularly in a noninvasive manner. Over the past decade, microfluidic techniques have been widely explored as a platform to segregate the rare cells in blood with the advantages of easy manipulation and low cost. Recently, we and several other groups have reported the successful development of cCPCs-specific microfluidic chip. However, there had been no comparative study of the performance of microfluidic chip to currently available clinical MRD prognostic tools, so we used our previously developed microfluidic platform to explore its clinical utility in comparison with existing methods of Multiparameter Flow Cytometry (MFC) and Serum Protein Electrophoresis analysis. The MRD test results from the 19 MM patients showed 89.47% of overall agreement between MFC and microfluidic chip. Through the monitoring of 2 MM patients before and after treatment, a similar fluctuation pattern was observed between the cCPCs level detected by microfluidic chip and the paraprotein level detected by Serum Protein Electrophoresis.

B-PRISM (Precision Intervention Smoldering Myeloma): A phase II trial of combination of daratumumab, bortezomib, lenalidomide, and dexamethasone in high-risk smoldering multiple myeloma.

8040 Background: Early therapeutic intervention with lenalidomide has shown to be effective in delaying progression in patients with high-risk smoldering multiple myeloma (HR-SMM) (Lonial J Clin Oncol 2020). Quadruplet regimen of daratumumab, bortezomib, lenalidomide and dexamethasone (D-RVD) has demonstrated significant activity in multiple myeloma, achieving high rates of minimal residual disease (MRD) negativity (Voorhees Blood 2020). Thus, we proposed to examine the activity and safety of D-RVD in patients with HR-SMM. Methods: This is a phase II single center, single-arm, open label study evaluating the combination of D-RVD in HR-SMM. Eligibility included high risk per Mayo 2018 “20-2-20” model and other previously established criteria including Mayo 2008 criteria, presence of immunoparesis, evolving type of SMM, and high risk FISH. Primary objective is rate of sustained MRD negativity at 2 years. Secondary objectives include PFS, response rates and safety. Treatment duration with modified D-RVD is for total of 2 years (24 cycles). Daratumumab is administered subcutaneously (SQ) per standard dose and schedule, bortezomib given weekly on days 1, 8, 15 for cycles 1-6 and then biweekly until completion of cycle 24. Lenalidomide is administered on days 1-21 and dexamethasone is administered weekly. All eligible patients will undergo stem cell collection after 6 cycles of therapy. Results: At the time of data cut off, 20 patients have been enrolled with a median follow up of 6 months. The median age is 58 years old (range 40-73). Sixteen out of 20 (80%) patients met high risk criteria per Mayo 2018 model with median plasmacytosis of 20%, median M protein value of 2.6 g/dl and median FLC ratio of 28.2. Seven patients had high-risk FISH: 5 with 1q duplication, 2 with t(4;14). Most common toxicities of any grade included neutropenia (65%), WBC decreased (55%) insomnia (50%), constipation (45%) and hypophosphatemia (45%). Most common grade 3 toxicities included neutropenia (15%), ALT increased (5%), thrombocytopenia (5%), hyperglycemia (5%), hypertension (5%), diarrhea (5%), syncope (5%). No patients discontinued therapy due to toxicity. The overall response rate is 90% with 40% PR, 25% VGPR and 25% CR. All patients have achieved at least a MR and 50% achieved VGPR or greater with responses deepening over time. No patients have progressed on treatment. MRD was evaluable in 16 patients and 8 patients have undergone MRD testing, with MRD negativity rate of 50% (4/8) and 25% (2/8) at thresholds of 10-5 and 10-6, respectively. Stem cells were successfully collected in all patients with mean stem cell yield of 5.78 x 106 CD34+/kg cells. Conclusions: D-RVD is well tolerated in patients with HR-SMM demonstrating significant early activity. Responses continue to deepen over time with patients achieving MRD negative disease. Clinical trial information: NCT04775550.

Optic neuropathy associated with GVHD after bone marrow allogeneic hematopoietic stem cell transplantation for B-Acute lymphoblastic leukemia: case report

