The Addition of Thalidomide to the Induction Treatment of Newly Presenting Myeloma Patients Increases the CR Rate Which Is Likely to Translate Into Improved PFS and OS.

Abstract Abstract 352 We present updated results from MRC Myeloma IX study evaluating the role of the addition of thalidomide to the induction and maintenance of patients with myeloma. The study ran from May 2003 – November 2007 and randomised 1,970 patients and now has a median follow up of more than 3.5 years giving it improved power to detect changes in outcome developing later after treatment. Projected median OS younger fitter patients 66 months, median OS older less fit patients 32 months. The trial comprised of 2 patient pathways, one for younger fitter patients comparing CTD (cyclophosphamide, thalidomide, dexamethasone) with CVAD (cyclophosphamide, vincristine, adriamycin, dexamethasone), all patients going on to receive an ASCT – median age 59 years. In older less fit patients, melphalan and prednisolone (MP) was compared to CTD attenuated – median age 73 years. In both pathways following initial treatment, eligible patients were randomised to low-dose thalidomide or no maintenance. Patient's response was monitored using electrophoresis, serum free light chain and multiparameter flow cytometry. Cytogenetics was availabel on up to 60% of cases and gene expression on a subset of these. CTD is a well tolerated regimen with a good safety profile giving excellent survivals in both groups of patients despite a small increase in risk of VTE. Using modified EBMT criteria, the addition of thalidomide to induction treatment increases both response rates and depth of response for all age groups. Preliminary results as follows: overall response: CTD vs CVAD: 91% v 82%; CR 21% v 14% and 100 days post-HDM, better responses were seen in CTD with CR rates 65% v 48%. Remission depth was also greater in CTD with more patients achieving minimal residual disease negativity by flow cytometry. The addition of thalidomide increases response rates overall, and particularly complete response (CR) rates (a 17% increase in CR rates post HDM, p=.006). In older/less fit patients CTDa vs MP: overall response 83% v 46%; CR 21% v 4%. Definitive results of these analyses will be presented as well as how they translate into PFS and OS and by cytogenetic subgroup. There is a substantial increase in response with the inclusion of thalidomide but at a median follow-up of three years we are not as yet seeing a substantial increase in survival in either of the two broad patient groups. We have collected data on treatment at relapse to explore how this confounds OS data. Importantly modelling analyses indicate when and to what extent, with further follow-up, the survival differences that should accrue from this increase in CR rate are likely to translate into a survival benefit. These results have a number of important implications. We show the benefit of the addition of thalidomide to myeloma treatment but also highlight the importance of later analysis of such trials because of the emergence of significant changes at these later time points. We will present full updated results from the study including the impact of thalidomide on cytogenetic subgroups and in the maintenance setting. Disclosures: No relevant conflicts of interest to declare.

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Assessment Of Minimal Residual Disease In Acute Myeloblastic Leukemia In Multiparameter Flow Cytometry

Blood ◽

10.1182/blood.v122.21.2613.2613 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2613-2613 ◽

Cited By ~ 1

Author(s):

Francis Lacombe ◽

Kaoutar Allou ◽

Christine Arnoulet ◽

Lydia Campos ◽

Adrienne de Labarthe ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Minimal Residual Disease ◽

