Prognostic Value of Minimal Residual Disease Assessed By WT1 Expression Level and Flow Cytometry in Acute Myeloid Leukemia Patients Undergoing Allogeneic Marrow Transplantation

Abstract Background: Allogeneic bone marrow transplantation (BMT) offers the greatest chance of cure for most patients affected by acute myeloid leukemia (AML). Persistence of disease or high levels of pre BMT minimal residual disease (MRD) have been reported to predict disease relapse after BMT. WT1 expression levels and multicolor flow cytometry (MFC) are widely used as markers of MRD. We recently reported that combined evaluation of MRD by WT1 and MFC after induction therapy can predict relapse risk in AML patients. Aims: The aim of the present study was to apply the same MRD assessment in pre BMT setting to evaluate its reliability in predicting relapse. Methods: We retrospectively analyzed BMT outcome of 66 AML patients with both WT1-based and MFC-based MRD evaluation on bone marrow samples before transplant. Median age at transplant was 44 years. Forty-one were transplanted in first and twenty-five in second or subsequent complete remission. Induction therapies included fludarabine-containing regimens or standard ara-C and daunorubicin schedule (3+7). Median follow-up was 44 months (range 0-119 months); pre-transplantation evaluations were performed at a median of one month before transplant (range 1-3). Disease-free survival (DFS) was calculated from the time of transplantation until last follow-up or documented leukemic relapse. Overall survival was calculated from the time of transplantation to the last follow-up or death for any cause. All causes of death not directly due to relapse or progression of leukemia were considered as non-relapse mortality. A positive MFC MRD was defined by the presence of no less than 25 clustered leukemic cells /105 total events (threshold of 2.5x10-4 residual leukemic cells) at four-color flow-cytometry. Real-time PCR for WT1 was performed on DNA Engine 2 (Opticon®, MJResearch®). WT1 copy number/Abl copy number 500x104 was used as cut-off value for high WT1 expression. Results: Twenty-five relapses (37.9%) were observed. Median DFS was 31 months. Our analysis shows that the probability of relapse was significantly influenced only by disease status (first or subsequent CR) and MRD status at transplantation. Specifically, MFC-MRD was the strongest predictor of longer disease free survival (p <0.001) since no relapses occurred in the eleven MFC-MRD negative patients. Among MFC-MRD positive patients a further stratification of relapse risk is obtained by the evaluation of WT1. Patients with double positive MRD had a significantly worse DFS compared with patients who were MRD positive by MFC but MRD negative by WT1 (p <0.01). The predictive value of MRD was independent from different induction schedules; furthermore the favorable prognostic value of achieving a negative MRD status was not affected by undergoing BMT in second or subsequent remission. Median OS was 26 months and was significantly influenced by disease status and MRD status at transplantation and by relapse after BMT. Cumulative non relapse mortality was 23% at 36 months and was not associated with pre-BMT status. Conclusion: pre BMT MRD evaluation by WT1 and MFC on bone marrow samples is a reliable tool to predict relapse risk. Patients with negative pre-BMT MRD have a significantly longer DFS and OS, while MRD positive patients by both methods display a higher risk of relapse. Patients at higher risk of poor outcome should undergo a more stringent program of post BMT evaluations, in order to detect disease relapse earlier and might be candidate for pre-emptive therapeutic interventions aimed at delaying or avoiding AML reoccurrence. Disclosures No relevant conflicts of interest to declare.

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High Predictive Value of Pre Transplant Minimal Residual Disease Assessment By Combining WT1 Expression and Flow Cytometry in Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v126.23.2029.2029 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2029-2029 ◽

Cited By ~ 1

Author(s):

Fabio Guolo ◽

Federica Galaverna ◽

Paola Minetto ◽

Livia Giannoni ◽

Chiara Ghiggi ◽

...