BackgroundGraft-versus-host disease (GVHD) is the most common complication after bone marrow allogeneic hematopoietic stem cell transplantation (allo-HSCT). The incidence of posterior segment complications was significantly lower than that of ocular surface lesions. Up to now, there has been no report about optic neuropathy associated with GVHD.Case presentationA 23-year-old man presented with visual acuity decline after allo-HSCT for B-acute lymphoblastic leukemia (B-ALL). Red rashes were found all over the body simultaneously. Visual field examination revealed central scotomas in both eyes. Visual evoked potential showed prolonged P100 latency and decreased P100 amplitude in both eyes. Other ocular examinations showed no obvious abnormality except for blunt pupillary light reflex. The minimal residual disease test was negative after transplantation, and no obvious abnormalities were found in optic nerve and brain by magnetic resonance imaging (MRI). After the multi-disciplinary consultation, the rashes and optic neuropathy were considered GVHD probably. As for the treatment, methylprednisolone and Ruxolitinib were suggested, supported by adjunctive neurotrophic therapy. Two months later, the rashes gradually subsided. However, the visual acuity was not significantly improved at latest follow-up.ConclusionsThe present case report demonstrated GVHD probably associated with optic neuropathy. Although extremely rare, optic nerve should be considered as a potential target of ocular GVHD, which could expand the dimensions of GVHD.

Is Quantification of Measurable Clonal Plasma Cells in Stem Cell Grafts (gMRD) Clinically Meaningful?

With the introduction of more effective novel therapies, the prognosis of multiple myeloma (MM) has improved significantly over the past decade, resulting with a significant proportion of patients achieving durable remissions that may reach even more than 10 years. Several studies demonstrated that the real prognostic value of complete remission (CR) relies on sustained undetectable minimal residual disease (MRD). Additionally, advances in MRD detection methods used for the detection of clonal plasma cells (cPC) inside or outside the bone marrow have also improved the value of MRD. The use of peripheral blood for MRD detection could be an effective method that overcomes the spatial heterogeneity and invasive intervention with recurrent bone marrow aspirations. During the last two decades, many groups have investigated the role of circulating plasma cells (CPCs) at diagnosis. As also presented by multiple groups during the recent ASH 2021 annual meeting, CPCs are becoming recognized as an independent prognostic factor. In addition, measurement of post-induction residual plasma cells in the stem cell graft is identified as another option for MRD assessment. Earlier studies in the era of less intensive induction regimens attempts to analyze the level of CPC contamination in the graft was shown to contribute to myeloma relapse and progression. According to these recent results, higher graft purity has been found to be in concordance with deeper responses. As expected, graft minimal residual disease (gMRD) may reflect the efficacy of induction as an additional response assessment tool. Although gMRD is a non-invasive approach, it has not gained sufficient support for routine use. In view of the hurdles related to monoclonal protein assessments, high-sensitivity cellular component measurement continues to possess its value as an end point for therapeutic efficacy. In this review, we will present a structural framework for MRD testing in peripheral blood stem cell autografts in MM and review the clinical integration into MM management.

Relevance of minimal residual disease testing in pediatric acute lymphoblastic leukemia; A tertiary care centre observational study

Relevance of minimal residual disease testing in pediatric acute lymphoblastic leukemia; A tertiary care centre observational study

Unrelated donor α/β T cell– and B cell–depleted HSCT for the treatment of pediatric acute leukemia

Unrelated donor (URD) hematopoietic stem cell transplant (HSCT) is associated with an increased risk of severe graft-versus-host disease (GVHD). TCRαβ/CD19 depletion may reduce this risk, whereas maintaining graft-versus-leukemia. Outcome data with TCRαβ/CD19 depletion generally describe haploidentical donors, with relatively few URDs. We hypothesized that TCRαβ/CD19-depletion would attenuate the risks of GVHD and relapse for URD HSCT. Sixty pediatric and young adult (YA) patients with hematologic malignancies who lacked a matched-related donor were enrolled at 2 large pediatric transplantation centers between October 2014 and September 2019. All patients with acute leukemia had minimal residual disease testing, and DP typing was available for 77%. All patients received myeloablative total body irradiation– or busulfan-based conditioning with no posttransplant immune suppression. Engraftment occurred in 98%. Four-year overall survival was 69% (95% confidence interval [CI], 52%-81%), and leukemia-free survival was 64% (95% CI, 48%-76%), with no difference between lymphoid and myeloid malignancies (P = .6297 and P = .5441, respectively). One patient (1.7%) experienced primary graft failure. Relapse occurred in 11 patients (3-year cumulative incidence, 21%; 95% CI, 11-34), and 8 patients (cumulative incidence, 15%; 95% CI, 6.7-26) experienced nonrelapse mortality. Grade III to IV acute GVHD was seen in 8 patients (13%), and 14 patients (26%) developed chronic GVHD, of which 6 (11%) had extensive disease. Nonpermissive DP mismatch was associated with higher likelihood of acute GVHD (odds ratio, 16.50; 95% CI, 1.67-163.42; P = .0166) but not with the development of chronic GVHD. URD TCRαβ/CD19-depleted peripheral HSCT is a safe and effective approach to transplantation for children/YAs with leukemia. This trial was registered at www.clinicaltrials.gov as #NCT02323867.