MRD Levels Research Articles

Hematopoietic stem cell transplantation for Ph positive acute lymphoblastic leukemia

The poor survival of Ph+ALL patients treated with chemotherapy alone has been substantially improved through the use of allogeneic hematopoietic stem cell transplantation in patients experiencing their first complete remission and, more recently, by combining tyrosine kinase inhibitors with induction and post-remission chemotherapy. However, disease relapse after transplantation remains a serious issue for Ph+ALL patients. This review summarizes the increasing knowledge gained by using TKIs for Ph+ALL treatment and considers how patients should be categorized according to their levels of MRD so as to be provided the most suitable therapy, in order to prevent Ph+ALL recurrence and improve survival.

First Line BR in Chronic Lymphocytic Leukemia Patients: Interim Results of Russian Norma Trial

CLL is the disease of the elderly. Many CLL pts have high comorbidity burden, which makes them ineligible for intensive chemotherapy regimens like FCR. Recently, DCLLSG CLL10 trial proved comparable efficacy of BR and FCR regimens, but better tolerability of BR. It is thus conceivable that BR is a good treatment alternative for slow-go pts, particularly those with advanced chronic kidney disease.In February 2012 a Russian prospective multicenter observational non-interventional clinical study of BR use in previously untreated CLL pts in routine clinical practice was initiated (BEN-001, NORMA). There are no strict incl./excl. criteria in this study, facilitating unbiased analysis of efficacy and toxicity under real world conditions. Here we report our preliminary results.Materials and methodsBy the time of writing (30 Jun 2014) 123 pts have been included by 25 participating centers. 68 pts have completed therapy and another 28 have completed at least 3 cycles. 55 pts continue treatment. Median duration of observation is 13 mos.Patient characteristics are as follows: median age 59 years (39–79); M-to-F ratio 1.7:1; Binet stage (А/B/C) - 13%/68%/19%; CD38-pos. 30%; IGHV-UM/M ratio 2.1:1; FISH (del13q/del11q/del17p/tri12) - 46%/41%/8%/17%; mut. NOTCH1 - 7%. Also, two FISH abnormalities were detected in 23% pts, three in 2% and four in 1%. IGHV stereotypy was found in 18% of analyzed cases. Median ECOG status was 1 (1-3). Median CIRS score was 3 (0-11), 20% pts had CIRS ≥6. CKD (stage II/III-IV) was documented in 53%/27% pts, 43% pts had GFR <70 ml/min. Enlargement of abdominal lymph nodes was seen in 52% pts.Rituximab and bendamustine were given in standard doses of 375/500 mg/m2 and 90 mg/m2 respectively. As per IWCLL recommendations remissions were confirmed by CT and BM MRD evaluation was done after 3 cycles and at least 2 mos after the last therapy cycle. MRD was evaluated by flow cytometry according to ERIC protocol. Data was entered to eCRF by investigators and centrally analyzed.ResultsData on response after 3 cycles is available for 79 pts. Of these 40.5% had CR and 53% had PR. Another 3 pts had progression and 1 ptn had SD. 14% of CRs were MRD-negative. All 4 refractory pts had del17p and IGHV-UM. Ptn with SD after 3 cycles developed progressive disease at final restaging.Of 68 pts who completed all 6 cycles data on response is currently available for 48 pts. Of these 42 (87.5%) had CR and 5 (10.4%) had PR. Among pts with confirmed CR 25 underwent MRD evaluation, giving 13 MRD-neg. and 12 MRD-pos. responses.MRD evaluation after 6 cycles of therapy was done in 33 pts. All MRD-negative pts (n=13) were in CR group, all of them having had MRD levels <10-2 after 3 cycles (4 pts had MRD-neg. already at that time). Among MRD-positive pts (n=20) 12 had CR and 8 had PR. Notably, all of them had MRD levels ≥10-2 (average 10-1) after 3 cycles. In general, the speed of tumor clone eradication, as measured by MRD level after 3 cycles of therapy, correlated well with further response to therapy (р=.0001). TableMRD levels<10-4≥10-4–< 10-2≥10-2After 3 cycles, N pts.4920After 6 cycles, N pts.13155There was no statistically significant difference in response rates among pts younger and older than 60 (р>.05), those with CIRS ≥6 and <6 (р>.05), those with early (A) and advanced (B+C) disease stages (р>.05); those with (GFR<70 ml/min) and without (GFR≥70 ml/min) meaningful renal dysfunction (р>.05).With regard to toxicity, only 4 cases of anemia grade II, 2 cases of thrombocytopenia grade II and 14 cases of neutropenia grade III-IV were reported. In addition there were 14 cases of infections with the need of systemic antibacterial or antiviral treatment, particularly one case of HBV reactivation. Mild to moderate skin reactions were seen in 7 pts. Two pts had grade II hepatotoxicity.ConclusionsOur preliminary results show outstanding clinical efficacy of BR regimen in 1st line treatment of CLL as the majority of our pts completed therapy with CR. Notably, response rates were not inferior in pts with advanced age, disease stage or high comorbidity burden. All refractory pts had known adverse biological factors like del17p and IGHV-UM haplotype. Reported toxicity was very low. Particularly, pts with renal dysfunction tolerated therapy equally well. One important consideration for the future is that MRD level <10-2 as early as after 3 treatment cycles is a robust predictor for MRD negativity after completion of therapy.ClinicalTrials.gov id NCT02110394 DisclosuresNo relevant conflicts of interest to declare.

