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Multi-parametric flow cytometry (MFC) has a well-established role in measurable residual disease (MRD) monitoring in patients with B-lymphoblastic leukemia (B-ALL). However, the optimal time-point (TP) for early MRD testing and associated prognostic impact remain undefined in adult B-ALL patients receiving Hyper-CVAD induction chemotherapy. To evaluate the utility of MRD analysis after one cycle (TP1) in comparison to MRD analysis after two cycles (TP2) of induction treatment with Hyper-CVAD chemotherapy, we studied 49 adult B-ALL patients over a 10-year period (2010-2020) who had available bone marrow samples for morphological and MFC MRD assessments at the two separate TPs. Median times to TP1 and TP2 relative to start of treatment were 21 and 45 days, respectively. When censored at transplant, achievement of MRD negativity at TP1 was not associated with a statistically significant improvement in either event-free survival (EFS) (p = .426) or overall survival (OS) (p = .335) when compared to patients with MRD positivity. In contrast, achieving MRD negativity at TP2 was associated with a statistically significant improvement in both EFS (p = ·005) and OS (p = .047) over patients who remained MRD positive. Multivariate analysis demonstrated that KMT2A-rearrangement and MRD positivity at TP2 were the only significant predictors of outcome, correlating with worse EFS and OS. Therefore, in the absence of residual morphologic disease, MRD analysis after one cycle of Hyper-CVAD induction chemotherapy did not provide additional benefit with regard to risk stratification or correlation with survival outcomes when compared to MRD testing after two cycles of Hyper-CVAD in adult B-ALL patients.

BACKGROUND: The delivery of education on pain neuroscience and the evidence for different treatment approaches has become a key component of contemporary persistent pain management. Chatbots, or more formally conversation agents, are increasingly being used in health care settings due to their versatility in providing interactive and individualized approaches to both capture and deliver information. Research focused on the acceptability of diverse chatbot formats can assist in developing a better understanding of the educational needs of target populations. OBJECTIVE: This study aims to detail the development and initial pilot testing of a multimodality pain education chatbot (Dolores) that can be used across different age groups and investigate whether acceptability and feedback were comparable across age groups following pilot testing. METHODS: Following an initial design phase involving software engineers (n=2) and expert clinicians (n=6), a total of 60 individuals with chronic pain who attended an outpatient clinic at 1 of 2 pain centers in Australia were recruited for pilot testing. The 60 individuals consisted of 20 (33%) adolescents (aged 10-18 years), 20 (33%) young adults (aged 19-35 years), and 20 (33%) adults (aged >35 years) with persistent pain. Participants spent 20 to 30 minutes completing interactive chatbot activities that enabled the Dolores app to gather a pain history and provide education about pain and pain treatments. After the chatbot activities, participants completed a custom-made feedback questionnaire measuring the acceptability constructs pertaining to health education chatbots. To determine the effect of age group on the acceptability ratings and feedback provided, a series of binomial logistic regression models and cumulative odds ordinal logistic regression models with proportional odds were generated. RESULTS: Overall, acceptability was high for the following constructs: engagement, perceived value, usability, accuracy, responsivenes

OBJECTIVES: Molecular biomarker tests can inform the clinical management of genomic heterogeneous hematological malignancies, yet their availability in routine care largely depends on the supporting health economic evidence. This study aims to systematically review the economic evidence for recent molecular biomarker tests in hematological malignancies. METHODS: We conducted a systematic search in five electronic databases for studies published between January 2010 and October 2020. Publications were independently screened by two reviewers. Clinical study characteristics, economic methodology, and results were extracted, and reporting quality was assessed. RESULTS: Fourteen studies were identified, of which half (n = 7; 50%) were full economic evaluations examining both health and economic outcomes. Studies were predominantly conducted in a first-line treatment setting (n = 7; 50%) and adopted a non-lifetime time horizon to measure health outcomes and costs (n = 7; 50%). Five studies reported that companion diagnostics for associated therapies were likely cost-effective for acute myeloid leukemia, chronic myeloid leukemia, diffuse large B-cell lymphoma, and multiple myeloma. Four studies suggested molecular biomarker tests for treatment monitoring in chronic myeloid leukemia were likely cost-saving. CONCLUSIONS: Although there is initial confirmation of the promising health economic results, the present research for molecular biomarker tests in hematological malignancies is sparse with many applications of technological advances yet to be evaluated.

