Minimal Residual Disease Testing Research Articles

Outcomes after Intermediate-Risk Relapse of Childhood B-Lymphoblastic Leukemia (B-ALL) and the Role of Allogeneic Stem Cell Transplantation (SCT): A Report from Children’s Oncology Group (COG) AALL0433

BACKGROUNDRelapsed childhood B-ALL has a poor prognosis, with time to and site of relapse being the best clinical predictors of outcome. The role of allogeneic SCT is unclear for patients with late bone marrow (BM) or isolated extramedullary (IEM) relapse. We recently reported initial results from the AALL0433 trial for intermediate-risk relapse of childhood B-ALL, which established a 0.1% end-induction minimal residual disease (MRD) threshold as the best predictor of outcome (Lew, ASCO 2014). We now report an updated analysis, including outcomes for patients receiving SCT vs. continued chemotherapy.METHODSAALL0433 included patients with early CNS/testicular (IEM) relapse (<18 mo. from diagnosis), or late BM/combined relapse (≥36 mo. from diagnosis) of B-ALL, enrolling 271 eligible patients between 3/2007 and 10/2013. Therapy was based upon the earlier COG AALL01P2 / P9412 platforms. BM MRD was measured by flow cytometry at the end of Induction block 1, and for this analysis was considered positive (MRD+) if ≥0.1%, or negative (MRD-) if <0.1%. MRD testing was performed centrally by a COG reference lab, with results blinded to local investigators. 48 patients underwent matched family donor SCT per protocol after 3 induction blocks. An additional 31 patients were removed from protocol therapy to pursue off-study alternative donor SCT. Donor sources for these patients were 21 unrelated BM, 9 unrelated cord blood, and 1 haploidentical BM. The remaining 192 patients received chemotherapy (plus irradiation for those with EM involvement at relapse). Event free and overall survival (EFS/OS) comparisons for patients receiving chemotherapy vs. SCT were adjusted to start from median time to SCT (138 days) or the actual time of SCT if <138d. Patients who had events or dropped out before the adjusted starting time were excluded from the survival analyses.RESULTSThe 3-yr. EFS/OS for the entire cohort of 271 patients were 61.4 ± 4.3% and 72.9 ± 3.9% respectively. Focusing on patients with BM/combined relapse, the 3-yr EFS/OS for the 175 patients with available MRD data showed EFS/OS of 80.4 ± 4.7% and 88.3 ± 3.8% respectively for MRD- patients, compared to 45.1 ± 8.4% and 60.1% ± 8.3% for those who were MRD+ (p<0.01) (FIGURE 1). Because outcomes for patients who received matched family donor or alternative donor SCT were highly similar, these were pooled in all analyses of chemotherapy vs. SCT. There was no significant difference in survival for MRD- patients; 3-yr EFS and OS for the chemotherapy group were 75.4 ± 6.7% / 91.8 ± 4.3%, vs. 80.1 ± 8.9% / 84.0 ± 8.1% in the SCT group (p>0.5). In MRD+ patients the 3-yr EFS was 42.2 ± 13.1% vs 62.8 ± 13.5% (p=0.30) for the chemotherapy and SCT groups respectively, corresponding to a 3-yr OS of and 57.6 ± 12.5% vs 78.1 ± 12.9% (p=0.14), with a trend toward improved survival after 3 years with SCT (FIGURE 2).Although numbers were small, there was a clear trend toward improved outcomes in IEM patients receiving SCT over chemotherapy (FIGURE 3). 3-yr EFS for the chemotherapy vs. SCT groups were 31.3% ± 25.9% vs. 71.4% ± 22% (p=0.16), with OS of 31.3% ± 25.9% vs. 77.8% ± 21.2% (p=0.08). No IEM relapse patients were MRD+ at end induction.CONCLUSIONSPatients with late BM relapse of B-ALL who are MRD- after induction have a relatively good outcome (3-yr EFS of 80.4 ± 4.7%) on COG AALL0433. While additional follow-up is needed, there was no obvious benefit of SCT over chemotherapy for these patients. Outcomes were worse for BM relapse patients who remained MRD+ after induction (3-yr EFS 45.1 ± 8.4%). There was a trend toward benefit for SCT over chemotherapy for MRD+ patients. In the small number with early IEM, there was also a clear trend toward superiority of SCT over chemotherapy + radiotherapy. These data support the approach of reserving SCT for patients with late BM relapse of childhood B-ALL who remain MRD+, and also for patients with early IEM relapse. [Display omitted] [Display omitted] [Display omitted] DisclosuresRheingold:Novartis: Consultancy. Whitlock:Glaxo-Smith-Kline: Research Funding. Borowitz:Becton Dickinson Biosciences: Research Funding. Hunger:Sigma Tau Pharmaceuticals: Honoraria; Jazz Pharmaceuticals: Honoraria.

Phase II Study of Cladribine and Low-Dose Cytarabine (AraC) Alternating with Decitabine in Older Patients with Acute Myeloid Leukemia (AML)

BackgroundIntensive chemotherapy is associated with poor tolerability and high early mortality in the treatment of AML in older patients. Lower intensity approaches using hypomethylating agents have been developed in patients with lower bone marrow (BM) blast counts, but more effective therapies are needed, particularly in those with higher BM blasts. Cladribine, which itself may have hypomethylating properties, has single-agent activity in AML. Cladribine has also been shown to improve survival in AML in combination with standard-dose araC (Holowiecki JCO 2012).MethodsWe developed a low-intensity, prolonged-maintenance treatment protocol studying the combination of cladribine and low-dose araC (LDAC) alternating with decitabine in patients aged ≥ 60. Patients with adequate organ function and newly diagnosed AML (except APL), including secondary- (s-AML) and therapy-related AML (t-AML), and high risk MDS were eligible. Induction consisted of cladribine 5 mg/m2 IV over 30 minutes on days 1-5 followed by araC 20mg SQ BID on days 1-10. Consolidation/maintenance consisted of 2 cycles of cladribine 5 mg/m2 IV over 30 minutes on days 1-3 + araC 20 mg SQ BID on days 1-10 alternating with 2 cycles of decitabine 20 mg/m2 on days 1-5, for a maximum of 18 cycles. One cycle was 4 weeks and up to 2 cycles of induction were allowed.ResultsA total of 74 patients have been enrolled with a median age of 69 years (range, 49-85), including 33 pts (45%) ≥ age 70. 41 pts (55%) had secondary- (s-AML) or therapy-related AML (t-AML) and 14 pts (19%) had therapy for an antecedent hematologic disorder. Patient characteristics are listed in table 1. Of the 74 pts evaluable for response, there were 42 CR (57%), 6 CRp (8%), and 3 PR (4%) for an overall response rate of 69% (51/74). 43 patients (58%) had BM blasts ≥ 30%, and they had a CR/CRp rate of 67%. Of the 43 patients who achieved a CR/CRp and had minimal residual disease testing (MRD) by flow cytometry, 28 patients (65%), achieved MRD negativity at the time of CR or later. AML mutation screening was performed prior to treatment. Ten of 10 patients (100%) with FLT3-mutated AML and 13/13 (100%) pts with an NPM1 mutation achieved a CR/CRp. With a median follow-up of 11+ months, the median OS is 12.7 months and the median CR duration is 13.1 months. (Figure 1) The 1-year OS estimate is 57%. The regimen was very well tolerated, with 1% 4-week mortality. There were no treatment-related grade 3/4 non-hematologic adverse events (AEs). Most common non-hematologic AEs were elevated bilirubin, nausea/vomiting, rash/itching, diarrhea and mucositis.ConclusionThe low intensity program of cladribine + LDAC alternating with decitabine is a highly effective, well-tolerated ambulatory regimen for older patients, including those with unfavorable-risk features. Patient samples are being analyzed for methylation changes on treatment.Table 1Patient Characteristics (N=74)CharacteristicN (%)Median age [Range]69 [49-85]CytogeneticsDiploid21 (28)Adverse30 (41)-5/-723 (31)Complex without -5/-77 (9)Misc. other17 (23)Insufficient6 (8)MutationsNPM113/69 (19)FLT3-ITD8/69 (12)RAS15/69 (22)Median BM Blast [Range]32 [8-95]Median WBC [Range]2.5 [0.5-72.3]Median Platelets [Range]40 [4-772]Median Peripheral Blasts [Range]6 [0-88]Median LDH [Range]742 [301-8425]Median Creatinine [Range]0.91 [0.46-1.94]Median Bilirubin [Range]0.6 [0.2-2] [Display omitted] DisclosuresKantarjian:ARIAD, Pfizer, Amgen: Research Funding.