Genetic Aberrations As Putative MRD Targets in Childhood Acute Myeloid Leukaemia

Background: Pediatric AML is considered as a heterogeneous disease affecting 15% of children with acute leukaemia. Outcome is still unsatisfactory with an overall survival around 70% and a relapse rate of 30-40% after primary treatment. The most important factors determining clinical outcome are genetic aberrations, and early response to treatment. Many studies have demonstrated the prognostic significance of early marrow response by bone marrow morphology, and more recently by flow cytometry for MRD quantification. All studies have demonstrated that rapid early clearance is associated with a better outcome. MRD-PCR is more sensitive than flow MRD but the applicability is lower since recurrent mutational targets, using conventional technology, are present in only 40-50% of patients. Furthermore, AML usually presents with multiple distinct clones at diagnosis varying in number and volume, varying in number between diagnosis and relapse and showing mutational losses or gains at time of relapse. Therefore, only few studies have investigated the significance of response by MRD-PCR. In this study we investigated the applicability and stability of various molecular aberrations as putative MRD targets.Methods: Dutch patients (n=86) included in the pediatric Dutch Belgium-AML-01 cohort were screened for various aberrations (CBFβ/MYH11; AML1/ETO; MLL/AF4; MLL/AF9; MLL/ENL; FLT3/ITDs; NPM-1exon 12). Patients positive for any of these mutations were followed during treatment using aberration specific quantitative MRD-PCR.Results: For 53% of the included patients (46/86) molecular aberrations could be identified. For the AML1/ETO gene fusion, 6/8 patients were followed for response to treatment by MRD-PCR until 17-51 weeks after diagnosis. None of these patients developed a hematological relapse in this period while low but stable MRD levels were still detectable in half of the tested patients (1.5E-05 to 8.9E-04). In case of CBFβ/MYH11 gene fusion, for 6/8 patients (4 CBFβ/MYH11 type A patients and 2 CBFβ/MYH11 type D patients) MRD-PCR was performed until 5-130 weeks after diagnosis. Three patients developed a hematological relapse (all type A) which was preceded by clear molecular relapse, in one case even 19 weeks preceding the clinical relapse. All tested patients with MLL fusions (1/1 MLL/AF4; 2/6 MLL/AF9; 0/0 MLL/ENL) showed no relapse until 54 weeks after diagnosis while MRD levels were all negative. For NPM-1, 7/9 patients were tested by MRD-PCR. Four patients developed a relapse also shown by MRD-PCR, indicating the stability of this marker. FLT3/ITD MRD-PCR was tested for 14/14 positive patients. This marker was not as stable as the other aberrations tested: we observed 1 patient with no FLT3/ITD at diagnosis and until week 8, while at hematological relapse (week 41) this marker was gained. In contrast, 2 patients showed presence of FLT3/ITD at diagnosis, with loss at relapse. Another patient showed 3 separate FLT3/ITD clones with different kinetics before and after relapse. Five other relapses (with positive FLT3/ITD at diagnosis) were also shown by MRD-PCR.Conclusions: In this study we show that, although numbers are low, selected genetic aberrations might be suitable as MRD targets in childhood AML. Thus, it is clear that response evaluation by MRD-PCR is feasible and carries significant prognostic information for a specific group of aberrations such as AML1/ETO, CBFβ/MYH11 and NPM-1. MRD-PCR in combination with MRD-flow will increase the number of patients suitable for MRD and subsequent earlier detection of imminent relapse. DisclosuresNo relevant conflicts of interest to declare.

EBF1-PDGFRΒ Fusion in Paediatric Acute Lymphoblastic Leukaemia (ALL): Genetic Profile and Clinical Implications