Keyword(s):

Flow Cytometry ◽

Myeloid Leukemia ◽

Residual Disease ◽

Risk Group ◽

Disease Status ◽

Leukemic Cells ◽

Free Survival ◽

Relapse Risk ◽

Risk Of Relapse

Abstract BACKGROUND AND AIMS Allogeneic bone marrow transplantation (BMT) offers the greatest chance of cure for patients with high-risk acute myeloid leukemia (AML). Persistence of disease or high levels of pre BMT minimal residual disease (MRD) have been reported to predict relapse risk after BMT. WT1 expression levels and multicolor flow cytometry (MFC) are the most common tools to evaluate MRD. We recently reported that combining WT1 expression and MFC for MRD detection after induction therapy strongly impacts on relapse risk in AML. The aim of this study was to analyze the role of pre-BMT MRD assessment as predictor for the post-transplant relapse risk. MATERIALS AND METHODS We retrospectively analyzed the outcome of 253 consecutive AML patients receiving allo-BMT. Pre-BMT marrow samples were analysed for WT1 expression and MFC as MRD evaluation . Median age at transplant was 45 years. Disease phase was CR1 in 161, CR2 in 63, and CR3 in 29 patients. One hundred eighty-two received myeloablative conditioning, whereas 71 patients received reduced intensity conditioning. Median follow-up was 59 months (95% CI 46.2 - 71.8 months). Relapse-free survival (RFS) was calculated from the time of transplantation until last follow-up or documented leukemic relapse. Overall Survival (OS) was calculated from the time of transplantation until death by any cause or last follow-up. A positive MFC MRD was defined by the presence of no less than 25 clustered leukemic cells/105 total events (threshold of 2.5x10-4 residual leukemic cells) at four-color flow-cytometry. Real-time PCR for WT1 was performed on DNA Engine 2 (Opticon®, MJ Research®). WT1 copy number/Abl copy number 500x104 was used as cut-off value for abnormal WT1 expression. RESULTS Relapse occurred in 81 patients (32%). Three-year estimate of RFS was 63.7% (median not reached). The probability of relapse was significantly affected by disease status (first or subsequent CR, p<0.01), occurrence of acute GVHD (grade 0-1 versus 2 or more, p <0.05), MRD status before transplantation, measured with any method (p <0.001 for WT1-based MRD, p<0.03 for MFC based MRD, p<0.0001 for combined MRD). Multivariate RFS analysis revealed that the combined MRD evaluation was the only independent predictor of RFS (p <0.001). Specifically, MFC-MRD was the strongest predictor of longer relapse free survival (p <0.001) since only two relapses occurred in the 25 MFC-MRD negative patients and 3-years RFS was 89.9%. Among MFC-MRD positive patients, WT1 MRD status stratified the risk of relapse as the 3-years RFS was 71.9% and 31.3%, respectively, for patients with normal or increase WT1, p <0.01, fig.1). The predictive value of MRD was independent from induction schedules, donor type, disease status at BMT and risk group, occurrence of acute or chronic GVHD. Similarly, MRD evaluation was a strong predictor of long term survival, as 3- years OS was 77.2% for MFC negative and 36.9% for double WT1 and MFC MRD positive patients, respectively, (p <0.001). Multivariate OS analysis showed that BMT year, disease status at BMT and combined MRD evaluation significantly influenced OS duration (p <0.001, <0.002 and <0.003, respectively) CONCLUSIONS Pre transplant MRD evaluation by WT1 and MFC on bone marrow samples is a reliable predictor of relapse risk. Patients with both negative pre-BMT MRD markers have a significantly longer RFS, while patients with both positive MRD markers display an higher risk of relapse. Identifying patients who have an higher risk of relapse could open the way to apply pre-emptive therapeutic strategies to prevent AML relapse, from donor lymphocyte infusion to other innovative approaches. Figure 1. RFS according to risk group Figure 1. RFS according to risk group Disclosures No relevant conflicts of interest to declare.

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Taperring Treatment According Minimal Residual Disease

Blood ◽

10.1182/blood.v126.23.4960.4960 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4960-4960 ◽

Cited By ~ 1

Author(s):

Ihab A. Eldessouki ◽

Eman Z Kandeel ◽

Shady Adnan ◽

Mohammed Ghareeb ◽

Ola Gaber ◽

...