Familial and sporadic pancreatic cancer share the same molecular pathogenesis.

Pancreatic ductal adenocarcinoma (PDAC) is nearly uniformly lethal, with a median overall survival in 2014 of only 6months. The genetic progression of sporadic PDAC (SPC) is well established, with common somatic alterations in KRAS, p16/CDKN2A, TP53, and SMAD4/DPC4. Up to 10% of all PDAC cases occur in families with two or more affected first-degree relatives (familial pancreatic cancer, FPC), but these cases do not appear to present at an obviously earlier age of onset. This is unusual because most familial cancer syndrome patients present at a substantially younger age than that of corresponding sporadic cases. Here we collated the reported age of onset for FPC and SPC from the literature. We then used an integrated approach including whole exomic sequencing, whole genome sequencing, RNA sequencing, and high density SNP microarrays to study a cohort of FPC cell lines and corresponding germline samples. We show that the four major SPC driver genes are also consistently altered in FPC and that each of the four detection strategies was able to detect the mutations in these genes, with one exception. We conclude that FPC undergoes a similar somatic molecular pathogenesis as SPC, and that the same gene targets can be used for early detection and minimal residual disease testing in FPC patients.

Myeloma genetics and genomics: practice implications and future directions.

Multiple myeloma (MM) is a heterogeneous, clonal disorder of the plasma cells originating from the B-cell line. The diagnosis and monitoring of MM requires routine measurement of biomarkers such as serum protein electrophoresis, urine protein electrophoresis, serum free light chains, among others. Prognostic models such as the Durie-Salmon staging system and International Staging System are available and account for the disease burden. Advanced biomarker and genetic testing includes cytogenetics, fluorescent in situ hybridization, and gene expression profiling to estimate the aggressiveness of the disease and personalize the patient's treatment. Future goals of therapy will be to achieve minimal residual disease (MRD), which incorporates biomarkers and genomic data. MRD testing might provide a better estimate of the depth of response to therapy and overall survival. A robust genomic program of research is still needed to provide additional information for the best MM care practices and to gain new strategies to treat the disease, in particular, in the relapsed and/or refractory setting.

Flow cytometry (FCM) in myeloid malignancies

Myelodysplasic syndromes (MDS) and acute myeloid leukaemia (AML) are the predominant myeloid neoplasms where FCM has both a diagnostic and prognostic role in addition to minimal residual disease (MRD) testing in AML. Criteria for the diagnosis and classification of MDS have been refined and proposed in a consensus International MDS Working Group publication (2007) with immunophenotyping recommended as a co-criterion for diagnosis. Utilising our understanding and delineation of normal antigen maturation and expression patterns of myelomonocytic precursors enables the detection of aberrancy in mature and immature populations. This forms the basis of reporting MDS and AML MRD FCM. There is a large body of literature supporting the routine utility of immunophenotyping in the diagnostic workup of MDS and provocative data in patients treated serially with hypomethy-lating agents. However, further validation and definitions of aberrancy and flow scoring systems are required to enable the introduction of immunophenotyping into mainstream diagnostic laboratories. Standardisation efforts are underway within the European LeukemiaNet. FCM is routinely used for the diagnosis of AML, however MRD testing requires significant expertise. Despite numerous publications demonstrating prognostic significance in MRD at both post induction and post consolidation time points, most are single institution studies highlighting the sophistication required by laboratories.

Monitoring of minimal residual disease in acute leukemia by multiparametric flow cytometry

In this review, we discuss methodological principles and clinical applications of minimal residual disease (MRD) assays based on multiparameter flow cytometry (MFC). The introduction of methods for MRD detection has revolutionized monitoring of treatment response in acute leukemia. Great progress has been made in the development of wide array of flow cytometric techniques for rare event detection. This advance was accompanied by increasingly greater understanding of the immunophenotypic features of leukemic and normal lymphoid cells, and of the antigenic differences that make MRD studies possible. Immunologic testing of MRD relies on "leukemia-associated" immunophenotypes which can be identified by MFC in the most of acute leukemia cases. The recent technical innovations in routine MFC (3 lasers and≥8 colors) and the new developments in software for data analysis make this technology the most attractive for MRD diagnostics. The importance of MFC methodology will be further strengthened by the ongoing international standardization efforts. Results of MRD testing provide unique and clinically important information. The systematic application of immunologic techniques to study MRD in clinical samples has demonstrated the prognostic significance of MRD in patients, leading to the use of MRD to regulate treatment intensity in many contemporary protocols. The identification of new markers of MRD should increase the sensitivity of MRD testing by MFC and is required to widen the applicability of MRD studies.

Minimal Residual Disease Testing in Acute Leukemia

SUMMARY Predictive/prognostic factors in acute leukemia continue to be sought, in order to refine treatment strategies. Minimal residual disease (MRD) testing has been shown to be a statistically significant factor by multivariate analysis in both acute lymphoblastic leukemia (ALL) and acute myeloid leukemia. Its utility in guiding therapy has been more extensively studied in pediatric ALL, with some protocols having instituted MRD testing into therapeutic algorithms. The clinical impact of MRD testing in ALL and acute myeloid leukemia will be presented, including both molecular and flow cytometric methodologies, with a more focused discussion of the strategy, methodology and interpretation of MRD testing by multiparametric flow cytometry.

Use Of Rearranged Immune Receptor Sequencing To Measure Minimal Residual Disease (MRD) In Bone Marrow, Cerebrospinal Fluid and Testes In Relapsed Childhood Acute Lymphoblastic Leukemia