BACKGROUND: Measurable residual disease (MRD) monitoring in acute lymphoblastic leukemia (ALL) is an important predictive factor for patient outcome and treatment intensification. Molecular monitoring, particularly with quantitative polymerase chain reaction (qPCR) to measure immunoglobin heavy or kappa chain (Ig) or T-cell receptor (TCR) gene rearrangements, offers high sensitivity but accessibility is limited by expertise, cost, and turnaround time. Flow cytometric assays are cheaper and more widely available, and sensitivity is improved with multi-parameter flow cytometry at eight or more colors. METHODS: We developed a 10-color single tube flow cytometry assay. Samples were subject to bulk ammonium chloride lysis to maximize cell yields with a target of 1 × 106 events. Once normal maturation patterns were established, patient samples were analyzed in parallel to standard molecular monitoring. RESULTS: Flow cytometry was performed on 114 samples. An informative immunophenotype was identifiable in all 22 patients who had a diagnostic sample. MRD analysis was performed on 87 samples. The median lower limits of detection and quantification were 0.004% (range 0.0005%-0.028%) and 0.01% (range 0.001%-0.07%) respectively. Sixty-five samples had concurrent molecular MRD testing, with good correlation (r = 0.83, p < 0.001). Results were concordant in 52 samples, and discordant in 13 samples, including one case where impending relapse was detected by flow cytometry but not Ig/TCR qPCR. CONCLUSIONS: Our 10-color flow cytometric MRD assay provided adequate sensitivity and good correlation with molecular assays. This technique offers rapid and affordable testing in B-ALL patients, including cases where a suitable molecular assay cannot be developed or has reduced sensitivity.

Background: Measurable residual disease (MRD) monitoring in acute lymphoblastic leukemia (ALL) is an important predictive factor for patient outcome and treatment intensification. Molecular monitoring, particularly with quantitative polymerase chain reaction (qPCR) to measure immunoglobin heavy or kappa chain (Ig) or T-cell receptor (TCR) gene rearrangements, offers high sensitivity but accessibility is limited by expertise, cost, and turnaround time. Flow cytometric assays are cheaper and more widely available, and sensitivity is improved with multi-parameter flow cytometry at eight or more colors. Method(s): We developed a 10-color single tube flow cytometry assay. Samples were subject to bulk ammonium chloride lysis to maximize cell yields with a target of 1 x 106 events. Once normal maturation patterns were established, patient samples were analyzed in parallel to standard molecular monitoring. Result(s): Flow cytometry was performed on 114 samples. An informative immunophenotype was identifiable in all 22 patients who had a diagnostic sample. MRD analysis was performed on 87 samples. The median lower limits of detection and quantification were 0.004% (range 0.0005%-0.028%) and 0.01% (range 0.001%-0.07%) respectively. Sixty-five samples had concurrent molecular MRD testing, with good correlation (r = 0.83, p < 0.001). Results were concordant in 52 samples, and discordant in 13 samples, including one case where impending relapse was detected by flow cytometry but not Ig/TCR qPCR. Conclusion(s): Our 10-color flow cytometric MRD assay provided adequate sensitivity and good correlation with molecular assays. This technique offers rapid and affordable testing in B-ALL patients, including cases where a suitable molecular assay cannot be developed or has reduced sensitivity.Copyright © 2021 International Clinical Cytometry Society.