Keyword(s):

Bone Marrow ◽

Bone Marrow Transplantation ◽

Marrow Transplantation ◽

Residual Disease ◽

Disease Free Survival ◽

Salvage Chemotherapy ◽

Free Survival ◽

Disease Free ◽

Minimal Residual

Abstract In spite its established prognostic role in ALL and being a powerful method for patient stratification, Minimal residual disease in AML is still an area of research need to be investigated to decide its value in AML treatment. In this is a retrospective study, 388 adult AML patients from period 2009-2014 in NCI Cairo University were included, comparing minimal residual disease to other prognostic factors to determine its value as an independent prognostic factor to stratify AML patients and to assess possibility of treatment tapering according MRD. We divided patients in to 3 groups according cytogenetics: favorable, intermediate, poor risk. (We considered patients having negative MRD: those having day 28 and day 42 BMA free for MRD less than 0.01) All patients with FLT3 were excluded prior start this study because we proved by other study its grave prognosis and it outweigh MRD as independent prognostic factor, and eventually those patients will relapse within a short period of time. 5 years disease free survival First group patient with favorable cytogenetics: included 156 patients. We found that 76 patients who become MRD negative post first cycle induction had significantly better disease free survival 64% and overall survival 61.7% compared to those having persistence MRD ( 80 patient) post first cycle of induction 24%, 14% respectively with p value 0.02. Out of 76 patients had negative MRD, 29 patients just took 2 cycles of chemotherapy one induction chemotherapy and one consolidation. Those patients continued to maintain CR in spite receiving 2 cycles of chemotherapy which confirm powerful prognostic impact of MRD with DFS : 61, OS 59.3% which showed no significant difference from those who completed their chemotherapy (p value : 0.07) Those patients didn't continue treatment due to medical problems or non compliance or insurance coverage problems. Those who had persistence MRD post first cycle of induction had prognosis resembling those of poor cytogenetics. Out of 80 patients having persistent MRD, 9 died prior relapse due to medical problems. 64 relapsed and took salvage chemotherapy then kept under follow up. 23 patient did allogenic bone marrow transplantation, 9 were in CR and were done due to persistence MRD and 14 patient did due to relapse and transplantation were done in second CR. patients who had did allogenic transplantation had better disease free survival and overall survival. Second group intermediate risk: 103 patients. We had 40 patients with negative MRD, whose DFS and OS were 59% and 55% respectively. Of those patients, 14 received only 2 cycles of chemotherapy and also showed favorable prognosis in spite being intermediate risk and retained CR. DFS : 57%, OS 55% with no statistical difference between those continued chemotherapy or not. 63 Patients had positive MRD, out of them 5 patients had lost follow up. DFS was13% and OS was 11%. 47 patients relapsed took salvage chemotherapy and kept under follow up out of which 16 patients did bone marrow transplantation. 11 patients did bone marrow transplantation due to persistence MRD and they had longer disease free survival compared to those had salvage chemotherapy and kept under follow up. Same disease free survival overall survival to those did BMT post second CR. Third group with poor risk cytogenetic included 127 patients. 32 patients got MRD negative (DFS: 38% OS: 8%). Out of which 9 didn't receive further chemotherapy post 2 cycles. Again with no significant p value between both groups (P: 0.08) We had 95 patients with persistent MRD post induction. 11 patients lost follow up. 65 relapsed and received salvage chemotherapy DFS 29% and OS: 5%. 19 patients did allogenic bone marrow transplantation. 8 patients did allogenic bone marrow transplantation due to persistence MRD. We found that poor risk cytogenetic outweighs MRD and only patients did BMT had favorable outcome regarding disease free survival 42% and overall survival 11%. Finally we conclude that minimal residual disease can be used as independent prognostic factor. Also MRD can be used as in stratifying patients and tailoring the treatment plan allowing the possibility to stop treatment at a less number of cycles and preventing further chemotherapy complications. Disclosures No relevant conflicts of interest to declare.

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Allogeneic or Autologous Stem Cell Transplantation (SCT) for the Treatment of Pediatric Patients with Non-Hodgkins Lymphoma (NHL).

Blood ◽

10.1182/blood.v106.11.2093.2093 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2093-2093

Author(s):

Rosanna Ricafort ◽

Tanya Trippett ◽

Nancy A. Kernan ◽

Trudy N. Small ◽

Susan Prockop ◽

...