BackgroundDetection of minimal residual disease (MRD) in pediatric acute lymphoblastic leukemia (ALL) is a strong predictor of outcome. In addition, MRD testing prior to stem cell transplant for ALL can inform on the risk of relapse. The ClonoSIGHT test uses deep sequencing of immunoglobulin and T-cell receptors to identify and monitor MRD. In retrospective cohorts, we have previously shown this technology is highly correlated with flow cytometry and PCR-based MRD methods, but has even greater sensitivity than both technologies (Faham et al, Blood 2012; Gawad et al, Blood 2012). Here we report on four clinical cases where we used the ClonoSIGHT assay to prospectively monitor MRD, in both the medullary and extramedullary compartments, to demonstrate the feasibility of this technology for MRD monitoring of children with relapsed ALL. MethodsUniversal primer sets were used to amplify rearranged variable (V), diversity (D), and joining (J) gene segments from the immunoglobulin heavy and kappa chain (IGH and IGK), as well as T-cell receptor beta, delta and gamma. The assay was performed on genomic DNA isolated from cells from the bone marrow, cerebrospinal fluid, or testes. The test was first done at the time of relapse to identify the malignant clonotype, which was monitored at subsequent time points. The patients were ineligible for clinical trials and concurrently underwent MRD testing using flow cytometry. The sequencing assays were performed to show feasibility of the approach. ResultsPatient one was a 14 y/o ALL relapse patient who was not in morphologic remission after standard re-induction therapy. The malignant clonotype was identified on a bone marrow aspirate from relapse; follow-up MRD tests were done using both flow cytometry and deep sequencing five times throughout salvage therapy with 5-aza-2'-deoxycytidine, suberoylanilide hydroxamic acid and high dose cytarabine over 75 days; the last two MRD data points showed 0.6% and 6% by ClonoSIGHT MRD and 0.4% and 1.3% by flow cytometry MRD. Morphologic remission with count recovery was used as the criteria to direct this patient to SCT. Patient two was a 9 y/o with ALL, for whom MRD was used to test for relapsed disease in multiple tissues. This patient experienced three isolated testicular relapses (M1 marrow and no CNS involvement) at the time of each relapse. The ClonoSIGHT assay was used on tissue from a testicular biopsy to identify the malignant clone(s). Testing of the bone marrow and cerebrospinal fluid did not detect the malignant clones in those sites. This patient underwent therapeutic orchiectomy and 4-week systemic re-induction resulting in a fourth complete remission and now is under evaluation for consolidation therapy with a SCT. A third patient was an 8 y/o with a combined bone marrow and testicular ALL relapse, who was in morphologic remission in the marrow after re-induction therapy and testicular radiotherapy. Prior to undergoing SCT the patient had negative MRD by flow cytometry but had 0.008% MRD using the ClonoSIGHT MRD assay. The fourth patient was a 15-yo with ALL relapse at 9 years from first remission, treated with a four-drug re-induction and Berlin-Frankfurt-Münster based consolidation and maintenance therapy. This patient was MRD negative by both flow cytometry and ClonoSIGHT MRD at end of re-induction as well as end of consolidation and remains in remission. ConclusionsWe have shown the feasibility of using sequencing-based tests for monitoring MRD in children with relapsed ALL in medullary (bone marrow) and extramedullary compartments (testes and CSF). Further studies are needed to establish the prognostic value of MRD detected by the ClonoSIGHT assay in both medullary and extramedullary sites that are below the limit of detection of PCR and flow cytometry. These sequencing-based tests may provide a useful tool to develop risk stratification schemas for drug development in relapsed childhood ALL. Disclosures:Weng:Sequenta, Inc.: Employment, Equity Ownership. Faham:Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Bfr (bendamustine, fludarabine, rituximab) Vs. FCR Nonmyeloablative Allogeneic Conditioning In Relapsed/Refractory Chronic Lymphocytic Leukemia (CLL): Outcome Results Including Minimal Residual Disease Analysis

BackgroundBendamustine is a novel active agent in CLL with favorable safety profile. We have previously reported the outcomes of allogeneic stem cell transplantation (alloSCT) in CLL after FCR conditioning (Khouri et al. Cancer 2013). More recently, we initiated a trial with BFR. Herein, we compare outcomes in relapsed/refractory CLL patients following alloSCT according to the conditioning regimen received. Patients and MethodsWe studied 101 CLL patients treated on 3 consecutive trials at our center between 1999 and 2012. 75 (74%) received FCR and 26 (26%) received BFR. The BFR regimen consisted of bendamustine 130 mg/m2 IV daily on days -5 to - 3 prior to transplantation, thus substituting the cyclophosphamide in the FCR regimen. The dose and schedule of fludarabine (30 mg/m2 IV daily x3) and rituximab (375 mg/m2 IV on day –13 and 1000 mg/m2 on days -6, +1, and +8) were similar in both regimens. Tacrolimus and mini-methotrexate were used for GVHD prophylaxis. In addition, thymoglobulin 1 mg/kg IV was given on days -2, and -1 in patients receiving an unrelated donor transplant. Patients were staged with CT, PET (whenever indicated) scans and marrow biopsies, every 3 months for the first year, every 6 months x 5 years, then yearly thereafter. Minimal residual disease (MRD) by multicolor flow cytometry using an international standard approach was also assessed from bone marrow samples since 2004. ResultsPatients characteristics were similar in both groups. This included median age (58 years in both), sex distribution, and median number of prior therapies (3 in both), β2-microglobulin >3 mg/L, refractory disease (38% in BFR vs. 46% in FCR, P=0.5), presence of 17p deletion [27% in BFR and 11/30 (37%) in FCR], and peripheral blood stem cell source (92% and 89%, respectively). More patients however received their transplants from unrelated donors in the BFR group than the FCR group (54% vs. 27%, respectively, P=0.01). Treatment-related mortality was similar within the 2 groups [4% and 7% at 6-months; 8% and 16% (P=0.3) at 1-year]. The incidence of acute grade 3 GVHD was 4% and 8% in the BFR and FCR groups, despite the higher % of unrelated transplants in BFR. None had acute grade 4 GVHD in either group. Median follow-up time for BFR and FCR groups were 14 (range 4-48), and 76 (range, 24-195) months, respectively. The 2-year progression rates in the BFR and FCR were 17% vs. 37%, respectively (P=0.04) (Figure), and the 2-year PFS rates were 75% vs. 40%, respectively (P=0.01). There was also a trend for a better 2-year OS rates (92% vs. 68%, P=0.1), favoring the BFR group. The conditioning regimen (P=0.04) and β2-microglobulin >3 mg/L (P=0.03) were the only predictors for progression by uni- and multivariate analysis undertaken in all 101 patients, whereas a number of prior therapies of > 3 was the only predictor for OS. Recent studies have suggested that MRD is an independent predictor of PFS and OS in CLL after conventional chemotherapy as well as after alloSCT. We assessed MRD in 61 patients following alloSCT. A 6-months landmark analysis showed that MRD-negativity was not a predictor for progression in these patients (P=0.6). Last MRD tests before clinical progression dates was available for 22 of 39 progressions in the FCR/BFR group. Last MRD test was found to be negative in 14/22 relapsed patients. Median duration between last MRD-negative test and clinical relapse was 92 days (range, 1-725). Median duration between last MRD-positive test and clinical relapse was 71 days (range, 21-330) [Display omitted] ConclusionOur results show a lower rate of progression and improved survival after BFR when compared to FCR regimen in alloSCT for CLL. MRD by multicolor flow cytometry was not able to predict risk of progression in these patients whose disease was also assessed by CT imaging following transplantation. Disclosures:Khouri:Cephalon: Research Funding. Off Label Use: Bendamustine in transplantation.

Updated Follow-Up In The CLL2007FMP Trial Supports The Non Superiority Of The Association Fludarabine-Cyclophosphamide (FC) Plus Campath Compared To FC Plus Rituximab and Points Out The Importance Of Minimal Residual Disease, By 6-Color Flow Cytometry Technique, On Progression Free Survival