Richter syndrome (RS), an aggressive lymphoma occurring in the context of chronic lymphocytic leukaemia/small lymphocytic lymphoma, is associated with poor prognosis when treated with conventional immunochemotherapy, therefore, improved treatments are required. Immune checkpoint blockade has shown efficacy in some B-cell malignancies and modest responses in early clinical trials for RS. We investigated the immune checkpoint profile of RS as a basis to inform rational therapeutic investigations in RS. Formalin-fixed, paraffin-embedded biopsies of RS (n = 19), de novo diffuse large B-cell lymphoma (DLBCL; n = 58), transformed indolent lymphomas (follicular [tFL], n = 16; marginal zone [tMZL], n = 24) and non-transformed small lymphocytic lymphoma (SLL; n = 15) underwent gene expression profiling using the NanoString Human Immunology panel. Copy number assessment was performed using next-generation sequencing. Immunohistochemistry (IHC) for LAG3 and PD-1 was performed. LAG3 gene expression was higher in RS compared to DLBCL (P = 0·0002, log2FC 1·96), tFL (P < 0·0001, log2FC 2·61), tMZL (P = 0·0004, log2FC 1·79) and SLL (P = 0·0057, log2FC 1·45). LAG3 gene expression correlated with the gene expression of human leukocyte antigen Class I and II, and related immune genes and immune checkpoints. IHC revealed LAG3 protein expression on both malignant RS cells and tumour-infiltrating lymphocytes. Our findings support the investigation of LAG3 inhibition to enhance anti-tumour responses in RS.

BACKGROUND: Minimal residual disease (MRD) assessment of hematopoietic neoplasia below 10-4 requires more leukocytes than is usually attainable by post-lysis preparation. However, not all laboratories are resourced for consensus Euroflow pre-lysis methodology. Our study aim was to validate a modified pre-lysis protocol against our standard post-lysis method for MRD detection of multiple myeloma (MM), chronic lymphocytic leukemia (CLL), and B-non Hodgkin lymphoma (B-NHL), to meet demand for deeper MRD assessment by flow cytometry. METHOD: Clinical samples for MRD assessment of MM, CLL, and B-NHL (50, 30, and 30 cases, respectively) were prepared in parallel by pre and post-lysis methods for the initial validation. Total leukocytes, MRD, and median fluorescence intensity of antigen expression were compared as measures of sensitivity and antigen stability. Lymphocyte and granulocyte composition were compared, assessing relative sample processing stability. Sensitivity of the pre-lysis assay was monitored post validation for a further 18 months. RESULTS: Pre-lysis achieved at least 10-4 sensitivity in 85% MM, 81% CLL, and 90% B-NHL samples versus 24%, 48%, and 26% by post-lysis, respectively, with stable antigen expression and leukocyte composition. Post validation over 18 months with technical expertise improving, pre-lysis permitted 10-5 MRD assessment in 69%, 86%, and 82% of the respective patient groups. CONCLUSION: This modified pre-lysis procedure provides a sensitive, robust, time efficient, and relatively cost-effective alternative for MRD testing by MFC at 10-5 , facilitating clinically meaningful deeper response assessment for MM, CLL, and B-NHL. This method adaptation may facilitate more widespread adoption of highly sensitive flow cytometry-based MRD assessment.

Background: Paroxysmal nocturnal haemoglobinuria (PNH) is a life-threatening clinical syndrome caused by acquired mutations in PIGA in the haemopoietic compartment. The clinicopathological features of PNH can overlap with aplastic anaemia (AA) and myelodysplastic syndrome (MDS) and these entities may represent different manifestations of a common underlying pathological process. To date, the landscape of acquired mutations in de novo PNH (i.e. not preceded by a formal diagnosis of AA/MDS) remains incompletely characterised. Aim(s): To characterise the spectrum of acquired mutations in the cellular and cell-free compartments in a cohort of patients with de novo PNH. Method(s): DNA was extracted from peripheral blood leukocytes (n = 19) or bone marrow (n = 2) from 21 patients with a clinicopathological diagnosis of PNH (defined as a detectable PNH clone by flow cytometry, active haemolysis and not meeting formal diagnostic criteria for MDS or AA). Patients with a pre-existing diagnosis of AA or MDS were excluded. Cell-free DNA (cfDNA) was also collected from 19 patients. DNA sequencing libraries were prepared from cellular DNA using both the Peter MacCallum Cancer Centre PanHaem hybridisation and Myeloid amplicon panels (sensitivity 5%) which includes sequence variant detection for recurrently mutated genes associated with haematological malignancy and whole genome copy number assessment. A custom panel using Anchored Multiplex PCR target enrichment chemistry and unique molecular barcodes was used for cfDNA sequencing (sensitivity <1%). Result(s): At time of sampling 18 of 21 patients were two or more years post diagnosis (median time since diagnosis of 8 years) with a median age of 41 years (range 23 - 82). 16 patients were being treated with complement inhibitor therapy. PIGA mutations were detected in 19 patients, with multiple mutations detected in half the cohort (11 patients, range 2 - 9 PIGA mutations per patient). 21 mutations were detected in the cellular DNA