Incorporating cytogenetics into risk stratification for the treatment of childhood ALL has contributed to increased survival rates. However 25% patients, the B-other subgroup, do not have any of the known major chromosomal abnormalities. Within this group, BCR-ABL1-like cases show a similar gene expression profile to those with BCR-ABL1 fusion and share the same high risk of relapse. BCR-ABL1-like cases are genetically heterogeneous and some harbour tyrosine kinase activating gene fusions e.g. EBF1-PDGFRB. Recent reports suggest patients with EBF1-PDGFRB ALL who are refractory to conventional therapy respond to the tyrosine kinase inhibitor (TKI), imatinib. Here we present 14 patients treated on ALL97 (n=3), UKALL2003 (n=10) or UKALL2011 (n=1) who were EBF1-PDGFRB positive including 1 patient treated with imatinib.Involvement of PDGFRB and EBF1 was confirmed using dual colour break-apart probes (normal signal pattern=0R0G2F). Eleven patients showed signal patterns (1R0G1F) consistent with a deletion of 5q33 having breakpoints within EBF1 and PDGFRB, as seen in previously reported cases of the fusion. The presence of the deletion was confirmed by single nucleotide polymorphism (SNP) array in 6 cases. Three cases showed signal patterns (1R1G1F) consistent with a balanced rearrangement resulting in the EBF1-PDGFRB fusion and a balanced t(5;5)(q33.1;q33.3) was seen in one of these cases. SNP array analysis for 2 of these cases showed no copy number abnormalities of chromosome 5. However, in the third case a series of deletions were seen along the long arm of chromosome 5, indicating that the EBF1-PDGFRB fusion was generated by a complex rearrangement such as chromothripsis. Multiplex Ligation-dependent Probe Amplification (MLPA) using the P335 IKZF1 kit (MRC Holland) was carried out in 11 cases. Deletions of the probe for exon 16 of EBF1 were seen in all cases with the 5q33 deletion. Loss of PAX5 was detected in 4 patients, while IKZF1 and CDKN2A/B were deleted in 3 cases each.Among the 14 patients there was a predominance of females (n=10). The median age was 12 years with 9 patients >10 years at diagnosis. The median white cell count (WCC) was 34.4 x109/L with 5 patients presenting with a WCC of >50×109/L. Two of 3 patients treated on ALL97 remain alive (one after relapse); both patients received regimen C and a transplant. The third patient, who was not treated as high risk, relapsed and died within 5 years.An unselected cohort of 276 B-other UKALL2003 patients revealed 8 (3%) cases of EBF1-PDGFRB (~0.6% of BCP-ALL). All 8 patients achieved CR but were MRD positive at day 29. Overall 4 patients relapsed, including 2/3 patients who received standard chemotherapy (regimen A/B) and 2/5 patients who received high risk chemotherapy (regimen C). Three relapsed patients were salvaged and remain in second CR 1-7 years post relapse. Targeted screening of 13 UKALL2003 patients who failed induction revealed 2 more patients with EBF1-PDGFRB; one subsequently died while the second remains in 1st CR 7 years later. None of the UKALL2003 or ALL97 EBF1-PDGFRB patients received TKI therapy.As a result of these findings, patients entered on UKALL2011 are screened for EBF1-PDGFRB fusion if they fail to achieve CR by day 29 or remain MRD positive (>0.5%) at week 14. A 5 year old girl who failed induction (50% blasts at day 35) tested positive for EBF1-PDGFRB. This prompted her withdrawal from UKALL2011 and she was treated according to EsPhALL with standard BFM consolidation plus imatinib (300mg/m2) followed by 3 intensive BFM HR blocks. After 4 weeks consolidation therapy her MRD levels had fallen to 6% and she was MRD negative by flow cytometry after 8 weeks. She continues in molecular remission 2 months after a transplant.In this largest cohort of EBF1-PDGFRB patients reported to date, we demonstrate the genetic mechanisms by which the fusion occurs and the spectrum of cooperating mutations. EBF1-PDGFRB fusion is associated with female sex, older age and a mixed outcome. Although these patients had high levels of MRD and a high relapse rate there was also evidence of durable remissions; especially with intensive chemotherapy. Evidence from this study and others suggest patients with this abnormality who fail standard chemotherapy may respond to imatinib. It is possible that treatment with imatinib might avoid the need for intensive chemotherapy to achieve a cure in these patients but that would need to be tested within a prospective trial. DisclosuresNo relevant conflicts of interest to declare.

Flow-Cytometry MRD Evaluation in Children with ALL; Can MRD Level on Day 33 Serve As a Surrogate Marker for Relapse?

Treatment-response assessment via quantification of MRD has become a corner-stone for risk stratification in childhood ALL. Although MRD has been shown to retain independent prognostic significance, the relationship with other prognostic variables has been incompletely explored. Moreover, determination of the most reliable treatment time-point for FCM-MRD evaluation that would serve as the best surrogate marker in predicting relapse, is still an issue of debate. To determine the utility of sequential measurements of FCM-MRD, with specific emphasis on day 33 and its correlation with known prognostic factors and outcome, we retrospectively analyzed the results of FCM-MRD study/data of 825 bone marrow samples of 133 children with ALL, at different treatment time-points, during induction and early consolidation (days 15, 33, 78, week 22-24, at start and at the end of maintenance therapy). All patients were homogeneously treated on BFM-based protocols and prognostic groups were defined by sex, WBC, prednisone response, risk-group allocation, immunophenotype and ETV6/RUNX1-presence. We investigated: a) the relationship between relapse probability and known prognostic factors like age, sex, WBC, immunophenotype, prednisone-response, ETV6/RUNX1 translocation, CDKN2A/2B deletion, hyperdiploidy, protocol risk-group, MRD(constant variable) and MRD positivity(≥0.1% vs <0.1%)(Kaplan-Meier and lïgrank test), b) the correlation between MRD(+) and outcome (OS and EFS, Kaplan-Meier). Additionally, multivariable Cox-models were used to determine the impact of MRD and WBC, sex, age, prednisone-response, on OS and EFS.Patients with MRD≥0.1% at the end of induction (day 33) were estimated with 5-year RFS rates of only 60.0±12.8%, compared to MRD low(<0.1%)/negative patients that enjoyed superior 5-year RFS rates of 91.3±3.0%. Both days 15 and 33 of FCM-MRD positivity maintained a significant prognostic impact on the incidence of relapse within all subgroups. MRD(+)d15 and MRD(+)d33 detection combined, appeared to have a statistically significant impact on survival probability (RFS 65.9%±10.6, p<0.001). Additionally, FCM-MRD on day 33 was notably more strongly associated with used prognostic factors, while we were able to demonstrate that FCM-MRD log-reduction rates between days 15 and 33 can also be added as an important relapse prediction marker, apart from absolute MRD quantifications.In our study, MRD(+)d15 and MRD(+)d33 in combination, strongly and statistically significant correlated with survival probability. Additionally, we propose the FCM-MRD log-reduction rate between days 15 and 33 and end-induction evaluation(day 33) as better predictors of survival and relapse probability, than only day 15 FCM-MRD estimations. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Nilotinib (Tasigna®) and Chemotherapy for First-Line Treatment in Elderly Patients with De Novo Philadelphia Chromosome/BCR-ABL1 Positive Acute Lymphoblastic Leukemia (ALL): A Trial of the European Working Group for Adult ALL (EWALL-PH-02)