▪ IntroductionCLL2007FMP (fit medically patients) is a Randomized Phase-III Trial conducted by the French Cooperative Group on CLL and WM (FCGCLL/WM) and the “Groupe Ouest-Est d'Etude des Leucémies et Autres Maladies du Sang” (GOELAMS), comparing FC plus Rituximab (FCR) to FC plus Campath (FCCam) in previously untreated fit patients with chronic lymphocytic leukemia (CLL). Early results showed that the FCCam regimen was associated with an unfavourable safety profile limiting significantly its use in this indication (Blood 2012). We present here the results of the extended follow up of the CLLFMP2007 trial, with particular emphasis on survival data, minimal residual disease (MRD) and late adverse events. MethodsIn this trial, 178 younger (<65) fit patients (pts) (cumulative illness rating scale (CIRS) score of up to 6), were enrolled between November 2007 and January 2009. Cases with del(17p) were excluded. Pts were randomly assigned to receive 6 oral courses of FCR (n=83) or FCCam arm (n=82). The primary endpoint of the study was 3-year progression-free survival (PFS). Secondary endpoints were safety, response to treatment, overall survival (OS) and MRD. MRD evaluation was performed by 6-color flow cytometry in an oligocentric manner. MRD testing was scheduled before therapy initiation and at final evaluation, (i.e. 3 months after completion of immunochemotherapy) where it was to be assessed for all responding patients in both peripheral blood (PB) and bone marrow (BM). Recruitment was interrupted in January 2009 after 165 pts had been randomized due to an excess of mortality in the FCCam arm. ResultsPFS and OS were not significantly different between the two arms. With a median follow-up of 55.5 months (interquartile range, 50-60), 57 pts in the FCCam arm were free of disease progression compared with 50 in the FCR arm, with a 3-year estimated PFS at 81% in both arms (p=0.80). Fourteen pts died in the FCCam arm (7 from progression and 7 from toxicity) and 9 died in the FCR arm (all from progression), with a 3-year estimated survival at 90% vs. 88% (p=0.85). PFS was significantly impacted by IGHV mutational status (p=0.001), Binet stage (p=0.0002) and MRD level. At final evaluation, MRD was established using the result in PB samples (available for 120 patients) and was determined in 103 pts by combining the results from blood and BM samples. Interpretation was based firstly on the use of the classical 10-4 threshold as reference and secondly on the limit of detection of the technique (0.7x10-5). In MRD-positive patients, the median PFS was 44.7 months (PB) whereas it was not reached in the group with MRD lower than 10-4 (p<0.0001, figure 1) ; similar data were found in MRD-positive PB+BM patients with a median PFS of 46 months whereas it was not reached in the group with MRD lower than 10-4 (p=0.002). No significant difference was found regarding OS but follow-up is still short for this evaluation. Similar results were observed when considering the limit of detection of the MRD technique (data not shown). Late toxicities (occurring after the final evaluation at 3 months after the end of treatment or at the ninth month when treatment was prematurely stopped) included : 1 bile duct cancer, 1 myelodysplastic syndrome, 1 transient ischemic attack, 1 lung adenocarcinoma and one prostate cancer in the FCR arm and 3 febrile neutropenia, 3 pneumonia (1 due to legionella), 1 pneumococcal sepsis, 1 bronchitis, 1 toxoplasma eye infection, 1 pyelonephritis, 2 herpes zoster, 1 acrodermatitis, 1 subdural hematoma, 1 autoimmune thrombocytopenia, 1 agranulocytosis, 1 autoimmune haemolytic anaemia in the FCCam arm. [Display omitted] ConclusionResults of this extended follow-up of the CLL2007FMP trial confirm the absence of superiority of the FCCam regimen on OS and PFS. Interestingly, longer follow-up did not reveal a higher rate of late toxicity in FCCam arm, notably in terms of secondary malignancies; Similarly to early toxicity, late was adverse events were mainly infectious. Finally, MRD status determined by 6-color technique in PB and/or BM at post-treatment evaluation was predictive of PFS whatever the treatment arm. This finding is in line with recent reports in other studies pointing out to the powerful value of MRD as prognostic factor, supporting its use as PFS surrogate primary endpoint in clinical trials. Disclosures:Feugier:roche: Honoraria. Cazin:roche: meeting invitation Other, Membership on an entity's Board of Directors or advisory committees; GSK: meeting invitation, meeting invitation Other, Membership on an entity's Board of Directors or advisory committees.

The Outcome Of Allogeneic Hematopoietic Cell Transplantation In Children With FLT3/ITD-Positive Acute Myelogenous Leukemia

FLT3 is a receptor tyrosine kinase expressed on hematopoietic progenitors that regulates hematopoietic development. FLT3 internal tandem duplication (FLT3/ITD) is a somatic mutations whose presence is associated with poor outcome in adults and children with AML. In children with FLT3/ITD who have high allelic ratio (ITD-AR), the progression free survival is estimated to be <20%. Hematopoietic stem-cell transplantation (HSCT) has been a recommended therapeutic option for children who are positive for FLT3. There is limited data on the post transplant outcome for children with FLT3/ITD.We conducted a retrospective study to determine the outcome for all children with FLT3/ITD positive AML who underwent allogeneic HSCT in 4 pediatric centers. Data was collected and analyzed to identify the potential risk factors for overall survival (OS) and event-free survival (EFS). The association with OS and EFS was evaluated by Cox proportional hazard regression with results reported using hazard ratio (95% CI). The survival curve and cumulative incidence were calculated using the Kaplan-Meier method.Between March 2007 and June of 2012, 29 AML patients with FLT3/ITD received HSCT. Median age at HSCT was10.2 years (range 4.5-21.9), 16 were males, and 26 were in first complete remission (CR), 2 with refractory disease and 1 in second CR. Among those with minimal residual disease testing, it was present in 3 (12%) patients in 1stCR. FLT3-ITD allelic ratio at the time of diagnosis was available in 25 patients and among those patient 14 (56%) the ratio was >0.4 (range 0.03-15.99). Eleven patients (38%) received stem-cells from HLA identical siblings while in 18 (62%) an alternative donors stem-cell was utilized (mismatched related donor (n=2), unrelated donor (n=7) or cord blood (n=9)). Eighteen patients (62%) received a total body irradiation (TBI) based preparative regimen while 11 (38%) received a busulfan-based regimen. Graft-versus-host disease (GVHD) prophylaxis consisted of calcineurin inhibitor and mycophenolate for recipients of cord blood HSCT and calcineurin inhibitor plus methotrexate for the rest. Three (10%) patients experienced primary graft failure following cord blood HSCT. All 3 patients received a second graft that resulted in sustained engraftment. The cumulative incidence of neutrophil engraftment at day 42 was 89.6%. Sixteen (55%) patients developed grade 2-4 GVHD and 11 (38%) developed chronic GVHD (6 with severe and 5 mild). None of the patients experienced transplant related mortality. Eleven (38%) patients experienced relapsed disease. The cumulative incidence of relapse at 2 years was 34.7% with 95% CI (20.4%, 54.9%). At 2-years the probability of EFS and OS was 65.3% with 95% CI (45.1%, 79.6%) and 82.2% with 95% CI (58.5%, 91.3%) respectively. Using univariable analysis there was no difference between the EFS of patients who received related donors versus alternative donors, (HR 2.64 (0.79-8.76) p=0.1) and those with residual disease at the time of transplant versus those in complete remission (HR 0.88(0.19-4.09, p=0.8). Patients with higher FLT3/ITD ration at diagnosis had significantly worse EFS (HR 1.42 (1.04-1.93) p=0.03). The use of TBI in the preparative regimen was associated with superior EFS and OS (HR 0.29 (0.08-0.99), p =0.04) and (HR 0.07 (0.01-0.62), p= 0.002) respectively.In conclusion the use of allogeneic HSCT is associated with improved EFS and OS in children with FLT3/ITD positive AML compared to what has been reported in those treated with chemotherapy alone. Disclosures:No relevant conflicts of interest to declare.