Internal Control ◽

Conflicts Of Interest ◽

Residual Disease ◽

Molecular Techniques ◽

Multiparameter Flow Cytometry ◽

Minimal Residual

Abstract Acute myeloid leukemias (AML) represent a vast and complex group of diseases where numerous molecular lesions have been and keep being described. From the immunophenotypic point of view, probably because of the variety of cells in the myeloid lineage, a rather large variety also exists. The detection of minimal residual disease (MRD) in such conditions therefore meets the challenge of tracking the proper anomaly and correctly separate leukemic cells from their normal counterparts. In an oligocentric project initiated in France in late 2006, ten cytometry platforms and six molecular biology laboratories collaborated to detect MRD concomitantly in flow and with molecular techniques. The flow cytometry part of this work is reported here. A total of 307 consecutive patients were tested at diagnosis with a comprehensive common panel allowing for the detection of immature markers and potential leukemia-associated immunophenotypic patterns. Follow-up samples were planned to be obtained after induction and at the end of treatment, with an optional control before the second consolidation. In fine, 274 patients had at least one point of follow up. All samples were tested in a technique of whole bone marrow lysis no wash, avoiding any cell loss. The flow cytometry panel comprised ten five-colors tubes, all containing CD45 as gating marker. A newly developed strategy was devised to analyse MRD data. The approach of the GTLLF (Arnoulet, Cytometry part B, 2011) was first applied to properly identify mature polymorphonuclears, monocytes and lymphocytes, allowing for a negative Boolean selection of immature cells in the region dubbed “bermudes” by this group. Focusing on this area, each combination of the four markers tested together with CD45 was then displayed in a total of six biparametric histograms. For each of them, on the diagnosis sample, quadrant gates were constructed so that the lower left one contained no blast cells. A Boolean operation was then designed to exclude all these six areas, thereby combining the positive blasts present in the three other parts of each quadrant. The resulting population was visualized on a CD45/side scatter biparametric histogram to check that the cells appeared as a focused cluster at a precise position. The same strategy was then applied for each patient’s consecutive samples, always checking whether any cells identified with this protocol displayed the scattered pattern of cells engaged in maturation (no MRD) or constituted a focused population without maturation (positive MRD). The amount of MRD was then calculated taking into account as denominator the whole population of nucleated cells in the sample (excluding debris on a live gate). As internal control a specific feature of the Kaluza software was used to merge samples obtained for a given patient in order to display on the same worksheet the diagnosis and follow up samples using the principal component separation provided by the “radar” tool of this software. This original method proved to be easily applicable and provide a consistent help for MRD interpretation. All patients could be assessed for MRD with only two of the ten tubes used. These contained the following combinations : CD15, CD13, CD33, CD34, CD45 and CD7, CD117, CD33, CD34, Cd45. At diagnosis, any combination of expression of CD13, CD33, CD117 and CD34 could be observed, the percentage of positive cases for each of these antigens being respectively 86%, 89%, 81% and 58%. As a whole, 93% of the follow-up samples (MRD) tested contained less than 5% of cells with an immunophenotype comparable to that of diagnosis. This figure was 77% for less than 1% and 43% for less than 0.1%. The strategy devised for files analysis was easily applicable for all patients except those with myelomonocytic leukemia. For some of them, separation of the blasts from the monocytic compartment could be problematic in regenerating bone marrow samples. In conclusion, the flow cytometry part of this multicenter study allowed to establish that the combination of CD45 with CD13, CD33, CD117 and CD34 with the additional information provided by CD5 and CD7 represents a quasi-universal panel, now easy to implement on instruments with 8 or 10 detectors, for the detection of MRD in multiparameter in flow cytometry. Moreover, a powerful strategy of listmodes analysis was developed allowing for the direct comparison of several samples from the same patients and/or of a given sample and normal (control) bone marrow. Disclosures: No relevant conflicts of interest to declare.

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Autologous Hematopoietic Stem Cell Transplantation (autoHSCT) in CLL: First Results of An EBMT Randomized Trial Comparing Autotransplant Versus Wait and Watch.

Blood ◽

10.1182/blood.v114.22.877.877 ◽

2009 ◽

Vol 114 (22) ◽

pp. 877-877

Author(s):

Mauricette Michallet ◽

Peter Dreger ◽

Laurent Sutton ◽

Ronald Brand ◽

Sue Richards ◽

...

Keyword(s):

Conflicts Of Interest ◽

Progression Free Survival ◽

Disease Status ◽

Primary Objective ◽

Phase Iii ◽

Induction Treatment ◽

Hematopoietic Stem ◽

Binet Stage ◽

The Impact

Abstract Abstract 877 This phase-III randomized EBMT-intergroup trial studied the impact of a consolidating autoHSCT vs no consolidation for patients with CLL in Binet stage A progressive, B or C , in CR, nodular PR or VGPR after first or second line therapy. The primary objective was to show that autoHSCT increased the 5-year progression-free survival (PFS) by 30%. Although it had been calculated that 270 patients were to be randomized, the study was terminated by the steering committee in July 2007 due to poor accrual. Here we present a first analysis based on 69% of expected follow-up forms. Results: Between November 2001 and July 2007, 223 patients were enrolled (SFGM-TC/FCLLG n=98, MRC n=62, GCLLSG n=32, SAKK n=10, other EBMT centers n=17). There were 74% males and 26% females. Binet stages were progressive A 13%, B 67%, C 20%; 59% were in CR, and 41% in very good or nodular PR. Of note, SFGM-TC/FCLLG included only patients in CR. 82% of the patients were enrolled in 1st, and 18% in 2nd remission. Patients were randomized between group 1 (autoHSCT n=112) and group 2 (observation n=111) after an induction treatment which was left at the discretion of the investigators. Median PFS was 43 months in the observation group but not reached in the autoHSCT group; 5-year PFS was 48% and 65%, respectively (p=0.005). Accordingly, autoHSCT halved the relapse risk (5-year relapse incidence 25% vs. 51%; HR 0.4 [0.23-0.71], p=0.002). Cox modeling for randomization arm, Binet stage, disease status, line of treatment, contributing group (country), and the interaction between randomization arm and contributing group confirmed that autoHSCT significantly improved PFS (HR 0.41 [0.23-0.75] p=0.004). The beneficial effect of autoHSCT was stable over all contributing groups although patients accrued by SFGM-TC/FCLLG overall had a significantly better PFS than patients from other countries (HR 0.2 [0.08-0.55], p=0.001). At 5 years, the probability of OS was 92% and 91% for autoHSCT and observation, respectively. Significant differences in terms of non-relapse death were not observed. At the last follow up, among 205 evaluable patients, 186 are alive (147CR, 39 relapse), 19 died (14 from relapse and 5 from non-relapse causes) . In conclusion, in patients with CLL in first or second remission, consolidating autoHSCT reduces the risk of progression (PFS) by more than 50%, but has no effect on overall survival. Disclosures: No relevant conflicts of interest to declare.

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Morphology Vs. Multiparameter Flow Cytometry in Evaluation of AML in Cerebrospinal Fluid (CSF).