▪Despite a high complete hematologic remission (CHR) rate with imatinib-based therapy, the prognosis of elderly patients (pts.) with Philadelphia positive (Ph+) acute lymphoblastic leukaemia (ALL) is poor, primarily due to relapse. Nilotinib (Tasigna®) is a potent ABL kinase inhibitor (TKI) approved for treatment of chronic and accelerated phase CML, but data on its efficacy in Ph+ ALL are limited. To study the activity of ABL-TKI in the front-line setting, the EWALL (European Working Group for Adult ALL) has developed a joint chemotherapeutic protocol for first-line therapy of elderly Ph+ ALL pts. (aged 55 years or more) that serves as a platform for the addition of targeted therapeutic agents. This chemotherapy backbone was used to test the efficacy and safety of the addition of nilotinib in elderly patients with newly diagnosed Ph+ ALL in a European, investigator-initiated trial.Male or female pts. > 55 years with Ph+ and/or BCR-ABL1 positive ALL were eligible if they had not been previously treated except with corticosteroids, single dose vincristine or three doses of cyclophosphamide, had a WHO performance status of 0-2, adequate organ function and had signed written informed consent. The trial was conducted in accordance with the principles of the Declaration of Helsinki and was approved by the ethics committees of all participating centres. The trial was registered under NCT01528085. After a pre-phase with dexamethasone (Dex) 10 mg/m² d-7 to d-3 (cyclophosphamide 200mg/m² i.v. d-3 to d-1 optional), nilotinib was administered at 400 mg BID starting with induction therapy and given continuously thereafter. During induction, nilotinib was combined with IV injections of vincristine (VCR) 1 mg and Dex 40 mg on 2 days (20 mg over 70y), repeated weekly for 4 weeks. Consolidation cycles consisted of nilotinib 400 mg BID, methotrexate 1000 mg/m² IV d1 (500 mg/m² over 70y) and asparaginase 10,000 UI/m² IV d2 (5,000 UI/m² over 70y) for cycles 1, 3 and 5 and cytarabine 1,000 mg/m²/12h IV d1, d3, d5 (500 mg/m² over 70y) for cycles 2, 4 and 6. Maintenance phase consisted of nilotinib, 6-MP QD and methotrexate once weekly over 1 month every other month, and Dex/VCR in 2 month intervals for up to 24 months of treatment. The primary end-point is the rate of pts. without an event (defined as relapse, death, SAE or study treatment discontinuation) at 12 months, secondary endpoints include the rate of CHR after induction, death during induction or in CHR, event free (and overall survival, the rate of major (MMR) or complete molecular response (CMR) defined by BCR-ABL/ABL ratios < 0.1% and < 0.001%, respectively.As of August 2014, 47 pts. (21 male, 26 female) have been enrolled. Median age is 66 years (55-85 years), twelve pts. are older than 70 years of age. To date, 43 pts. are evaluable for safety and 36 pts. are evaluable for efficacy. The CHR rate is 97% (35 of 36 pts. evaluable for response), one patient was refractory (3%). No patient died during induction therapy. With a median follow-up of 211 days, 31 of 35 evaluable pts. are in CCR and four pts. relapsed, two of whom had discontinued study treatment to undergo allogeneic SCT. 13 of the 36 pts. with documented induction response have discontinued study treatment prematurely, primarily because of transfer to allogeneic SCT (n=7), as explicitly permitted by the protocol. Six pts. discontinued for various other reasons. Eight of 35 CR pts. have completed the consolidation cycles and have entered maintenance phase, five pts. have completed protocol therapy. The rate of complete molecular remission (CMR) after induction (32 pts. evaluable) was 30%, with 2 pts. having undetectable BCR-ABL1 transcripts. During consolidation, 13 of 31 pts. (42%) had a CMR, and BCR-ABL transcripts were undetectable in 9 of 31 pts. (29%). Tolerability has been acceptable, with thirty-four SAEs reported so far, 11 during induction (of 43 pts.), 16 during consolidation (of 37 pts.), 6 during the maintenance phase and one following study discontinuation. Infectious events and neutropenic fever predominated, individual SAEs included metabolic, cardiovascular, neurologic, renal and hepatic events.In conclusion, nilotinib in conjunction with chemotherapy according to the EWALL-PH-02 protocol is well tolerated and highly effective with a 97% CR in elderly pts. with newly diagnosed Ph+ ALL. Molecular response rates are high and MRD levels in responding pts. continue to decrease with time. DisclosuresOttmann:Novartis: Consultancy, Honoraria, Research Funding. Ribera:Novartis: Research Funding. Rousselot:Novartis: Research Funding.

The Clinical Significance of Minimal Residual Disease Detected By Multiparametric Flow Cytometry in Acute Leukemia

PurposeMinimal residual disease (MRD) detected by multiparametric flow cytometry is an important method for predicting relapse. According to the dynamic changes of MRD, through the first clinical treatment to prevent relapse in patients with elevated MRD levels.MethodWe retrospectively analyze dynamic level of MRD of 75 patients with acute leukemia in our department of hematology from January 2011 to June 2013. According to the retrospective data, to analyze the expression of LAIP and discuss the relationship between changes of MRD and prognosis.ResultIn this study there were 75 patients, including 20 patients with acute lymphocytic leukemia (ALL) and 55 patients with acute myeloid leukemia (AML). The specific LAIP were detected in 49 patients with AML, and the cross lineage and non-synchronous expression were majority. In cross lineage expression, detected frequency of CD45/CD34/CD33/CD13/CD56 was highest, accounted for 67.3% (33/49). In non-synchronous expression, CD45/CD34/CD117/CD33/CD64 accounted for 57.1% (28/49). In 20 patients with ALL, there were 16 patients with B-ALL and 4 patients with T-ALL. The specific LAIP were detected in all of the patients. In patients with B-ALL, detected frequency of CD45/CD19/CD22/CD10/CD13 was highest, accounting for 43.8% (7/16). In abnormal quantity of antigens, CD45/CD19/CD22/CD10/CD38 was the most common type, accounting for 50%. The abnormal expression of CD7/TDT was detected in four patients with T-ALL. In monitoring period 7 patients relapsed. We analyzed the relationship between the clinical data and replase. The results showed that level of peripheral hemoglobin at diagnosis and the times of remission induction were significantly associated with replase (P=0.021 and P=0.017, respectively). To analyze the relationship between change of MRD and prognosis, the results showed that 0.05% as the threshold was significantly related with recurrence. 20 patients of persistent MRD≥0.05% had significant differences with 28 patients of persistent MRD<0.05% in replase free survival (P=0.005).ConclusionIt has important significance to predict relapse in patients with acute leukemia by detecting minimal residual disease. Patients of MRD ≥ 0.05% should receive early intensive therapy or hematopoietic stem cell transplantation. Even patients without relapse, it should be closely monitor dynamic levels of MRD and highly vigilant extramedullary relapse. DisclosuresNo relevant conflicts of interest to declare.