Evaluation Of IKZF1 Δ4-7 Deletion As a Suitable Marker For Minimal Residual Disease Monitoring; A Study Of 161 Consecutive Acute Lymphoblastic Leukaemia (ALL) Patients On The On-Going UKALL14 Trial

Minimal residual disease (MRD) testing is vital to risk assignment in acute lymphoblastic leukaemia (ALL). Quantification of patient-specific rearrangements of immunoglobulin and T-cell receptor (Ig/TCR) genes is the most standardised method in current use. Recent studies demonstrated that deletion in the IKZF1 gene is common and is prognostic of poor outcome. IKZF1 targets could offer potential for MRD monitoring in BCR-ABLnegative (neg) ALL. The 5' and 3' break points of this deletion are highly conserved, making it possible to design a universal PCR assay to amplify the fusion genomic sequence created by the deletion.With the initial aim of developing an MRD assay we studied the incidence of IKZF1 Δ 4-7 in 161 consecutive patients enrolled on the on-going adult ALL trial UKALL14, aged 25-59 years. We carried out PCR using breakpoint-specific primers designed as close as possible to the breakpoint. In order to ensure that the screening PCR enabled sensitive detection of the deletions, serial dilution of a positive control SUP-B15 cell line diluted into non-tumoral DNA was analysed. IKZF1 Δ 4-7 could be detected with a sensitivity of at least 10-4 (0.01%). PCR bands could be readily allocated to “strong” or “weak” on visual inspection; all PCR positive signals were confirmed as IKZF1 Δ 4-7 by Sanger sequencing of the PCR products and the breakpoints mapped. The frequency of the deletion in the total population studied was 80/161 (50%); 23/35 (66%) in the BCR-ABL positive (pos) subgroup and strikingly 57/126 (45%) in the BCR-ABLneg subgroup. The high rate of IKZF1 Δ 4-7 in the BCR-ABLneg patients was unexpected, being approximately double the reported incidence BCR-ABLneg adults.Real time quantitative (RQ)-PCR analyses to investigate the suitability of IKZF1 quantitation as the basis for an MRD assay was performed on 26 BCR-ABLneg cases, selected only on the basis of availability of follow-up material. Assays were assessed using the same criteria applied by EuroMRD for Ig/TCR quantification. On that basis, “limited testing” of PCR amplifications at the 10-2 (1%) and 10-4(0.01%) dilution points was performed on all 26 samples. To our surprise, only 5 samples gave acceptable data to qualify for a quantitative MRD assay. Notably, all the 21 cases that failed “limited testing” had yielded a weak PCR signal on initial screening, suggesting the PCR assay have detected intragenic deletions restricted to minor subclones.Although subclonal IKZF1 gene alterations are well described, the apparent high frequency of these events in our cohort (21/26 tested for RQ-PCR assay) was surprising. To further analyse this, we performed MRD-based genomic quantification of all 26 diagnostic specimens using SUP-B15 dilution series. The percentage of ALL cells bearing IKZF1 Δ 4-7 in the putative 21 subclonal cases was <0.01% compared to >90% in the putative 5 major clonal cases. In total, 82% of BCR-ABLneg cases gave a weak PCR band at initial screening, suggesting that most of the Δ 4-7 in BCR-ABLneg adult ALL are subclonal.In the 5 cases where a quantifiable IKZF1 Δ 4-7-based MRD assay could be developed, MRD analysis was performed on 12 follow-up samples and sensitivity and quantitative range of at least 10-4 (0.01%) and 5×10-4 (0.05%) were obtained, respectively. These parameters compare favourably with the performance of standard Ig/TCR assays. Notably in one sample MRD could only be quantified by the IKZF1 deletion approach (level= 7.79E-05, ∼ 0.008%).IKZF1 lesions are not leukaemia-initiating events, hence the stability of this alteration during the history of ALL is not clear. To address this, 14 diagnosis and relapse paired samples were analysed. Three different patterns emerged: in 5 cases the IKZF1 Δ 4-7 at diagnosis was preserved at relapse; in 3 cases the lesion was newly acquired at relapse; and in 3 cases the deletion was lost at relapse, notably all these 3 cases had showed a weak PCR signal, suggesting the deletion which was lost at relapse was subclonal at diagnosis. Theoretically this observation suggests that IKZF1deletion-based MRD monitoring carries a risk of missing a resurgent clone.In conclusion, IKZF1 Δ 4-7 can provide highly sensitive MRD assays and could be a potential candidate to add to the repertoire of currently available MRD markers. However, we have shown limited applicability due to many IKZF1 deletions occurring in putative subclones. The stability of these deletions in major clones remains to be determined. Disclosures:No relevant conflicts of interest to declare.

Carfilzomib and Bortezomib Containing Regimens Yield High Rates Of MRD Negative Autologous Hematopoietic Progenitor Cell (HPC) Grafts In Multiple Myeloma (MM) and High Risk Smoldering Myeloma (SMM)

▪ BackgroundRegimens incorporating modern anti-myeloma drugs, such as carfilzomib (CFZ) and bortezomib (BOR), produce rapid, deep and durable responses in newly diagnosed myeloma patients but their effect on collection of autologous HPC is not well known, including minimal residual disease (MRD) testing of stem cell grafts. Employing older induction regimens (such as VAD), less sensitive flow cytometry techniques detected circulating myeloma cells in 38-46% of autologous HPC grafts (Stewart, et al. JCO. 2001 and Bourhis, et al. Haematologica. 2007). We hypothesized that the use of modern CRd combination therapy including Carfilzomib (CFZ)-Lenalidomide (LEN)-Dexamethasone (DEX) would significantly lower the rates of HPC product contamination. MethodsThirty-six patients, including 29 with MM and 7 with high-risk SMM, underwent HPC mobilization and collection following induction with CRd (n=30), LEN-BOR-DEX (RVd, n=4), Cyclophosphamide-BOR-DEX (CyBorD, n=1) and Cyclophosphamide-BOR-Prednisone (CyBorP, n=1). For HPC mobilization, all patients received 5 days of filgrastim at 10-16 mcg/kg/dose. A combination of the patient's weight and a peripheral blood CD34 count after 4 doses was used to determine the likelihood of collecting > 4 x106 CD34+ cells/ kg in a single apheresis procedure after a fifth filgrastim dose, according to a previously published algorithm from our institution. Only subjects predicted to require > 1 apheresis by the algorithm received Plerixafor (PLX) at 240 mcg/kg/dose on the fifth day along with the fifth filgrastim dose. HPC collection occurred on day 6, 8 hours after the last mobilizing agent(s) administration. Product contamination with myeloma cells (i.e. MRD status) was evaluated using multi-parameter flow cytometry with a minimum of 3 x 106 events obtained (sensitivity detection rate 1 x 10-5) to examine expression of 9 antigens by the plasma cells. ResultsThe median age at mobilization was 56.2 years (range 40-73) and 19 (53%) were male. At the time of HPC collection, 20 (55%) patients were in sCR/CR/nCR, 11 (30%) had VGPR with 4 PR (11%) and 1 SD (3%). The mean CD34+ cells in the peripheral blood were 33/uL on day 5 and 55/uL on day 6 for the whole cohort. Thirteen (36%) patients did not need PLX. Interestingly, the mean CD34+ count dropped by a mean of 2% from D5 to D6 in patients not receiving PLX while, as expected, it increased by 304% in those who did. The median number of CD34+ cells collected was 6.86 million/kg (range 2.6-12.5) for the whole cohort, (6.6 million/kg without PLX and 7.52 million with PLX p=0.46). Thirty-three of 36 patients (92%) achieved a collection of > 4 million cells /kg in a single apheresis procedure. The 30 patients treated with CRd had a median of 5 (range = 3-7) prior cycles containing LEN with a median of 12 days (range 1-34) between mobilization and last LEN dose. Only 2 of 36 (5%) products were found to have evidence of tumor cell contamination (i.e. MRD positive) using sensitive multiparameter flow cytometry, one patient in PR after 6 cycles of CRd and a second patient in CR after 5 cycles of RVd. ConclusionsModern anti-myeloma therapies, such as CRd and RVd, allow adequate HPC collection in a single apheresis procedure in most cases and improve the quality of the HPC product with greatly reduced tumor cell contamination compared to historical controls. Indeed, 34/36 (94%) patients treated with modern anti-myeloma therapy collected an MRD negative HPC product. Future prospective studies are needed to assess whether autologous stem cell transplants (ASCT) using tumor-free HPC products collected in the era of modern induction therapies have better outcomes. Disclosures:No relevant conflicts of interest to declare.