Blood ◽

10.1182/blood.v120.21.2499.2499 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2499-2499

Author(s):

Keith R. Loeb ◽

Sindhu Cherian ◽

John M. Pagel ◽

Pamela S Becker ◽

Frederick R. Appelbaum ◽

...

Keyword(s):

Central Nervous System ◽

Flow Cytometry ◽

Conflicts Of Interest ◽

False Negative ◽

Multiparameter Flow Cytometry ◽

Single Tube ◽

Hla Dr ◽

Atypical Cells ◽

The Central Nervous System

Abstract Abstract 2499 Background and Methods: Multiparameter flow cytometry (MFC) is increasingly used in AML diagnostics as a supplement to morphologic examination. Here we retrospectively examine the concordance between MFC and morphologic examination for the detection of AML in CSF. Between 2005 and 2012, CSF from 604 sequential patients with AML or high-risk MDS was obtained at diagnosis or in follow-up of treatment for AML involving the central nervous system. Morphologic examination was done using conventional Wright-Giemsa stained cytospin preparations. MFC was done using a standard single-tube myeloid panel (HLA-DR/CD15/CD33/CD19/CD117/ CD13/ CD38/CD34/CD71/CD45) and/or a panel targeting the prior AML immunophenotype. Results: Results detecting any evidence of AML in the CSF by both techniques is noted in the Table below: The concordance between MFC and morphology (excluding atypical morphology) was high (97%; 539/558), in large part due to the predominance of uninvolved samples using both techniques. However, of the samples positive by MFC, 13% were negative by morphology and another 15% showed atypical cells insufficient for positivity by morphology. Of samples positive by morphology, only a single sample (1.4%) was negative by MFC. In addition, 65% of cases in which morphology was judged atypical were MFC negative, suggesting suboptimal specificity. Patients typically received intrathecal treatment based on positive MFC making it difficult to know whether the disease in the CSF would have expanded without treatment in patients who were MFC positive/morphology negative. Conclusion: Subject to the limitation that the morphology may not have been interpreted entirely independently of the MFC result, the results suggest morphology yields a false negative result in between 13% and 28% of samples and may not be sufficiently sensitive for routine screening of CSF. To follow-up our findings we are examining whether there are features that distinguish patients who were MFC positive/ morphology negative or vice versa and performing a study in which morphology and MFC are being independently assessed. Disclosures: No relevant conflicts of interest to declare.

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Impact Of Age On Toxicity Associated With Chemotherapy For Acute Lymphoblastic Leukaemia (ALL): Results From The UK Prospective Study UKALL2003

Blood ◽

10.1182/blood.v122.21.840.840 ◽

2013 ◽

Vol 122 (21) ◽

pp. 840-840

Author(s):

Rachael E. Hough ◽

Clare Rowntree ◽

Rachel Wade ◽

Nicholas Goulden ◽

Chris Mitchell ◽

...

Keyword(s):

Conflicts Of Interest ◽

Residual Disease ◽

Age Groups ◽

Serious Adverse Events ◽

Number Of Patients ◽

Teenagers And Young Adults ◽

The Uk ◽

Cure Rates ◽

The Impact

Abstract Despite the substantial improvements made in the outcomes of paediatric ALL, with ‘cure' rates now in excess of 90%, survival in teenage and young adult (TYA) patients has remained inferior. The reasons for this are likely multifactorial, including tumour biology, toxicity, compliance, access to clinical trials and protocol (adult or paediatric) used. We report the toxicity profiles observed in children, teenagers and young adults treated on the UK intensive, minimal residual disease (MRD) directed ALL protocol, UKALL2003. Of a total of 3126 patients treated, 1520 patients were under 5 years old, 767 were aged 5-9 years, 610 aged 10-15 years and 229 aged 16-24 years, with a median overall follow-up of 4 year and 10 months. The risk of serious adverse events (SAEs) was higher in patients older than 10 years (56% in 10-15 year olds, 53% in 16-24 year olds) compared to those aged 9 or younger (30% in under 5 years and 31% in 5-9 years)(p<0.0001), with no difference in the those aged 16-24 compared to younger teenagers (p=0.5). The incidence (per number of patients in each group) and distribution of toxicities according to age group is summarised in the table.Table 1Age in years<55-910-1516-24AllTotal number of patients1520767610229 NB: 56 pts≥20 years3126Infection n (%)328 (21.6%)130 (17.0%)145 (23.8%)72 (31.4%)675 (21.6%)Asaparaginase n (%)57 (3.8%)57 (7.4%)64 (10.5%)31 (13.5%)209 (6.7%)Methotrexate n (%)100 (6.6%)74 (9.6%)123 (20.2%)33 (14.4%)330 (10.6%)Steroid n (%)54 (3.6%)37 (4.8%)141 (23.1%)52 (22.7%)284 (9.0%)Vincristine n (%)34 (2.2%)11 (1.4%)22 (3.6%)7 (3.0%)74 (2.4%)Other SAEs94 (6.2%)42 (5.5%)90 (14.8%)25 (10.9%)251 (8.0%) The incidence of certain toxicities including viral infection (5.3%), asparaginase hypersensitivity (1.9%) and vincristine neurotoxicity (2.1%) appeared equivalent across all age groups. Avacular necrosis was seen predominantly in adolescents (83% of 147 events in 10-19 year olds) and was rare in those younger than 10 years (n=18) or older than 20 years (n=7). Asparaginase thrombotic events increased in frequency with increasing age (1.5% in under 5 years, 3.3% in 5-9 years, 4.4% in 10-15 years and 8.3% in 16-24 year olds)(p<0.0001). All other toxicities were more frequently observed in over 10 year olds compared to patients aged 9 or younger, with no difference between 16-24 year olds and 10-15 year olds. The impact of age on SAEs associated with intensive ALL chemotherapy varies according to specific toxicities. In general, toxicity is higher in those over 10 years compared to younger patients, with no excess toxicity in those aged 16-24 compared to 10-15 years. However, specific toxicities may increase with increasing age (thrombosis), be restricted to adolescence (AVN) or be unrelated to age (vincristine neurotoxicity, asparaginase hypersensitivity). Disclosures: No relevant conflicts of interest to declare.