Keyword(s):

Pediatric Patients ◽

Residual Disease ◽

Significant Proportion ◽

Disease Free Survival ◽

Disease Status ◽

Entire Group ◽

Free Survival ◽

Entire Cohort ◽

Disease Free

Abstract Thirty six patients (pts) with NHL, aged 3.5–20.9 years (median 14.7 years) received an allo-SCT (n=21) or auto-SCT (n=15) at our institution between 12/82 and 12/04. Pathologic NHL classification included: lymphoblastic (n=12), Burkitt’s (n=5), Large Cell (n=17) including ki-1 + (n=11), peripheral NHL (n=1), and undifferentiated NHL (n=1). Disease status at SCT was: first remission (CR1) (n=1), CR2 (n=15), CR3 (n=5), partial remission (n=7), relapse (Rel) or refractory (Ref) (n=8). Cytoreductive regimens were total body irradiation (TBI)-based for all but 3 pts in each of the allo-SCT and auto-SCT group. For the allo-SCT pts, donors were: matched related (n=15), mismatched related (n=1), and unrelated (n=5); graft-versus-host disease (GvHD) prophylaxis included T-cell depletion (TCD) (n=10) or post-BMT immunosuppression (n=11). Only one pt received a non-myeloablative allo-SCT. With one pt in the allo-SCT group lost to follow-up, 35 of 36 pts in the entire cohort were evaluable. The median follow up of the entire group was 21.5 months; it was 34 months and 20 months respectively for the allo-SCT and the auto-SCT groups. The overall survival (OS) and disease-free survival (DFS) were 56% and 54%, respectively. Ten of the 11 pts with ki-1 positive NHL are alive, disease-free after either allo-SCT (n=4) or auto-SCT (n=7) with a median follow-up of 26 months. The DFS for those patients transplanted in CR was 60% compared to 49% for those transplanted in either PR or Rel/Ref disease. Of the eight pts transplanted with progressive disease, 2 are alive, disease-free at 110 and 115 months; both pts post allo-SCT. For the allo-SCT group, the DFS was 52%. Seven pts relapsed, 4 of whom had Rel/Ref disease at the time of SCT. Four pts died of SCT-related complications. Five pts developed Grade II–IV aGvHD. For the auto-SCT group, the DFS was 53%. Two pts relapsed, and 2 pts died of toxicity. In summary, both allo-SCT and auto-SCT offer the prospect of durable disease-free survival for a significant proportion of pediatric patients with NHL after a first relapse or disease progression. In particular, those pts with ki-1-positive NHL had the most favorable outcome. As expected, pts transplanted in minimal residual disease achieved superior DFS. Results N CR/PR Rel/Ref Relapse TRM DFS Allo-SCT 21 11/4 6 33% 20% 52% Auto-SCT 15 10/3 2 13% 13% 53%

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Busulfan/etoposide--initial experience with a new preparatory regimen for autologous bone marrow transplantation in patients with acute nonlymphoblastic leukemia

Blood ◽

10.1182/blood.v81.2.319.319 ◽

1993 ◽

Vol 81 (2) ◽

pp. 319-323 ◽

Cited By ~ 47

Author(s):

NJ Chao ◽

AS Stein ◽

GD Long ◽

RS Negrin ◽

MD Amylon ◽

...

Keyword(s):