Utility Of 9-Color, 11-Parameter Flow Cytometry For Detection Of Plasma Cell Neoplasms: A Comparison With Bone Marrow Morphologic Findings and Concurrent M-Protein Studies In Serum and Urine

BackgroundMultiparameter flow cytometry (MFC) is a widely available laboratory platform used for primary diagnosis of mature B-cell and plasma cell (PC) neoplasms. Ongoing advances in myeloma therapeutics have drawn attention to the need for advanced laboratory methods for monitoring disease response and defining disease eradication. Recent literature indicates that achievement of remission based on sensitive MFC criteria is prognostically superior to remissions defined by older criteria based on monitoring of paraproteins in blood and urine. As such, the International Myeloma Working Group (IMWG) recommends MFC for minimal residual disease (MRD) testing during clinical trials for multiple myeloma (MM). This study validates a sensitive 9-color, 11-parameter, two-tube MFC assay suitable for both the initial diagnosis of patients with paraproteinemias and MRD monitoring in MM. MethodWe established and validated a 9-color, 11-parameter MFC assay in our laboratory. The 9-color tube contained cytoplasmic κ/cytoplasmic λ/CD45/CD38/CD56/CD138/CD19/CD117/CD20. Results of the MFC were compared to bone marrow microscopic examinations, immunohistochemical (IHC) studies and serum/urine M-protein measurements ordered on 363 samples from patients with documented or suspected PC neoplasms and IgM paraproteinemias. ResultsTotal preparation time is approximately 90 minutes (“hands-on” time under 30 minutes) for up to 3 concurrent specimens. The assay is performed in two tubes (control and PC panel) with analysis of up to 1.8 x 106 total bone marrow cells/tube. The mean instrument analysis time was ∼5 minutes/tube (range 1-9 minutes). MFC detected clonal PCs in 19% and 23% of cases in which IHC or morphologic evaluation, respectively, failed to show a clonal plasma cell population. The MFC assay consistently detected clonal PC in bone marrow aspirates when serum M-protein levels were above 1g/dl. The frequency of clonal PC detection by MFC fell in concert with M-protein levels. However, in 11% of patients, MFC detected clonal PC after serum and urine immunofixation studies turned negative. ConclusionWe conclude that the assay described herein is suitable when immunologic studies are indicated for either primary diagnosis or MRD detection in PC neoplasms. It equals or exceeds sensitivities reported in the literature and it is readily integrated into a high volume, multipurpose clinical flow cytometry laboratory. Disclosures:Keren:Sebia, Inc (Norcross, GA). : Consultancy.

Combined ROR1 and CD160 Detection For Improved Minimal Residual Disease In Patients With Chronic Lymphocytic Leukemia (CLL)

IntroductionOver the past decade, treatments for patients with chronic lymphocytic leukemia (CLL) have produced complete remissions (CR) without evidence for minimal residual disease (MRD), particularly for younger and/or fitter patients. In this setting, achieving an MRD-negative CR has prognostic implications, yielding longer progression free survival (PFS) and overall survival (OS) than for patients who achieve a CR with persistent MRD. Complicating efforts to incorporate testing for MRD in clinical practice has been the lack of a defined consensus on the methods of MRD detection. In this study, we report on a novel combination of mAbs for MRD detection by flow cytometry based on two antigens: the NK-cell receptor and tumor specific antigen, CD160; and the tumor associated antigen, receptor tyrosine kinase-like orphan receptor 1 (ROR-1). ObjectiveTo compare a novel single-tube, tumor-specific (CD160+ROR1) targeted approach to MRD detection against the previously published CD160 flow cytometric assay (CD160FCA) (Farren et al, 2011) and the new, single-tube 8-color ERIC assay. MethodsBetween October 2012 and July 2013, prospective assessment of MRD was performed on peripheral blood in 56 patients (86 samples). We developed a flow cytometric assay using mAbs specific for CD160 or ROR1 (Fukuda et al, 2008). For this we used the following mAb from BD Biosciences: CD2 FITC, CD5 Pe-Cy7, CD19 PerCP5.5, CD45V500, CD160PE, ROR-1 AF647 (“ROR-160FCA”) and a sequential gating strategy. This was compared with CD160FCA and the 8-color ERIC consortium protocol (unpublished). Light chain analysis (LCR) was performed in all cases and reported where detectable. A proof of concept spiking experiment simulating MRD was prepared by mixing CLL and normal peripheral blood leukocytes in a serial dilution to a level of 10-5(n=3). Statistical analysis was performed using Spearman Rank correlation coefficients, Mann-Whitney t-test, and Bland-Altman method comparison. Significance was set at <0.05%. ResultsTo establish the proof of principle, MRD levels ranging from 0.001% to 100% (Neat CLL) were prepared by serial dilutions, in which MRD levels could be established by the ROR-160FCA to 10-5 (n=3). Assessment of the observed incidence against expected incidence of CLL MRD demonstrated a highly significant correlation (R2=0.96, p=0.01). In the study, the range of detectable disease went from <0.01% to 38.59%, of which 37% of samples had levels below <0.01%. Analyzing all flow cytometric methods, a highly significant correlation was observed between all three: CD160FCA vs ERIC: Spearman R=0.96 (95%CI: 0.93 - 0.97, p<0.001); CD160FCA vs ROR-160FCA: Spearman R=0.97 (95%CI: 0.96 – 0.99, p<0.001); ROR-160FCA vsERIC: Spearman R=0.97 (95%CI: 0.94 – 0.98, p<0.001).54 samples had levels of disease <1%. Bland-Altman assay comparison in these patients again demonstrated significant associations between the assays (CD160FCA vs ERIC: mean 0.08 ±0.15; CD160FCA vs ROR-160FCA: mean 0.04 ±0.19; ROR-160FCA vs ERIC: mean 0.03 ±0.25). Light chain restriction was detectable in 24 patients (size of the restricted population ranged from 0.2% to 47% of all cellular events). This sub-group of patients also demonstrated excellent correlation between level of LCR and detectable disease by CD160FCA (Spearman R=0.96, 95%CI: 0.92-0.98, p<0.001), ERIC (R=0.95, 95%CI: 0.92-0.98, p<0.01) and ROR-160FCA (R=0.97, 95%CI 0.93-0.98, p<0.001). ConclusionMonitoring minimal residual disease in CLL is a key focus for clinical trials, as MRD is an important prognostic marker in CLL in terms of PFS and OS. Here we provide a single tube assay, ROR-160FCA, which is unique by targeting two antigens restricted to malignant B-cells, CD160 and ROR1. ROR-160FCA is equivalent in MRD detection compared to both CD160FCA and the current ERIC assay under development. The two tumor-specific antigens give ROR-160FCA the potential for improved sensitivity, particularly where limited sample is available. Furthermore, it only requires a simple sequential gating strategy, is rapid, and appears more cost effective than other Methods.ReferencesFarren TW, Giustiniani J, Liu FT et al. Blood. 2011;118 (8):2174-2183.Fukuda T, Chen L, Endo T et al. Proc Natl Acad Sci U S A. 2008;105 (8):3047-3052. Disclosures:Farren:BD Biosciences: Research Funding. Warner:BD Biosciences: Employment, Research Funding. Agrawal:BD Biosciences: Research Funding.