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Bortezomib, Dexamethasone, Thalidomide and Melphalan (VDT-Mel) Preparative Regimen in Autologous Stem Cell Transplant (ASCT) Results in a Very High Stringent CR (sCR) Rate in Multiple Myeloma (MM)

Blood ◽

10.1182/blood.v126.23.1971.1971 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1971-1971

Author(s):

Kalyan Nadiminti ◽

Kamal Kant Singh Abbi ◽

Annick Tricot ◽

Allyson Schultz ◽

Lindsay Dozeman ◽

...

Keyword(s):

Flow Cytometry ◽

Mortality Rate ◽

Conflicts Of Interest ◽

Residual Disease ◽

High Dose ◽

Cell Transplant ◽

Color Flow ◽

Preparative Regimen ◽

Very High

Abstract Background: Melphalan 200mg/m2 is the standard preparative regimen in MM and addition of other cytotoxic drugs has not been found to result in superior activity. The novel agents have improved outcome in MM significantly, but data on their role in preparative regimens are scarce. The purpose of this study was to understand the toxicity and efficacy of triple therapy with VDT in combination with high-dose melphalan. Methods: An IRB approved retrospective analysis was performed on all patients who received an ASCT with the VDT-Mel during 2012-2014. Mel: 100 mg/m2 was given on days -4 and -1; V: 1 mg/m2 on days -4, -1, +2 and +5; T: 100 mg daily from -5 to +5; and D: 20 mg/day from -4 to -1 and +2 to +5. End points were treatment-related toxicity during the first 100 days and quality of response at 6 months post-transplant; 98 patients had follow-up ≥ 6 months. Patients in sCR were also minimal residual disease negative (MRD-) by 10-color flow cytometry with a sensitivity of 10-4. Results: 100 patients received 153 transplants; 47 patients underwent single and 53 had tandem transplants (TT); 64 patients received early (≤ 12 months of induction therapy) and 36 salvage transplantation. Median age was 61 y; median followup was 16.2 months. Only 1patient had achieved a sCR and 11 a CR prior to transplantation. Best responses at 6 months were 53% sCR (and MRD-), 24% CR, and 9% VGPR. The sCR rate after single transplant was 47% (overall) and 54% (early transplant) vs 59% and 60% after TT. Grade 3-5 non-hematologic toxicities were almost entirely related to infections (38% and 53% in single and TT, respectively); the 100-day mortality rate was 2.6% (4/153), 1.8% for early transplants and 4.5% for salvage transplants. Median time to ANC recovery > 500/µL was 12 days in both early and salvage transplantation. Conclusion: VDT-Mel is well-tolerated and resulted in minimal additional toxicity and a similar mortality rate when compared to historic data of MEL alone. Importantly, the sCR rate with MRD- by flow cytometry at 6 months in our study was very high compared to published reports. The ultimate sCR rate will be higher as at this time an additional 13 patients attained a sCR during further follow up past 6 months for a total of 66% sCR. Since both sCR and MRD- are proven early surrogate markers for progression-free and overall survival, it appears highly likely that this regimen will be superior to Mel alone and should become the new standard for ASCT in myeloma. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

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Immunophenotypic Response After Allografting In Multiple Myeloma

Blood ◽

10.1182/blood.v122.21.3371.3371 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3371-3371 ◽

Cited By ~ 1

Author(s):

Luisa Giaccone ◽

Lucia Brunello ◽

Roberto Passera ◽

Moreno Festuccia ◽

Milena Gilestro ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Flow Cytometry ◽