Next-Generation Sequencing Reveals Clonal Evolution at the Immunoglobulin Loci in Chronic Lymphocytic Leukemia

Background: Immunoglobulin (Ig) gene rearrangement is a hallmark of early B-cell development. Chronic lymphocytic leukemia (CLL) is typically considered a malignancy of mature B-cells and is thought to originate from the oncogenic transformation of a single pre- or post-germinal B-cell. Activation-induced deaminase (AID), an enzyme that induces somatic hypermutation (SHM) at the heavy and light chain Ig loci, has been shown to be active in CLL cells in vitro (Patten et al., Blood 2012). Previous studies suggest that multiple CLL-specific Ig clonotypes related by SHM may be present in patients (pts) with dominant CLL clones possessing somatically mutated or unmutated Ig loci (Logan et al., PNAS 2011; Campbell et al., PNAS 2008). To our knowledge, evolution of the dominant CLL-specific Ig clonotype over the course of treatment has not been demonstrated. Here we utilized the LymphoSIGHT™ method, a next-generation sequencing-based method for lymphocyte characterization and quantification, to quantify clonal evolution at the Ig heavy and kappa chain (IGH and IGK) loci in 63 pts with CLL.Methods: Samples were collected at Stanford University and the Dana-Farber Cancer Institute. Peripheral blood mononuclear cells were isolated, and genomic DNA was extracted. Using unbiased universal primer sets, we amplified IGH and IGK variable, diversity, and joining gene segments. Amplified products were sequenced and analyzed using standardized algorithms for clonotype determination (Faham et al., Blood 2012). CLL-specific clonotypes were identified for each patient based on their high frequency (>5%) within the B-cell repertoire of a diagnostic (dx) sample. The highest frequency CLL clonotype identified in a dx sample is termed the “index clonotype”. Dx and post-treatment peripheral blood samples were assessed for evidence of evolved CLL clonotypes using LymphoSIGHT. A clonotype was considered “evolved” based on CDR3 sequence homology to the dx “index clonotype.”Results: CLL clonotypes were identified in dx samples from 63 pts (51 unmutated IGHV; 12 mutated), and we assessed post-treatment samples for the presence of CLL clonotype-associated oligoclonality. Two of 63 pts exhibited clonal evolution in post-treatment samples. One patient with unmutated CLL was MRD negative for over 7 years following allogeneic hematopoietic cell transplant (HCT), and subsequently became MRD positive with the evolved clonotype (differing by 1 nucleotide from the index clonotype) leading to clinical relapse 9 months after MRD positivity, while the original index clone remained undetectable. The patient was treated with ibrutinib upon clinical relapse and continues to have detectable MRD with the same evolved CLL clonotype (Fig 1A). In a second patient with mutated IGHV, we observed several evolved clonotypes in the dx sample. Multiple evolved clonotypes, including 5 that exhibited a significant increase in their frequency relative to the index clonotype, were present in the follow-up sample after treatment with fludarabine and rituximab (Fig 1B). These evolved clonotypes differed from the index clonotype by 1-4 nucleotides, but otherwise shared CDR3 identity, excluding independently arisen B cell clonotypes.Conclusions: We observed evidence of clonal evolution at Ig loci in a small subset (3.2%) of pts with CLL undergoing treatment. The presence of evolution in pts with CLL indicates that either the SHM mechanism, including the AID enzyme, remains active after neoplastic transformation, or the evolved clonotypes arose through a mechanism distinct from SHM. These evolved CLL clonotypes may have a selective advantage, and may be useful as surrogate markers for other oncogenic mutations providing resistance to therapy. Additional cases are under investigation and updated results will be presented.Figure 1. CLL clonal evolution during therapy. MRD levels of two related Ig clonotypes, expressed as leukemia molecules per million leukocytes in peripheral blood, are shown at multiple time points following allogeneic HCT (A). In another patient undergoing conventional treatment, the level of each individual evolved clonotype as a fraction of the total CLL molecules is plotted at dx and post treatment time points. The index clone, evolved clones with increasing levels post-treatment, and evolved clones with decreasing levels post-treatment are shown in red, blue, and white, respectively (B). [Display omitted] [Display omitted] DisclosuresMoorhead:Sequenta, Inc.: Employment, Equity Ownership. Carlton:Sequenta, Inc.: Employment, Equity Ownership. Faham:Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

How I determine if and when to recommend stopping tyrosine kinase inhibitor treatment for chronic myeloid leukaemia.