Development Of Minimal Residual Disease (MRD) Analysis In Childhood Acute Lymphoblastic Leukaemia (ALL) By Next Generation Sequencing (NGS)

Detection of sub-microscopic levels of disease (minimal residual disease; MRD) in childhood acute lymphoblastic leukaemia (ALL) during treatment is an important prognostic factor. Currently, stratification of therapy for the new frontline trial in childhood ALL (UKALL 2011) is provided by MRD analysis using real time quantitative PCR (RQ-PCR) to identify and quantitate the patient specific rearrangements of the immunoglobulin (Ig) and T-cell receptor (TCR) genes. The current methodology is expensive, time-consuming and complex to perform. Although MRD has proven to be a powerful and essential tool in stratification of ALL patients, 8% of individuals in the current UKALL 2011 trial do not have an informative MRD result. Recently, Next Generation Sequencing (NGS) has led to the opportunity to improve the sensitivity and specificity of Ig/TCR based MRD analysis.In this study, we focussed on the IgH locus using BIOMED 2 primers (van Dongen et al., 2003) modified to allow target identification and quantitation by deep sequencing on the Illumina MiSeq platform. We developed a novel pipeline to automate the clustering and classification of sequencing reads leading to characterisation of the clonal subtypes present. In a sample of 12 patients, the method correctly identified all the major clones revealed by current methodologies, and also detected many related and unrelated low-frequency clones. Additional targets were also identified in patients in which no IgH targets were detectable by current methodologies. These NGS-identified targets were subsequently used to monitor MRD by RQ-PCR to the desired quantitative range required for stratification of therapy according to UKALL 2011 guidelines (Figure 1). In addition, we were able to delineate patterns of IgH rearrangements in two patients previously shown to have oligoclonal (>2) rearrangements. Such patients represent a time consuming and technical challenge for current technologies as it is important that all targets at the locus are followed by RQ-PCR to provide an informative and robust MRD result. Furthermore, by clustering similar sequences, we identified diagnostic samples where multiple V regions are attached to the same N1-D-N2-J region. This may allow for the study of clonal evolution in follow-up samples. Altogether, NGS sequencing has the potential to significantly reduce false negative results, as multiple evolved clones can be identified. This methodology also represents a significant time saving (5-7 days) in comparison to established methods (3-4 weeks). [Display omitted] Having established NGS for identifying clonal targets in ALL, we are currently assessing the ability of the method and pipeline to quantify disease levels in end of induction and relapse samples, previously analysed by RQ-PCR, to determine the concordance between the methodologies. Indeed, logarithmic dilution series of patient DNA in a normal background revealed that stratification based on a clinical threshold of 1 in 1,000,000 is possible using this methodology. Further investigation into the clinical utility of NGS for MRD analysis will focus on analysing earlier time points in treatment and studying the potential use of blood rather than bone marrow. Altogether, this will further improve the predictive value and specificity of MRD testing. Disclosures:No relevant conflicts of interest to declare.

DNA Sequencing-Based Monitoring Of Serum Predicts Clinical Relapse Before CT Imaging in Diffuse Large B-Cell Lymphoma

BackgroundDiffuse large B-cell lymphoma (DLBCL) is the most common lymphoma with >40,000 cases annually in the US. While most patients are cured by frontline immunochemotherapy, up to 20% recur and salvage therapy is poor. Relapse detection relies on CT imaging, which is hampered by radiation exposure, high costs, high false positive rates, and infrequently detects relapse prior to symptoms. Novel methods to detect early relapse may improve outcomes. The LymphoSIGHT™ platform is a high-throughput DNA sequencing method that can detect minimal residual disease (MRD) of lymphoid malignancies in peripheral blood (PB) (Faham et al. Blood 2012) with a sensitivity of one lymphoma cell per million leukocytes. We showed it can also detect circulating tumor DNA in the PB of DLBCL pts (Armand Br J Haematol 2013). We assessed the sensitivity and specificity of LymphoSIGHT™ to detect circulating tumor DNA in serum after frontline therapy in DLBCL. MethodsAll pts were newly diagnosed DLBCL undergoing frontline EPOCH-based chemotherapy. Post-therapy surveillance included clinic visits, PB samples, and contrast CT scans at 3, 4, 6, 12 and 12 mos intervals for the first 5 years, respectively. Pre-treatment lymph node biopsies and PB samples collected at initial diagnosis and during surveillance visits were sent for VDJ amplification and MRD sequencing assessment. Using universal primer sets, we amplified immunoglobulin heavy and kappa chain (IGH and IGK) variable, diversity, and joining gene segments from genomic DNA in tumor biopsy and PB samples. Amplified products were sequenced, and tumor-specific clonotypes were identified for each pt based on their high frequency within the B-cell repertoire in the lymph node. The presence of the tumor-specific clonotype was then quantitated in PB samples. ResultsTwenty-seven pts, treated between July 1996 and July 2005, had adequate DNA from pre-treatment FFPE lymph node biopsies, and 25 (92.6%) had a high-frequency clonal rearrangement (MRD) detected. The median pt age was 49 years (20-75) and an IPI risk groups included 8 low, 3 low-intermediate, 11 high-intermediate and 3 high. The pts had a median of 12 surveillance CT scans (range 1-13) and 221 serum samples analyzed for MRD. Two pts progressed early after cycles 1 and 6. The first pt had progressive disease by CT scan after cycle 1 and remained MRD positive. The second pt remained MRD positive at the end of cycle 6, at which time a CT showed CR-unconfirmed, and progressed one month later. In the remaining 23 pts who achieved CR, seven relapsed at a median of 20.3 (range 5.6-151.6) months. The 16 pts in sustained CR had a median follow-up of 99.7 months (range 3.0-136.3) and none were MRD positive post-treatment (specificity 100%; 88.9-100%). There were 0 false positives in 200 post-treatment samples tested (specificity 100%, 99-100%). Of 7 relapsing pts, 6 had serum samples within 1 year of recurrence. Of these, 5 pts had a positive MRD (range 3.2 to 11.2 months) (sensitivity 83%; 44-98%) at a median of 9.2 months (range 3.2 to 11.2 months) prior to a positive CT scan (Figure). One relapsing pt only had a serum sample obtained 18 mos before CT recurrence and it was MRD negative. ConclusionsOur data suggest that a DNA sequencing-based MRD test of serum can predict clinical relapse with high sensitivity and specificity in DLBCL prior to CT progression. MRD assay detection may significantly reduce the cost, inconvenience and toxicity of surveillance. Importantly, early detection of recurrence may significantly improve pt outcomes. Additional pts are being analyzed and updated results will be available.This work was supported by the Intramural Research Program of NCI at NIH and Sequenta, Inc.▪ Disclosures:Kong:Sequenta: Employment, Equity Ownership. Zheng:Sequenta: Employment, Equity Ownership. Willis:Sequenta: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Faham:Sequenta: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Detection of Circulating Tumor Cells of Nonepithelial Origin: A Bench to Bedside Approach