Conflicts Of Interest ◽

Univariate Analysis ◽

Conditioning Regimen ◽

First Line ◽

Significant Difference ◽

The Impact

Abstract Background Minimal residual disease (MRD) by multiparameter flow-cytometry recently showed a promising role in predicting outcomes in patients with multiple myeloma. However, data on immunophenotypic response (IR) after allografting are lacking. Aim To evaluate the impact of IR and compare it to conventional complete remission (CR) following allografting in myeloma patients. Methods Sixty-six consecutive patients, median age 54 years (35-66), who underwent an allograft between January 2000 and December 2011 with a follow-up of at least 3 months were included. Disease response was evaluated by serum and urine electrophoresis, and bone marrow aspirate at baseline, 3, 6, 12, 18, 24 months after transplant and yearly thereafter. Skeletal survey or MRI were performed yearly or as clinically indicated (overt relapse or complaints of bone pain). Bone marrow aspirates had to contain at least 13000 cells/µL for flow-cytometry studies and IR was defined as absence of monoclonal plasma-cells detected by 4 or 6-colour staining with the following antibodies: CD38, CD138, CD56, CD19, CD45, cyKappa, cyLambda. CR was defined according to standard criteria (Durie et al, Leukemia 2006; 20:1467-73). Results Conditioning regimen was non-myeloablative 2Gy TBI-based in 55 patients, reduced intensity (fludarabine-melphalan-based) in 10 and myeloablative in 1 patient. Post-grafting immunosuppression consisted of cyclosporine with mycophenolate mofetil or methotrexate. Donors were HLA identical siblings in 58 patients and unrelated in 8. Only 1 patient received bone marrow as source of stem cells. Thirty-five/66 (53%) received the allograft as part of the first line treatment, whereas the remaining 31/66, (47%) were transplanted at relapse. At the time of transplant, 5/66 were both in IR and CR, 16 were only in IR and 4 patients were only in clinical CR. All 21 patients in IR at the time of transplant maintained it, while 26/45 (58%) entered IR after the allograft. Among patients surviving at least 3 months, overall treatment related mortality was 10.6% at 3 years. After a median follow-up of 69 months (range 19-147), the incidence of acute and chronic graft-versus-host disease was 45.6% and 49.3% without significant difference between responsive and non-responsive patients. At follow-up, overall, 24 patients achieved CR and IR (CR/IR group), 21 achieved IR but not CR because of persistence of urine/serum M-component (noCR/IR group), and 21 did not achieve either CR or IR (noCR/noIR group). Interestingly, none achieved CR without IR. Median overall survival (OS) and event-free survival (EFS) in patients who achieved IR were 96 and 55 months versus 36 and 7 months in those who did not (p<0.001). Median OS and EFS were not reached and 59 months in the CR/IR group, 77 and 15 months in the noCR/IR, and 30 and 5 months in the noCR/noIR respectively (p<0.001 for both EFS and OS-fig.1). In univariate analysis, being in the CR/IR group was the only significant predictor for prolonged OS and EFS (p<0.001). Of note, cumulative incidence of extra-medullary disease at first relapse after the allograft was 4% in the CR/IR, 32% in the noCR/IR and 15% in the noCR/noIR groups respectively (p<0.001). Receiving the allograft as first line therapy or later during the disease course did not significantly impact on OS and EFS. Conclusion The achievement of IR confers a favorable impact on OS and EFS after allografting. A higher incidence of extra-medullary in the noCR/IR group (some 30% of our patient cohort) may suggest that myeloma cells escape immune control outside the bone marrow. In this group, imaging studies such as positron emission tomography may clinically be indicated during follow-up to detect early relapse. Disclosures: No relevant conflicts of interest to declare.

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Patient-Tailored Analysis of Minimal Residual Disease in Acute Myeloid Leukemia Using Next Generation Sequencing

Blood ◽

10.1182/blood.v126.23.3841.3841 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3841-3841

Author(s):

Erik Malmberg ◽

Sara Ståhlman ◽

Anna Rehammar ◽

Tore Samuelsson ◽

Sofie J Alm ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Exome Sequencing ◽