Prevention of cancer-associated thromboembolism (TE) remains a significant clinical challenge and priority world-wide safety initiative. In this prospective non-small cell lung cancer (NSCLC) cohort, longitudinal TE risk profiling (clinical and biomarker) was undertaken to develop risk stratification models for targeted TE prevention. These were compared with published models from Khorana, CATS, PROTECHT, CONKO, and CATS/MICA. The NSCLC cohort of 129 patients, median follow-up 22.0 months (range 5.6-31.3), demonstrated a hypercoagulable profile in >75% patients and TE incidence of 19%. High TE risk patients were those receiving chemotherapy with baseline fibrinogen ≥ 4 g/L and d-dimer ≥ 0.5 mg/L; or baseline d-dimer ≥ 1.5 mg/L; or month 1 d-dimer ≥ 1.5 mg/L. The model predicted TE with 100% sensitivity and 34% specificity (c-index 0.67), with TE incidence 27% vs. 0% for high vs. low-risk. A comparison using the Khorana, PROTECHT, and CONKO methods were not discriminatory; TE incidence 17⁻25% vs. 14⁻19% for high vs. low-risk (c-index 0.51⁻0.59). Continuous d-dimer (CATS/MICA model) was also not predictive of TE. Independent of tumour stage, high TE risk was associated with cancer progression (HR 1.9, p = 0.01) and mortality (HR 2.2, p = 0.02). The model was tested for scalability in a prospective gastrointestinal cancer cohort with equipotency demonstrated; 80% sensitivity and 39% specificity. This proposed TE risk prediction model is simple, practical, potent and can be used in the clinic for real-time, decision-making for targeted thromboprophylaxis. Validation in a multicentre randomised interventional study is underway (ACTRN12618000811202).

Background: MRD monitoring in ALL is a strong predictive factor and a stratification tool for treatment intensification. The currently accepted standard of molecular monitoring with either immunoglobin heavy or kappa chain (IG) or T-cell receptor (TCR) quantitative PCR (qPCR) in Philadelphia negative (Ph-) ALL offers high sensitivity, but accessibility is limited by expertise, cost and turnaround time. Flow cytometric assays are increasingly utilised and improved sensitivity is seen with multi-parameter flow cytometry at 8 or more colours. Method(s): We developed a 10-colour single tube flow cytometry assay (Figure 1). Samples were subject to bulk ammonium chloride lysis to maximise cell yields with a target of 1 x 106 events. Once normal maturation patterns were established, patient samples were analysed in parallel to standard molecular monitoring with either IG/TCR qPCR in Ph-disease or BCR-ABL qRT-PCR in Ph+ disease. Statistical analysis was performed in Graphpad Prism v7.0. Result(s): Flow cytometry was performed on 47 samples from 16 patients. 13 samples were at diagnosis or morphologic relapse. An informative immunophenotype was identifiable in all patients; however a molecular assay could not able to be developed in one patient. 38 samples were tested for MRD by flow cytometry (Figure 2). In 2 samples, flow cyto-metric MRD was detected despite blinatumomab (anti-CD19) therapy. 27 samples were tested concurrently for MRD by both molecular and flow cytometric methods (Figure 3A and 3B). There was a strong correlation between molecular and flow cytometric MRD (R2=0.909, p<0.001; Figure 3B). Correlation was strong with both IG/TCR-based (n=16; R2=0.955, p<0.001) and BCR-ABL-based (n= 11; R2=0.957, p<0.001) (Figure Presented) assays. The cost was significantly lower than IG/TCR qPCR (eg. Cost for four time-points per patient approximately $1200 vs $3700). Conclusion(s): Our 10-colour flow cytometric MRD assay attained sensitivity of <=0.01% in 87% of samples, and