Treatment-free remission (TFR) has recently emerged as a goal of treatment in chronic myeloid leukaemia (CML). Molecular remission is sustained in around 30% of imatinib-treated patients who stop treatment after ≥2 years with undetectable minimal residual disease (UMRD) by conventional real-time reverse transcription polymerase chain reaction. An additional 20-30% of patients will lose UMRD, but remain in stable major molecular remission off treatment. Most patients with molecular recurrence have a significant increase in BCR-ABL1 within the first 6 months off treatment, but there are also rare late relapses. As re-treatment with imatinib restores control, a trial of TFR is safe so long as careful molecular monitoring is provided to enable prompt re-treatment. The minimum eligibility criteria for a trial of TFR are not yet defined, but the available data support a MRD level of around a molecular response of 4.5 log for at least 2 years. Factors associated with a higher probability of TFR include low risk Sokal score, prior interferon treatment, longer total duration of imatinib treatment and higher numbers of natural killer cells at the time of imatinib discontinuation. Preliminary data suggest that the rate of TFR in patients treated with more potent tyrosine kinase inhibitors will probably be higher. The biology that underlies TFR is an area of active investigation.

Host genetic variants of ABCB1 and IL15 influence treatment outcome in paediatric acute lymphoblastic leukaemia

Background:Host germline variations and their potential prognostic importance is an emerging area of interest in paediatric ALL.Methods:We investigated the associations between 20 germline variations and various clinical end points in 463 children with ALL.Results:After adjusting for known prognostic factors, variants in two genes were found to be independently associated with poorer EFS: ABCB1 T/T at either 2677 (rs2032582) or 3435 (rs1045642) position (P=0.003) and IL15 67276493G/G (rs17015014; P=0.022). These variants showed a strong additive effect affecting outcome (P<0.001), whereby patients with both risk genotypes had the worst EFS (P=0.001), even after adjusting for MRD levels at the end of remission induction. The adverse effect of ABCB1 T/T genotypes was most pronounced in patients with favourable cytogenetics (P=0.011) while the IL15 67276493G/G genotype mainly affected patients without common chromosomal abnormalities (P=0.022). In both cytogenetic subgroups, increasing number of such risk genotypes still predicted worsening outcome (P<0.001 and=0.009, respectively).Conclusion:These results point to the prognostic importance of host genetic variants, although the specific mechanisms remain unclarified. Inclusion of ABCB1 and IL15 variants may help improve risk assignment strategies in paediatric ALL.

Role Of Day-15 Peripheral Blood MRD Assessment In Pediatric B-ALL Patients

IntroductionMinimal residual disease (MRD) has emerged as an independent prognostic factor for patients of acute lymphoblastic leukemia (ALL). There is a strong correlation between MRD levels in bone marrow and the risk of relapse in childhood & adult leukemias 1, 2. Bone marrow MRD (BM-MRD) level of ≥ 0.01% is considered as positive and a mid-induction MRD of ≥ 1% is associated with high risk of relapse 3. Recently, the concept of peripheral blood MRD (PB-MRD), as a replacement for BM-MRD, has hit the lime light. In pediatric B-ALL, presence of PB-MRD is associated with a high relapse rate in comparison to cases which are PB-MRD negative 4, 5.This study was aimed to compare the levels of mid-induction (day 15) MRD levels in bone marrow and peripheral blood of pediatric B-ALL patients with a hypothesis that PB-MRD levels correlate with BM-MRD levels, and thus can predict BM-MRD levels for further management of the patient. MethodsForty newly diagnosed CD19+CD10+CD34+/- pediatric B-ALL patients under Vincristine, L-Asparaginase and Dexamethasone, were assessed for MRD levels on their paired day 15 PB & BM samples using six colour flow cytometry. With informed consent, both the samples were collected in EDTA vacutainers and lyse-stain-wash technique was used to prepare a single six colour tube comprising of SYTO 13/ CD34PE/ CD20PerCP/ CD19 PECy7/ CD10APC/ CD45APCH7 for each sample. The processed samples were run on BD FACS Canto II with acquisition of 1 million events or till the tubes were empty. Analysis was done using BD FACS Diva software and MRD of ≥ 0.01% was considered positive. ResultsAmong 40 pairs of day 15 PB and BM samples, 25 (62.5%) were BM-MRD positive. Sixteen pairs (40%) had PB-MRD and BM-MRD co-positivity, 9 pairs (22.5%) had isolated BM-MRD positivity and 15 pairs (37.5%) were MRD negative in both PB and BM samples. In other words, among the 25 BM-MRD positive cases, simultaneous PB-MRD was positive in 16 patients (64%) and none of the samples had isolated PB-MRD positivity. Overall analysis of MRD positive cases showed a direct correlation between PB-MRD and BM-MRD (ρ = +0.684, p < 0.000) and BM-MRD levels were 7 times higher than the PB-MRD. In addition, ROC analysis with PB-MRD of ≥ 0.01% as a cut-off, revealed that, the most likelihood of PB-MRD being positive was when BM-MRD was ≥ 0.31%. ConclusionsIn contrast to the sparsely available literature, our study shows a significant correlation between PB & BM-MRD levels in day 15 paired samples of B-ALL cases. The MRD levels were 7 times higher in BM as compared to PB and PB-MRD was mostly positive with BM-MRD of ≥0.31%. In other words, day 15 PB-MRD positivity indirectly indicates that there is a minimum BM-MRD of 0.31%. Since literature reports prognostic significance of mid-induction BM-MRD at levels ≥1%, on day 15, an assessment of peripheral blood MRD alone, might yield clinically relevant prognostic information. A paired analysis at different time points might also establish a similar correlation as seen in the present study, eliminating the need of BM-MRD during further follow ups of the patient. This will help in avoiding an invasive procedure and improve patient compliance.