Deep Sequencing ◽

Myeloid Leukemia ◽

Residual Disease ◽

Leukemic Cells ◽

Multiparameter Flow Cytometry ◽

Targeted Deep Sequencing

Abstract Background and aim: The importance of sensitive minimal residual disease (MRD) analysis for determination of response to treatment in acute myeloid leukemia (AML) is becoming increasingly evident. Routinely, this analysis is performed using multiparameter flow cytometry, and in select cases with fusion transcripts using reverse transcription polymerase chain reaction. The drawback with flow cytometry is that it is associated with false negativity due to immunophenotypic shifts during treatment and in pending relapse. In addition, leukemia immunophenotypes often overlap with the normal regenerating bone marrow cell populations. Therefore, other means of identifying remaining leukemic cells are warranted. Leukemic cells in AML are characterized by somatic mutations in recurrently mutated genes as well as in random genes, in most cases as single nucleotide variations (SNVs). We have previously reported that leukemia-specific mutations can be readily identified at the time of diagnosis of AML using exome sequencing of high purity sorted leukemic cells and lymphocytes. The aim here was to show that leukemia-specific mutations identified with exome sequencing at diagnosis can serve as markers for MRD, quantified with targeted deep sequencing, during follow-up. Method: Seventeen cases of AML, age 2-71 years old, were included in the study. Leukemic cells and lymphocytes were sorted using fluorescence activated cell sorting (FACS), from blood or bone marrow at diagnosis of AML. Exome sequencing of sorted cell populations was performed on the Illumina platform. Variant calling was performed with Mutect for SNVs and with Strelka and Varscan for short insertions/deletions. The data was subjected to an in-house statistical algorithm to identify variants present in all leukemic cells and thus suitable for MRD analysis. For targeted deep sequencing, the Truseq-library system was used for in-house PCR and sequencing on the Illumina Miseq platform (2x150 bp). The acquired reads were stitched using PEAR, aligned to the human reference genome and the resulting alignments were analyzed with in-house scripts with respect to specific SNVs and NPM1 insertion. Results: Exome sequencing of the paired leukemia/lymphocyte samples identified 240 leukemia-specific SNVs (14 (0-29) per case (median, range) and 22 small insertions and deletions (1 (0-5) per case). The most common type of mutation was, as expected, substitution of cytosine to thymine (CàT). The number of leukemia specific SNVs correlated with age (r=0.76, p<0.001). Mutations suitable for MRD analysis were identified in all but one of the investigated AML cases. Targeted deep sequencing of leukemic cells in serial dilutions established linearity down to a determined variant allele frequency (VAF) of 0.025% for SNVs and of 0.016% for insertion in NPM1. The level of detection (mean+3SD of normal samples) was VAF 0.025% for SNVs and VAF 0.007% for insertion in NPM1. Targeted deep sequencing was then performed on DNA prepared from follow-up bone marrow slides from a patient with AML with mutations suitable for MRD analysis according to our algorithm. Targeted deep sequencing of three SNVs (in the genes CPS1, ITGB7 and FAM193A) and NPM1 type A mutation could detect mutations at all eight time points tested. There were strong correlations between the detected mutation load of the SNVs and the NPM1 type A mutation and all four mutations were present at relapse 10 months after diagnosis. Targeted deep sequencing of SNVs was in this case more sensitive and robust than multiparameter flow cytometry, which could not detect leukemic cells (<0.1% of all cells) at two of the tested time points (5 and 8 months after diagnosis) and showed a completely switched immunophenotype of leukemic cells at relapse. Conclusions: Exome sequencing of high purity sorted leukemic cells and lymphocytes at the time of diagnosis of AML can identify leukemia-specific mutations suitable for MRD analysis. With targeted deep sequencing of leukemia-specific SNVs identified in this manner, leukemic cell burden can be estimated with high sensitivity during follow-up. The method could be used for patient-tailored MRD analysis in AML. Disclosures No relevant conflicts of interest to declare.

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Thalidomide Combinations Improve Response Rates; Results from the MRC IX Study.

Blood ◽

10.1182/blood.v110.11.3593.3593 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3593-3593 ◽

Cited By ~ 27

Author(s):

Gareth J. Morgan ◽

Faith E. Davies ◽

Roger G. Owen ◽

Andrew C. Rawstron ◽

Sue Bell ◽

...

Keyword(s):

Plasma Cells ◽

Qualitative Difference ◽

Bone Marrow Aspiration ◽

Response Rates ◽

Multiparameter Flow Cytometry ◽

Serum Free Light Chain ◽

Definition Of ◽

New Treatments ◽

The Impact

Abstract Improving response rates represents an important way to improve PFS and OS in myeloma. In the MRC Myeloma VII study, now with a median follow up of 8 years, we showed the importance of achieving CR. The addition of thalidomide represents a way of increasing response rates. In the MRC Myeloma IX study, which has recruited 1800 patients, in two randomisations embracing younger and older patients, we have compared CTD with MP and CTD + HDM + ASCT with CVAD + HDM + ASCT. In addition to giving insight as to the impact of thalidomide on ORR and CR in both settings, we can assess the effects of HDM + ASCT following CTD induction. We have used a number of techniques to assess response in this study including protein electrophoresis, immunofixation and serum free light chain estimation combined with bone marrow aspiration and trephine assessment. This approach has been supplemented by sensitive multiparameter flow cytometry MRD analysis able to quantitate low levels of residual myeloma plasma cells. In a preliminary analysis, a thalidomide containing combination is associated with improved response rates. Post induction rates are CTD: ORR 95.7%, CR 20.3%; CVAD: ORR 83.4%, CR 11.7% and these differences are maintained at 100 days post HDM, CTD + HDM: ORR 98.7%, CR 58.2%; CVAD + HDM: ORR 95.7, CR 41%, showing that the ORR rate is higher and that the percentage of CR is doubled before HDM and 20% more following the HDM. The depth of CR is also greater in the cases receiving thalidomide. We are updating these results as well as the results from the MP vs CTD comparison and will present these data. There are some discrepancies between each of the disease monitoring approaches and optimally all should be used. At d+100 it is possible to have true CR measured by flow MRD assessment but still have detectable paraprotein. The opposite also occurs: apparent CR by paraprotein estimation with MRD positivity by flow. This has important implications for the definition of remission as well as for the assessment of new treatments and their use. Our findings suggest that thalidomide combinations significantly improve ORR, taking more patients to response levels which render them eligible for HDM + ASCT. CR rates are increased and there is a qualitative difference in the depth of CR obtained. The additive value of HDM + ASCT, even when thalidomide is used initially, is suggested by the further increase in the number of responses above that achieved by CTD alone. We await the follow up of patients entered into this large study to assess whether these increased and enhanced responses translate into improved PFS and OS.