Breast implant-associated anaplastic large cell lymphoma (BIA-ALCL) is a rare form of T-cell lymphoma that occurs after implantation of breast prostheses. We performed comprehensive next generation sequencing based genomic characterization of 11 cases of BIA-ALCL including sequence variant detection on 180 genes frequently mutated in haematological malignancy, genome-wide copy number assessment, structural variant detection involving the T-cell receptor loci and TRB deep-sequencing. We observed sequence variants leading to JAK/STAT activation in 10 out of 11 patients. We also observed germline TP53 mutations in two cases. In addition we detected a recurrent copy number loss involving RPL5 as well as copy number amplifications involving TNFRSF11A [RANK] (in 2 cases), MYC, P2RX7, TMEM119 and PDGFRA. In summary, our comprehensive genomic characterisation of 11 cases of BIA-ALCL has provided insight into potential pathobiological mechanisms (JAK/STAT, MYC and TP53) as well as identifying targets for future therapeutic intervention (TNFRSF11A, PDGFRA) in this rare entity.

Background: Minimal residual disease (MRD) monitoring in Acute Lymphoblastic Leukemia (ALL) is an accepted standard of care in both adult and pediatric patients as one of the strongest predictive factors for disease outcome and as a stratification tool for treatment intensification and allogeneic stem cell transplant. The currently accepted standard of molecular monitoring with either immunoglobin heavy or kappa chain (IG) or T-cell receptor (TCR) quantitative PCR (qPCR) in Philadelphia negative ALL allows for sensitive monitoring of MRD, but requires a high degree of expertise, and factors such as cost and turnaround time may limit generalized applicability of this technique. Flow cytometric MRD monitoring is utilized in many centers, with increased sensitivity seen with implementation of multi-parameter flow cytometry at 8-colours or more. Aim(s): We sought to compare a 10-color flow cytometry assay for detecting MRD in B-ALL with standard molecular monitoring. Method(s): To facilitate rapid identification of MRD in patients with B-ALL, we developed a 10-colour single tube flow cytometry assay utilizing CD19, CD22, CD20, CD38, CD58, CD13/33, CD66c, CD10, CD45 and CD34 as markers. These markers were selected to provide at least two targets for identification of B-lineage cells, and to include the most frequently aberrant markers in precursor B-lineage ALL. Samples were subject to bulk ammonium chloride lysis to maximize cell yields with a target of 1 x 106 events. Once normal maturation patterns were established, patient samples were analyzed in parallel to standard of care molecular monitoring with either IG/TCR qPCR in Philadelphia negative (Ph-) disease and BCR-ABL qRT-PCR in Philadelphia positive (Ph+) disease. Statistical correlation was performed in Graphpad Prism version 7.0 for linear regression and calculation of correlation co-efficient. (Table Presented) Results: 33 samples at different time points from 13 patients were analyzed by flow cytometry. 9 samp

Several novel therapeutics are poised to change the natural history of chronic lymphocytic leukaemia (CLL) and the increasing use of these therapies has highlighted limitations of traditional disease monitoring methods. Here we demonstrate that circulating tumour DNA (ctDNA) is readily detectable in patients with CLL. Importantly, ctDNA does not simply mirror the genomic information contained within circulating malignant lymphocytes but instead parallels changes across different disease compartments following treatment with novel therapies. Serial ctDNA analysis allows clonal dynamics to be monitored over time and identifies the emergence of genomic changes associated with Richter's syndrome (RS). In addition to conventional disease monitoring, ctDNA provides a unique opportunity for non-invasive serial analysis of CLL for molecular disease monitoring.