Minimal Residual Diease Evaluation By Flow Cytometry Is a Complementary Tool To Cytogenetics For Treatment Decision In Acute Myeloid Leukemia

▪The two main criteria for risk-adapted treatment in AML are the presence of adverse cytogenetic or molecular features, and the response to induction treatment assessed by morphology, in which only failure to respond is truly informative. Therefore, more sensitive techniques are needed to evaluate the response. However, the potential value of MRD detection within different cytogenetic subgroups in AML has not yet been defined. This raises the question of whether a negative MRD result could counterbalance the adverse effect of poor-risk cytogenetics, or whether high MRD levels after induction modify the outcome of patients with otherwise favourable feature. In addition, it is not known whether the modality of intensification therapy modifies the influence of the level of MRD assessed after induction therapy.We analyzed the prognostic impact of MRD level on the BM at CR after induction therapy using MFC in 306 non-APL AML patients. First, we have validated the prognostic value of MRD thresholds we had previously proposed (≥0.1%; ≥0.01-0.1%; and <0.01), with a 5-year RFS of 38%, 50% and 71%, respectively (p=0.002). The second aim was to investigate if MRD is independent of cytogenetics. Intermediate risk cytogenetics represent a grey zone for risk stratification, and our data show that MRD evaluation by MFC discriminate three prognostic subgroups, with a 5-year RFS of 38%, 50% and 70% for patients with high (n = 86), intermediate (n = 83), and low MRD levels (n = 18) (p = 0.03). In addition, MRD assessments yielded relevant information on favourable (poor prognosis for high MRD levels) and adverse (undetectable MRD overcomes adverse prognosis) cytogenetic groups. Regarding the role of different intensification approaches, we have observed that MRD levels (evaluated at the time of mCR) allowed the identification of three risk groups among the 118 patients who underwent autologous transplantation (p = 0.02), with 5-year RFS of 40%, 57% and 83% for patients with high, intermediate and low MRD, respectively. In patients intensified with allogeneic transplantation (n = 83), MRD levels also discriminated three subgroups with 5-year RFS of 53%, 62%, and 75% for cases with high, intermediate and low MRD levels (not statistically significant differences). In patients intensified with chemotherapy alone (n = 105), only cases that achieved low MRD had an acceptable outcome, with a 5-year RFS of 65%, whereas those with intermediate or high MRD levels had a very poor outcome (5-year RFS of 11% and 28%; p = 0.020). In addition, considering patients with intermediate or high risk MRD levels (n=169), intensification with transplant improved the outcome as compared with intensification with chemotherapy (p < 0.001), with a 5-year RFS of 75%, 43% and 20% for allogeneic transplanted patients, autologous transplantation, and chemotherapy treated patients, respectively. When only patients with high MRD levels (n = 86) were considered, those receiving autologous transplantation and chemotherapy had similarly very poor results (5-year RFS of 35% and 30%), and only allogeneic transplantation offered a favourable outcome for these patients, with a 5-year RFS of 66% (p = 0.06). By contrast, the type of intensification therapy did not influenced the outcome of patients with low MRD levels. Multivariate analysis (Cox regression) revealed an independent prognostic value for flow-MRD levels at the time of mCR, whether considered as a continuous (p=0.03) or categorical variable (p < 0.001), together with the cytogenetic classification (p = 0.001), and patient age (p = 0.002). A scoring system, easy to apply in clinical practice, was generated based on MRD level and cytogenetics, assigning 0 points if the variable fell in the low-risk category, 1 point for intermediate levels of MRD or medium-risk cytogenetics, and 2 points for high MRD levels or high-risk cytogenetics. Based on these scores, five significantly different AML risk groups could be defined for scores of 0 to 4. These groups had 5-year RFS of 100% (n = 4), 70% (n = 30), 54% (n = 102), 35% (n = 103) and 19% (n = 19), respectively (p < 0.001).We conclude that immunophenotypic evaluation of MRD is a useful prognostic factor that could be used together with cytogenetics for the prognostic stratification of AML patients. Moreover, allogeneic transplant is the preferable option for patients with high MRD levels after induction therapy independently of the cytogenetic signature. Disclosures:Hernandez:Roche Diagnostics: Research Support Other.

Prognostic Value Of Deep Sequencing Approach For Minimal Residual Disease (MRD) Detection In Multiple Myeloma Patients