Bone Marrow ◽

Complete Remission ◽

Median Time ◽

Relapse Rate ◽

Disease Free Survival ◽

Autologous Bone Marrow ◽

Free Survival ◽

Acute Nonlymphoblastic Leukemia ◽

Disease Free

Abstract Current intensive chemotherapy for acute nonlymphoblastic leukemia (ANLL) results in a complete remission in the majority of patients. Unfortunately, the duration of remission is short and most of the patients will experience a relapse of their underlying disease. Autologous bone marrow (BM) transplantation is being explored as a treatment modality designed to improve relapse-free survival. We have conducted a phase II trial exploring the combination of busulfan (16 mg/kg) and etoposide (60 mg/kg) in an attempt to improve antitumor efficacy using this novel preparative regimen. To date, 50 patients (48 with ANLL and 2 patients with biphenotypic acute leukemia) have been treated. The first 20 patients received unmanipulated BM; 28 patients subsequently received 4-hydroperoxycyclophosphamide (4–HC) (60 micrograms/mL)-purged bone marrow, and 2 patients with biphenotypic acute leukemia received both 4–HC (60 micrograms/mL) and etoposide (5 micrograms/mL)-purged BM. Thirty-four patients were in first complete remission (CR1), 12 patients in second complete remission (CR2), and 4 patients in relapse. The median time from first complete remission to BM harvest was 3 months (range, 0.8 to 4) compared with median time of 2 months (range, 1.5 to 5.0) for patients in second complete remission. The median time from harvest to transplant was 1 month for both groups (range, 0.4 to 36). A median of 0.7 x 10(8) (range, 0.2 to 1.4) mononuclear cells were infused. Patients achieved an absolute neutrophil count of > or = 500/microL at a median of 26 days (range, 13 to 96), an untransfused platelet count > or = 20,000/microL at a median of 56 days (range, 15 to 278) and a sustained hematocrit > or = 30% at a median of 50 days (range, 19 to 116). Twenty-six patients are alive and in continued CR. Follow-up of the surviving patients ranged from 6 months to 66 months with a median follow-up of 31 months. Patients receiving purged BM have an actuarial disease-free survival of 57% with a relapse rate of 28% compared with patients receiving unpurged BM whose actuarial disease-free survival is 32% with a relapse rate of 62% (P = .06 for relapse rate). The most significant extramedullary toxicities for this regimen are hepatic and cutaneous (including mucositis). The BU/VP-16 regimen is associated with a significant proportion of patients surviving disease free, especially in the group receiving purged BM. Whether this regimen offers a substantial improvement in disease-free survival over currently used regimens will require a prospective randomized study.

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Monitoring AML Disease Status with Next Generation Sequencing

Blood ◽

10.1182/blood.v128.22.5261.5261 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5261-5261

Author(s):

Zach Liu ◽

Nikolay Dimov

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Residual Disease ◽

Disease Status ◽

Newly Diagnosed ◽

Fish Cytogenetics ◽

Bone Marrow Morphology ◽

Work Up ◽

Generation Sequencing

Abstract Recent advance in next generation sequencing (NGS) have confirmed that AML is a heterogeneous malignancy harboring may many genetic mutations. These mutations have been studied for leukemia genesis, diagnosis and therapeutic targets. Monitoring minimal residual diseases has also been studied recently. We summarized our experience with NGS in morning AML disease status. NGS data during 2014 and 2016 from patient with newly diagnosed or AML and/or AML follow-up patients along with bone marrow biopsy, FISH/cytogenetics, flow cytometric results were reviewed. Targeted sequencing was performed with customized panel (34 genes) on Ion PGM platform from Life Technology Inc. 41 AML patients with complete bone marrow work-up with bone marrow morphology, flow cytometry, FISH/cytogenetics (MFFC) and NGS were collected. At least one sample with complete work-up for each patient was included. Majority of the patients had several studies (2-8 samples). 15 out of 41 (36.6%) has complete remission based on bone marrow morphology, flow cytometry, FISH/cytogenetic studies. No mutations were detected among these 15 patients. 17 patients (41%) showed concordant result with other technologies, i.e. when the patient was in remission based on MFFC, No mutations were detected. When patient had recurrent AML or residual disease, mutations were detected. It worth to point out that 2 patients showed positive mutation without detectable increase in myeloblasts. These 2 patients had relapsed AML within 3 months. Different subclones were detected at different intervals in 1 patient. 2 (0.5%) patients (1 with newly diagnosed AML and 1 with early recurrent AML) showed no detectable mutations. Mutations were detected in 5 patients (12%) with AML remission by MFFC, additional follow-up is need for these patients. The most common mutations included TET2, ASXL1, DNMT3A, RUNX1, IDH1 and TP53. NGS is valuable to assess the AML status despite of heterogeneous genetic abnormalities. Although the NGS results were concordant with bone marrow morphology, FISH/cytogenetics and flow cytometry in most of the cases (87.5%), persistent mutations may be detectable in cases without detectable residual AML by other modalities, which may be associated with minimal residual disease or early relapse, and need further evaluation. Clonal evaluation may occur at molecular level occasionally. Disclosures No relevant conflicts of interest to declare.