Purpose/Objective(s): No validated biomarkers exist for minimal residual disease (MRD) detection in non-small cell lung cancer (NSCLC). Tumor DNA is continually shed into the circulation, where it can be accessed for analysis by next-generation sequencing (NGS). We aimed to design a NGS-based method for quantifying circulating tumor-derived DNA in NSCLC and other cancers. Materials/Methods: We developed CAPP-Seq (CAncer Personalized Profiling by deep Sequencing), a novel method for noninvasive and sensitive measurement of cancer burden from cfDNA. Our strategy leverages hybrid affinity capture to enrich recurrently mutated genomic regions from tumor genomic DNA and plasma cfDNA. We used mixtures of NSCLC cell line DNA in cfDNA to benchmark CAPP-Seq. We then analyzed a set of NSCLC tumor/normal genomic DNA pairs. Finally, we studied serial plasma samples collected from patients before and after treatment with radiation therapy, chemotherapy, and/or surgery. Results: We show that CAPP-Seq detects multiple types of cancer-specific somatic mutations within NSCLC DNA, including single nucleotide variants, insertions/deletions, and rearrangements. Benchmarking analyses demonstrate that CAPP-Seq accurately enumerates somatic mutations within cfDNA at a fractional abundance of 0.025%. Applying CAPP-Seq to tumor/normal genomic DNA pairs from 11 NSCLC patients uncovered a median of 3 somatic mutations per patient (range, 1-16). Fifteen plasma samples from 7 patients were profiled by CAPP-Seq. The abundance of patient-specific cancer markers within plasma cfDNA was readily measured by CAPP-Seq in both earlyand late-stage NSCLC. Importantly, circulating tumor DNA responded appropriately to anti-cancer therapies, becoming undetectable in 2 patients (after surgery and chemoradiation therapy, respectively) and declining but remaining detectable in 5 patients (after chemotherapy in 4 patients and surgery in 1 patient). CAPP-Seq measurements correlated with tumor volume (R Z 0.97) and allowed for the detection of resistance mutations in patients being treated with targeted therapies. Conclusions: In this study, we described CAPP-Seq, a novel NGS-based approach to MRD testing in cancer patients. CAPP-Seq could have clinical utility for monitoring disease burden and response to therapy, early detection of relapse, distinguishing radiation pneumonitis from relapse, and risk stratification in early-stage NSCLC following surgery or stereotactic body radiation therapy. Ultimately, we envision CAPP-Seq becoming a routine clinical assay for measuring tumor burden in diverse cancers from plasma and other body fluids. Author Disclosure: S.V. Bratman: None. A.M. Newman: None. J. To: None. J.F. Wynne: None. J.W. Neal: None. H. Wakelee: None. J. Shrager: None. B.W. Loo: None. A.A. Alizadeh: None. M. Diehn: None.

Clinical significance of minimal residual disease in patients with acute leukaemia undergoing haematopoietic stem cell transplantation

In patients with acute leukaemia, the relative risk of relapse influences the choice between chemotherapy and haematopoietic stem cell transplantation (HSCT). The demonstration that minimal residual disease (MRD) is the strongest overall prognostic indicator and can identify patients who are unlikely to be cured by standard chemotherapy has added a powerful new factor to consider when making this decision. There is substantial data indicating that the likelihood of relapse after transplant is directly correlated with levels of MRD before transplant. This knowledge can be used to adjust the timing of HSCT, and guide the selection of donor, conditioning regimen, and post-HSCT strategies to maximize the graft-versus-leukaemia effect. Because MRD emerging post-transplant carries a dire prognosis, its detection can trigger withdrawal of immunosuppression, additional cellular and molecular therapies, or preparations for a second HSCT. Although it is not yet clear whether any of these actions will significantly improve outcome, it is likely that they will be most effective for patients with a relatively low tumour burden, who can be identified only through MRD testing. In this article, we review the clinical significance of MRD in the context of autologous and allogeneic HSCT.

Flow Cytometric Minimal Residual Disease Levels After First Inducton Can Define T-Acute Lymphoblastic Leukemia Patients with High Risk of Relapse

AbstractAbstract 4817T-cell acute lymphoblastic leukemia (T-ALL) patients have increased risk for treatment resistance and early relapse. The precise bone marrow evaluation for the presence of minimal residual disease (MRD) is essential for guiding treatment options. This requires techniques more sensitive than the level of sensitivity of light microscopic technique such as multicolour flow cytometry (FCM). Immunophenotypic alterations called leukemia associated immunophenotypic patterns (LAIP) (i.e.aberrant myeloid markers) and ectopic phenotypic expression (i.e. appearance of immature phenotypes such as TdT, CD1a and CD3 outside their normal site in the thymus) are of benefit to track the residual leukemic cells in T-ALL.A retrospective data analysis of MRD was done comprising T-ALL patients diagnosed and followed-up at the Institute of Cancer Research/Royal Marsden Hospital by means of 3-colour flow cytometry (3C FCM).The aim was to answer a question whether the 3C FCM can reliably split patients into two groups (positive, MRD+ and negative, MRD-) and predict a subsequent relapse and to define a right time point for performing MRD tests. Eight patients were enrolled in the study following the inclusion criteria: (i) complete remission after 1st induction phase of chemotherapy, (ii) presence of LAIP or an ectopic phenotypic expression, and (iii) monitored at defined time points after initial treatment. MRD was measured during the first year of treatment as follows: at the end of phase 1 induction (day 29–35, MRD1), before the start of consolidation (3 months, MRD2), after consolidation (MRD3), during the maintenance therapy (12 months, MRD4). Immunophenotyping was performed on lysed-washed bone marrow samples using CD45 gating strategy and originally defined blast gates at diagnosis. The phenotypes to be followed-up included: TdT+/CytCD3+, CD34+/CYTCD3+, TdT+/CD2+, CD8+/CD10+, CD2+/CD10+, CD7+/CD10+, CD7+/CD33+, CD7+CD34+. Patients were divided into 2 groups in relation to subsequent relapse. Group 1 included 6 patients without relapse. Patient characteristics of the group were: male:female 5:1, mean age 17.7 years, overall survival (OS) 59 months, relapse free survival (RFS) 85 months. Group 2, relapsed patients, included 2 men, mean age 56 years, OS 13 months, RFS 8.5 months. According to the EGIL classification system the 2 men in Group 2 were with an early T-precursor phenotype, whilst Group 1 was heterogenous but cortical-T-ALL predominated. Cytogenetics/FISH and RQ-PCR studies were performed at diagnosis and showed normal karyotype in only one of the Group 2 patients.MRD results showed a difference between the two groups as regards MRD1 and MRD2 time points. Group 1 patients had negative or low MRD levels (below 0.18%) in their MRD1 bone marrow - MRD-, n=4 and MRD+,n=2 (0.18% and 0.12% respectively, sensitivity 0.04%). Those of them who were tested at MRD2 and MRD3 were negative. Both patients in Group 2 showed higher levels of MRD positivity at MRD1 (1% of total bone marrow cells), the first one of them also being positive at MRD2 and the second one becoming MRD+ at MRD4 time point. Although turning to MRD- at MRD3 time point both Group 2 patients relapsed 2.5 and 4.5 months, respectively, after the end of consolidation treatment. Additionally, Group 1 patients had a significantly longer RFS than Group 2 (median 58 months RFS vs. 8.5 months; P <0.001).Conclusions: Reliable detection of MRD in T-ALL is possible by 3C FCM using a combination of TdT and a T cell marker (cytCD3 or mCD3) as such a combination is normally found exclusively in the thymus. The higher MRD-positive levels in Group 2 reflect the more resistant disease in this group and higher probability of early relapse and shortened overall survival. Early T-cell precursor phenotype in these patients appeared to be a subtype at very high risk for treatment failure irrespective of the lack or the presence of genetic lesions. Based on MRD positivity above 0.18% at time points MRD1 or both MRD1 and MRD2 these patients need reassessment of treatment options and more intensive therapy has to be considered for relapse prevention. Finally, the results of our retrospective study suggest the usefulness of implementation of MRD testing by FCM for taking clinical decisions in the prospective clinical trials for novel therapies for T-ALL. Acknowledgments:The study was supported by the Union for International Cancer Control, Geneva, Switzerland (Grant ICRETT-080–2011) Disclosures:No relevant conflicts of interest to declare.