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Analysis of Immunophenotypic Response (IR) by Multiparameter Flow Cytometry In 516 Myeloma Patients Included In Three Consecutive Spanish Trials

Blood ◽

10.1182/blood.v116.21.1910.1910 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1910-1910 ◽

Cited By ~ 2

Author(s):

Bruno Paiva ◽

María-Belén Vidriales ◽

María-Angeles Montalbán ◽

María-Victoria Mateos ◽

Laura Rosiñol ◽

...

Keyword(s):

Flow Cytometry ◽

Board Of Directors ◽

Residual Disease ◽

Young Patients ◽

Novel Agents ◽

Multiparameter Flow Cytometry ◽

High Dose ◽

Newly Diagnosed ◽

Advisory Committees ◽

The Impact

Abstract Abstract 1910 The outcome of multiple myeloma (MM) patients has markedly improved in the last decade. Thus, overall response rates between 85%-95%, with 30%-50% complete remission (CR) rates are now being reported in young patients treated with novel agents plus high-dose therapy/autologous stem cell transplantation (HDT/ASCT). A similar scenario is also emerging in the elderly (non-transplant candidates) population. Accordingly, more sensitive techniques are needed to assess patients’ response; these may contribute to compare the efficacy of different treatment schemas, to monitor minimal residual disease (MRD) and for prognostication. In the present study we have assessed the frequency and the prognostic value of IR by multiparameter flow cytometry in a total of 516 newly diagnosed MM patients included in three consecutive PETHEMA/GEM Spanish trials: two designed for transplant candidate patients - GEM 2000 (n=157) and GEM2005<65y (n=206) - and one for elderly patients - GEM2005>65y (n=153). The GEM2000 trial was based on 6 induction cycles of VBMCP/VBAD followed by HDT/ASCT; the GEM2005<65y included three arms with 6 cycles each (Thalidomide/Dexamethasone -TD-, Bortezomib/Thalidomide/Dexamethasone -VTD- and, VBMCP/VBAD with Bortezomib in the two final cycles -VBMCP/VBAD/Bortezomib) followed by HDT/ASCT; and the GEM2005>65y compared 6 cycles of Bortezomib/Melphalan/Prednisone -VMP- vs. Bortezomib/Thalidomide/Prednisone -VTP-. All three trials had in common that patients received 6 induction cycles and IR was evaluated at this time point. In addition, IR was assessed on day +100 after HDT/ASCT in the first two trials. Patients were defined to be in IR when myelomatous plasma cells (MM-PCs) were undetectable by MFC or when less than one phenotypically aberrant PC was detected among 104 cells analyzed. Patients were referred for MRD studies if they were mainly in CR or VGPR. The IR rates reported here were calculated on intention to treat analysis. Figure 1 summarizes the IR rates after induction. The lowest IR rates corresponded to the VBMCP/VBAD and TD schemes (5% and 6%, respectively) while with the bortezomib-based regimens an approximately 3-fold increment in the IR rates was observed: VTP (12%), VBMCP/VBAD/Bortezomib (15%), VMP (16%) and VTD (17%). After HDT/ASCT, IR rates were found to be significantly increased (p<.001) in the GEM2000 protocol (14%) and in all arms of the GEM2005<65y trial: TD (18%), VBMCP/VBAD/Bortezomib (30%) and VTD (34%). Thus, a minimum 2-fold increment of IR rates was further achieved after HDT/ASCT. In addition, IR rates achieved after HDT/ASCT in patients included in all three arms of the GEM2005<65y trial were significantly superior (p≤.008) to cases treated according to the GEM2000 protocol, indicating that induction regimens with novel agents improved post-transplantation rates of IR. Moreover, bortezomib-based regimens vs. TD were associated with increased IR rates not only before but also after HDT/ACSCT (p=.06 and p=.02 for VBMCP/VBAD/Bortezomib and VTD, respectively). We further compared the impact of achieving an IR after induction and at day+100 after HDT/ASCT in the progression-free (PFS) and overall survival (OS) within the three protocols. Patients in IR status after an induction regimen according to the GEM2000, GEM2005<65y and GEM2005>65y protocols showed significantly longer (p<.001) 3-year PFS rates (100%, 100% and 90%, respectively) compared to patients in a no-IR status (61%, 59% and 35%, respectively). Similarly, 3-year OS rates were significantly longer (p=.01) in IR vs. no-IR patients status (100%, 100% and 94% vs. 84%, 90% and 76% for the GEM2000, GEM2005<65y and GEM05>65y protocols, respectively). Likewise, an IR vs. no-IR status after HDT/ASCT in both the GEM2000 and GEM05<65y trials was also associated with significantly increased 3-year PFS (p<.001) and OS (p=.007) rates. In summary, this study demonstrates that the achievement of an IR is a strong prognostic factor regardless of the type of treatment; thus, higher IR rates may help to identify optimal therapeutical schemes. In this sense, HDT/ASCT is able to markedly increase IR rates after induction even in the era of novel agents, and this translates into extended survival. Disclosures: Off Label Use: VTP is not approved for the treatment of newly diagnosed myeloma patients and VT and VP are not approved for maintenance therapy. None of the combinations proposed, VBCMP/VBAD plus bortezomib, VT and VTD are approved as induction therapy in newly diagnosed myeloma patients. Mateos:Janssen Cilag: Honoraria; Celgene: Honoraria. Rosiñol:Janssen-Cilag: Honoraria; Celgene: Honoraria. Cibeira:Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Oriol:Janssen-Cilag: Honoraria; Celgene: Honoraria. de Arriba:Janssen-Cilag: Honoraria; Celgene: Honoraria. Palomera:Janssen Cilag: Honoraria. De La Rubia:Janssen-Cilag: Honoraria; Celgene: Honoraria. Díaz-Mediavilla:Janssen-Cilag: Honoraria; Celgene: Honoraria. Garcia-Laraña:Janssen Cilag: Honoraria; Celgene: Honoraria. Sureda:Janssen-Cilag: Honoraria; Celgene: Honoraria. Alegre:Janssen-Cilag: Honoraria; Celgene: Honoraria. Blade:Janssen cilag: Honoraria; Celgene: Honoraria. Lahuerta:Janssen-Cilag: Honoraria; Celgene: Honoraria. San Miguel:Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees.