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Autologous bone marrow transplantation for high-grade lymphoid malignancy using melphalan/irradiation conditioning without marrow purging or cryopreservation. The Northern Regional Bone Marrow Transplant Group

Blood ◽

10.1182/blood.v77.7.1593.bloodjournal7771593 ◽

1991 ◽

Vol 77 (7) ◽

pp. 1593-1598

Author(s):

PJ Carey ◽

SJ Proctor ◽

P Taylor ◽

PJ Hamilton

Keyword(s):

Bone Marrow ◽

Disease Free Survival ◽

Autologous Bone Marrow ◽

Marrow Transplant ◽

High Grade ◽

Lymphoid Malignancy ◽

Free Survival ◽

First Remission ◽

Disease Free

We report the safety and efficacy of 34 consecutive autologous bone marrow transplant (ABMT) procedures performed in adult patients with high-grade lymphoid malignancy after remission induction therapy. Fifteen patients with acute lymphoblastic leukemia (ALL) and six with high-grade non-Hodgkin's lymphoma (NHL) received pretransplant conditioning with intravenous (IV) melphalan and fractionated total body irradiation (TBI). Thirteen other patients with NHL were conditioned with melphalan alone, having previously received local involved field radiotherapy. Unmanipulated noncryopreserved autologous marrow was reinfused within 48 hours of harvesting. Engraftment occurred in all patients with medians of 10 days of neutropenia (neutrophils less than 0.5 x 10(9)/L), 4-day platelet transfusion requirement, 3 U packed RBC transfusion, and 18 days in hospital posttransplant. There were no procedure-related deaths. Actuarial disease-free survival in the 13 patients with ALL receiving autotransplant early in first remission is 48% with a median follow-up of 3 years. Two other ALL patients who had autotransplants after a period of maintenance therapy also remain in complete remission (CR). These results compare favorably with our 34% disease-free survival (DFS) in 15 allogeneic ALL transplant patients and 21% DFS in 19 patients on standard maintenance after a common induction schedule. No relapses have occurred in the 17 NHL patients transplanted in remission (median follow-up 2 years), but the two NHL patients who developed recurrent disease before ABMT died of progressive disease after temporary responses. We conclude that this method of ABMT results in rapid reengraftment with lack of toxicity and that the conditioning treatment used shows good efficacy against disease. It is applicable in high-grade lymphoid malignancy in first remission, and our results call into question the need for marrow purging in ALL and NHL patients transplanted in first remission.

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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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90Y-Ibritumomab Tiuxetan (Zevalin®) in Combination with High-Dose Therapy (HDT) Followed by Autologous Stem Cell Transplant (ASCT) May Improve Survival in Patients with Poor-Risk Follicular Lymphoma (FL) and Diffuse Large B-Cell Lymphoma (DLBCL): Results of a Retrospective Comparative Analysis.

Blood ◽

10.1182/blood.v108.11.327.327 ◽

2006 ◽

Vol 108 (11) ◽

pp. 327-327 ◽

Cited By ~ 4

Author(s):

Auayporn P. Nademanee ◽

Amrita Krishnan ◽

Nicole Tsai ◽

Joycelynne Palmer ◽

Arturo Molina ◽

...

Keyword(s):

B Cell ◽

Performance Status ◽

Disease Free Survival ◽

Disease Status ◽

High Dose ◽

Free Survival ◽

Poor Risk ◽

Disease Free

Abstract Since NHL is radiosensitive, total body irradiation (TBI) has been used as part of HDT and ASCT for NHL. However, due to short-term and long-term toxicity associated with TBI, alternative regimens have been developed. We have reported that Zevalin at conventional and high doses can be given in combination with HDT and ASCT in patients (pts) with poor-risk or relapsed B-cell NHL without additional toxicity. Given the efficacy of Zevalin in FL and DLBCL, we retrospectively evaluated the outcome of HDT and ASCT in pts with FL and DLBCL who received Zevalin-based HDT regimens (Z-ASCT) and compared to those receiving TBI-based regimen (TBI-ASCT)Between 1/2000 to 1/2006, 187 pts with FL grade I/II (30), FL grade III (20) and DLBCL (137) underwent HDT and ASCT, 62 received Z-ASCT while 125 received TBI-ASCT. For Z-ASCT, pts < 60 years old without prior radiotherapy (RT) received high-dose Zevalin in combination with high-dose etoposide and cyclophosphamide while pt > 60 yrs or with prior RT received conventional dose Zevalin plus high-dose BEAM. TBI-ASCT was performed during the same period for the following reasons: ineligible for Z-ASCT, pt refusal, physician preference and protocol closure. The pt characteristics between the two groups were similar with respect to histology, disease status, prior regimens, bulky disease, B symptoms and performance status. However, the median age was younger for TBI-ASCT (49 vs. 53, p=0.01) and there were more chemo-resistant pts in the Z-ASCT group (p=0.01). Results: At a median follow-up of 28 months (range 2–64) for Z-ASCT and 38 months (range 1–78) for TBI-ASCT, the 2-year overall survival (OS) and disease-free survival (DFS) were 91% (95% CI, 82–96) and 74% (95% CI, 64–82), respectively for Z-ASCT, and 76% (95% CI, 69–80) and 72% (95% CI, 65–77), respectively for TBI-ASCT(Figure 1). OS remained significantly different when first complete remission pts were excluded from analysis (p=0.019). Multivariate models were generated for the primary endpoints of the study (OS and DFS). The results of these analyses showed that the risk of death and/or relapse was less among the Z-ASCT pts after adjusting for baseline differences (ie. Age, performance status and chemosensitivity status at transplant), and other factors (i.e., disease status at transplant, number of previous chemotherapies) previously shown to be associated with survival/disease free survival post transplant (OS: p<0.01 | DFS: p<0.10). Conclusion: Zevalin in combination with HDT followed by ASCT was associated with significantly improved survival in pts with poor-risk or relapsed/refractory FL and DLCBL when compared to TBI-ASCT. Further studies and longer follow-up are required to evaluate the long-term efficacy and safety of Zevalin in the HDT/ASCT setting. Figure Figure

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Busulfan/etoposide--initial experience with a new preparatory regimen for autologous bone marrow transplantation in patients with acute nonlymphoblastic leukemia

Blood ◽

10.1182/blood.v81.2.319.bloodjournal812319 ◽

1993 ◽

Vol 81 (2) ◽

pp. 319-323 ◽

Cited By ~ 1

Author(s):

NJ Chao ◽

AS Stein ◽

GD Long ◽

RS Negrin ◽

MD Amylon ◽

...

Keyword(s):

Bone Marrow ◽

Complete Remission ◽

Median Time ◽

Relapse Rate ◽

Disease Free Survival ◽

Autologous Bone Marrow ◽

Free Survival ◽

Acute Nonlymphoblastic Leukemia ◽

Disease Free

Current intensive chemotherapy for acute nonlymphoblastic leukemia (ANLL) results in a complete remission in the majority of patients. Unfortunately, the duration of remission is short and most of the patients will experience a relapse of their underlying disease. Autologous bone marrow (BM) transplantation is being explored as a treatment modality designed to improve relapse-free survival. We have conducted a phase II trial exploring the combination of busulfan (16 mg/kg) and etoposide (60 mg/kg) in an attempt to improve antitumor efficacy using this novel preparative regimen. To date, 50 patients (48 with ANLL and 2 patients with biphenotypic acute leukemia) have been treated. The first 20 patients received unmanipulated BM; 28 patients subsequently received 4-hydroperoxycyclophosphamide (4–HC) (60 micrograms/mL)-purged bone marrow, and 2 patients with biphenotypic acute leukemia received both 4–HC (60 micrograms/mL) and etoposide (5 micrograms/mL)-purged BM. Thirty-four patients were in first complete remission (CR1), 12 patients in second complete remission (CR2), and 4 patients in relapse. The median time from first complete remission to BM harvest was 3 months (range, 0.8 to 4) compared with median time of 2 months (range, 1.5 to 5.0) for patients in second complete remission. The median time from harvest to transplant was 1 month for both groups (range, 0.4 to 36). A median of 0.7 x 10(8) (range, 0.2 to 1.4) mononuclear cells were infused. Patients achieved an absolute neutrophil count of > or = 500/microL at a median of 26 days (range, 13 to 96), an untransfused platelet count > or = 20,000/microL at a median of 56 days (range, 15 to 278) and a sustained hematocrit > or = 30% at a median of 50 days (range, 19 to 116). Twenty-six patients are alive and in continued CR. Follow-up of the surviving patients ranged from 6 months to 66 months with a median follow-up of 31 months. Patients receiving purged BM have an actuarial disease-free survival of 57% with a relapse rate of 28% compared with patients receiving unpurged BM whose actuarial disease-free survival is 32% with a relapse rate of 62% (P = .06 for relapse rate). The most significant extramedullary toxicities for this regimen are hepatic and cutaneous (including mucositis). The BU/VP-16 regimen is associated with a significant proportion of patients surviving disease free, especially in the group receiving purged BM. Whether this regimen offers a substantial improvement in disease-free survival over currently used regimens will require a prospective randomized study.