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Validation of MRD Quantification By Flow Cytometry for Pediatric BCP ALL Relapsed Patients Treated on the Intreall Protocol

Blood ◽

10.1182/blood.v126.23.1414.1414 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1414-1414 ◽

Cited By ~ 1

Author(s):

Ester Mejstrikova ◽

Julie Irving ◽

Leonid Karawajew ◽

Marian Case ◽

Jenny Jesson ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Clinical Decision Making ◽

Conflicts Of Interest ◽

Residual Disease ◽

Lymphoblastic Leukemia ◽

Induction Treatment ◽

High Dose ◽

Risk Patients ◽

To Receive

Abstract At acute lymphoblastic leukemia (ALL) relapse, about 40% of children can be salvaged with intensified multi-agent, high dose chemotherapy and in very high risk patients, with additional stem cell transplantation (SCT). To improve on this, the International BFM Study Group has led a consortium of 19 countries to develop the world's largest trial for relapsed ALL (IntReALL). Standard risk patients will be randomized to receive the ALL-REZ BFM 2002 or UK ALL R3 therapy and post induction will be randomised to receive the additional targeted anti-CD22 drug, Epratuzumab, during consolidation, to clear residual disease. Children with end of re-induction MRD positive bone marrow will undergo SCT following consolidation. Both ALL-REZ BFM 2002 and UK ALL R3 used MRD PCR-based quantification of clonal Ig/TCR rearrangements, with different cut offs (10-3 for ALL-REZ BFM 2002 and 10-4 for UK ALL R3) and this is the reference assay for IntReALL. However, flow MRD may also play a role for patients without PCR targets. Flow MRD relies on the discrimination of leukaemic blasts from hematogones and this can be hampered depending on the degree of haematopoietic regeneration which varies depending on the treatment protocol and is especially important after intensive induction treatment in relapsed protocols. Thus, prospective MRD quantification of patients entered onto the UKALLR3 and ALL-REZ BFM 2002 clinical trials was performed by a standardised, quality assured, 4-8colour Flow MRD assay in end of re-induction bone marrow aspirates, by laboratories in the IBFM FLOW consortium (n=221). Flow MRD in both treatment protocols was classed as a prospective biological add on study and not used for clinical decision making. Median MRD levels were 0.026 +/-9.9% SD for BFM versus 0.027+/-18% SD for UK protocols, with comparative MRD positivity rates of 45% versus 54%, respectively. Comparison with MRD levels as assessed by molecular analysis of antigen receptor gene rearrangements was performed in 170 samples (BFM,128; UK R3, 42). The Spearman rank correlation of all samples was 0.90 (p<0.0001) for patients treated on the BFM protocol, compared to 0.82 (p<0.0001) for those on UK ALL R3. Risk category concordance was 88% (ALL-REZ BFM) and 88% (UKALLR3). For the 21 discordant samples, 5 were MRD positive by flow but negative by PCR and 17 were negative by flow and positive by PCR. When analysing the accuracy, with which flow MRD classified specimens identically as PCR, the sensitivity of flow MRD in the ALL-REZ BFM protocol was 81% (cut off 0.1%) and in UK ALL R3 was 79% (cut off 0.01%). Specificity values were 93% versus 100%, respectively. Although sample processing and quantification of MRD differ between PCR and FC MRD, in both re-induction protocols, there was good correlation of MRD levels assessed by flow cytometry and PCR, validating the use of Flow MRD as a method of choice in patients without PCR targets in the IntReALL trial. Flow MRD also has the advantage of enabling levels of CD22 to be assayed on MRD cells, prior to treatment with Epratuzumab. This research has received funding from the European Union's Seventh Framework Programme for research, technological development and demonstration under grant agreement no 278514 - IntReALL", Deutsche Kinderkrebsstiftung for its funding support of the ALL-REZ BFM 2002 clinical trial and the minimal residual disease studies by PCR and the Deutsche Jose Carreras Leukämiestiftung for support of the international principal investigator, Leukaemia and Lymphoma Research and North East Children's Cancer Research Fund, NT 13462-4, NV15-28525A, NV15-26588A, UNCE 204012. Disclosures No relevant conflicts of interest to declare.

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