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Allogeneic Transplantation for Adult Acute Lymphoblastic Leukemia (ALL) with Rituximab or Campath I-H.

Blood ◽

10.1182/blood.v104.11.5132.5132 ◽

2004 ◽

Vol 104 (11) ◽

pp. 5132-5132

Author(s):

Issa F. Khouri ◽

Rima M. Saliba ◽

Partow Kebriaei ◽

Carrie Ma ◽

Cindy Ippoliti ◽

...

Keyword(s):

Median Time ◽

Acute Gvhd ◽

Disease Free Survival ◽

Disease Status ◽

Allogeneic Transplantation ◽

Study Group ◽

Free Survival ◽

The Impact ◽

Disease Free

Abstract Because of potential synergy with chemotherapy and non-overlapping toxicity, we investigated the addition of Rituximab or Campath 1-H to the standard myeloablative conditioning regimen of cyclophosphamide (60 mg/kg daily x 2) and total body irradiation (12.0 Gy in four fractions) prior to allogeneic transplantation for ALL. Transplantation was performed on day 0. Rituximab was added if patients’ disease expressed CD20+ > 20% by flow-cytometry. It was administered (375 mg/m2 ) on days −6, −1, +7 and +14. Campath I-H (10 mg daily intravenously, days −6 to −2) was added if patients’ CD20 expression was <20% and CD52 >20%. Thirty-two adult consecutive patients were studied. Eleven were in first remission with poor prognostic features, 11 in 2nd remission, and 10 were ≥ 3rd remission, or in relapse. Twenty-nine patients had B-cell, two had T-cell and one had an undifferentiated phenotyping. The study group included 19 males and 13 females of median age 35 yrs (range, 19–57). Median # of prior chemoregimens received was 2 (range, 1–6). In both groups of patients, prophylaxis for GVHD consisted of a combination of tacrolimus and methotrexate. Pharmacokinetic studies in patients who received Campath I-H showed no detectable level of the antibody one-day prior to- or after the infusion of the donor graft. Median follow-up for survivors was 19 months. Outcomes were: Campath-study group Rituximab-study group P -value Prior Chemoregimens (range) 2(1–6) 2(1–3) 0.04 Donor Type Matched unrelated 3(28%) 8(38%) 0.2 Matched sibling 7(63%) 12(57%) Mismatched sibling 1(9%) 1(5%) Cell Source PB 8(73%) 11(52%) 0.2 Marrow 3(27%) 10(48%) Disease Status CR1/CR2 5(45%) 17(81%) 0.05 Others 6(55%) 4(19%) Median time ANC >500 13 12 0.07 (range) (11–17) (10–24) Median time Platelets >20K 13 13 0.8 (range) (6 – 31) (7 – 34) Day 100 TRM 0 1(5%) Acute GVHD II–IV (N,% kM) 2 (18%) 5 (24%) 0.7 Acute GVHD III–IV (N, % kM) 0 2 (9%) Chronic extensive GVHD (N, cumulative incidence) 3 (27%) 9 (54%) 0.4 Overall Survival (18 mos) (95% CI) 53% (21 – 77) 52% (26 – 73) 0.9 Disease-free survival (18 mos) (95% CI) 54% (23 – 75) 37% (15 – 60) 0.8 No prognostic factor was found to be of significance for survival, disease-free survival, or relapse. This included: age (<35 vs ≥ 35), source of graft, disease status at transplant, # prior regimens (<2 vs ≥ 2), acute or chronic GVHD, use of Rituximab or Campath. Our results indicate that the addition of Rituximab or Campath I-H in allogeneic transplantation for ALL is safe. There was no delay in engraftment and no added toxicity or risk of mortality. Longer follow-up is needed to evaluate the impact of this strategy upon survival and relapse.

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