Background and AimMost multiple myeloma (MM) patients will experience relapse due to the persistence of residual tumor cells, or MRD. We compared the prognostic value of traditional response criteria with MRD measured by three different methods: a sequencing-based method, termed the LymphoSIGHT™ platform, multiparameter flow cytometry (MFC) and ASO-PCR of Ig genes in a cohort of 133 uniformly-treated MM patients from the Spanish Myeloma Group trials. MethodsBone marrow samples were obtained from 133 patients at diagnostic and post-treatment time points on GEM clinical trials (GEM00 and GEM05). All 133 patients were either in CR or VGPR at the post-treatment time point. Using the sequencing assay, we identified clonal rearrangements of immunoglobulin (IGH-VDJ, IGH-DJ, and IGK) genes in diagnostic samples. We then assessed MRD in follow-up samples, analyzing concordance between: sequencing, MFC and ASO-PCR of Ig genes MRD results, and comparing the prognostic value of each method with traditional response criteria. ResultsThe sequencing assay detected the presence of a myeloma-specific gene rearrangement in diagnostic samples from 121 of 133 (91%) patients. We tested MRD in follow-up time points in 109 of the 121 patients.Of the 109 patients, 79 were positive by sequencing at MRD levels of 10-5 or higher and 30 were MRD negative. The Time to Tumor Progression (TTP) and Overall Survival (OS) were significantly longer in the MRD negative group compared with the MRD positive group by sequencing (TTP, median 80 vs. 31 months, p<0.0001; and OS, median not reached vs. 80 months p=0.02) (Figures 1A and 1B). All patients in VGPR, with the exception of 4 cases, were MRD positive by sequencing (92%). Three of the 4 cases with MRD negative and positive immunofixation became immunofixation negative after maintenance treatment. When restricting the analysis to the 61 patients that were in conventional CR (negative immunofixation), 35 of 60 patients were positive by sequencing at MRD levels at 10-5 and higher and 26 were MRD negative. There was a significantly improved TTP in the MRD negative group compared with the MRD positive group (median 131 vs. 35 months, p=0.001) (Figure 1C).A high correlation between MFC and sequencing MRD results was observed (r2=0.87); as well as between ASO-PCR of Ig genes and sequencing (r2=0.89). Discordant results between FCM and sequencing (SEQ) included: 14 cases who were positive by SEQ and negative by FCM, and 5 cases negative by SEQ but positive by FCM; while the remaining cases were concordant: 63 cases were positive by both methods and 16 were negative by both methods. Correlation between ASO-PCR and SEQ was possible in 45 patients: 10 were discordant (9PCR-SEQ+ and 1 PCR+SEQ-) while 35 were concordant (20 PCR+SEQ+, 15 PCR-SEQ-). [Display omitted] ConclusionsThe sequencing applicability for MRD studies in MM is 91%, which is essentially equivalent with that of MFC (96% Rawstron AC et al, JCO 2013). There is a high correlation between MRD levels by sequencing, ASO-PCR and MFC. Based on these results, MRD assessment by sequencing is a useful method for patient risk stratification and can be used to determine molecular CR in MM. Disclosures:Pepin:Sequenta, Inc.: Employment, Equity Ownership. Weng:Sequenta, Inc.: Employment, Equity Ownership. Faham:Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Minimal Residual Disease Monitoring In t(8;21) Acute Myeloid Leukemia Based On RUNX1-RUNX1T1 Fusion Quantification On Genomic DNA

BackgroundAcute myeloid leukemia (AML) with t(8;21) chromosomal translocation, leading to the RUNX1-RUNX1T1 fusion, belong to the favorable risk AML subset. However, relapse incidence may reach 30-40% in these patients. Minimal residual disease monitoring (MRD) based on the quantification of RUNX1-RUNX1T1 fusion transcript by real-time quantitative PCR (RQ-PCR) has been reported to be an independent prognostic factor for the risk of relapse. The specificity of the RUNX1-RUNX1T1 fusion and the high sensitivity of RQ-PCR techniques have made RUNX1-RUNX1T1 an ideal marker to assess treatment response in t(8;21) AML. Undetectable MRD could mean either that tumor cells persist in a latent state without RNA expression or that MRD level is below the sensitivity threshold. Studies in chronic myeloid leukemia showed that BCR-ABL DNA was still detectable in patients in long-term complete molecular response with undetectable BCR-ABL fusion transcript. Using a similar approach, we investigated the use of RUNX1-RUNX1T1 DNA as a MRD marker in t(8;21) AML, instead of RUNX1-RUNX1T1 mRNA. This approach allows linking results directly to the amount of leukemic cells, since each leukemic cell contains one copy of the RUNX1-RUNX1T1 sequence, while the level of RUNX1-RUNX1T1 mRNA may vary from a patient to another. MethodsThis study focuses on 17 patients with t(8;21) AML included in the CBF-2006 trial and for whom frozen material was available for further molecular analysis. Bone marrow and blood samples were collected at AML diagnosis and during follow-up, as defined in the CBF-2006 trial. Eight patients relapsed during follow-up and 9 were still in complete remission at the end of the study. Interestingly, 3 patients relapsed with a previously undetectable MRD (in blood and bone marrow samples). First, we identified the breakpoints in the RUNX1 and RUNX1T1 genes for each patient using long-range PCR approaches, coupled with next-generation sequencing (NGS) on Personal Genome Machine™ (PGM). The stability of the RUNX1-RUNX1T1 rearrangement at relapse was checked by Sanger sequencing. Then, we performed quantification of RUNX1-RUNX1T1 DNA by RQ-PCR using Taqman technology. For each patient, a primer pair and a probe were designed using the patient's unique RUNX1-RUNX1T1 breakpoint sequence. The forward and reverse primers were located in RUNX1 and RUNX1T1 genes, respectively, and the probe was located at the RUNX1-RUNX1T1 junction. Calibration curves were established using 10-fold dilutions of the diagnostic DNA of each patient in normal control DNA. Results were given as a ratio of chimeric DNA amount in the follow-up sample to chimeric DNA amount at diagnosis. ResultsChromosomal breakpoints were located in RUNX1 intron 5 for all patients. RUNX1T1 breakpoints were located in intron 1b for 15 patients, and in intron 1a for 2 patients (Fig. 1). Quantification failed for 1 patient which was further leave up. Between 2 and 7 follow-up samples were studied for the other patients (median 4.5). DNA monitoring was strongly correlated with RNA monitoring (Fig. 2). Sensitivity threshold, determined by the lowest diagnostic sample dilution which gives a signal, was 10-5 for 7 patients, 10-4 for 6 patients, and only 5.10-4 for 3 patients. MRD was detectable in 31 samples and undetectable in 30 samples by both methods, whereas MRD was detectable only on RNA in 7 samples, probably because of a lack of sensitivity of the RQ-PCR assay. Interestingly, RUNX1T1-RUNX1 DNA was detected in 3 samples from 2 patients who relapsed and for whom RUNX1T1-RUNX1 transcript was undetectable, despite a good RNA quality. [Display omitted] [Display omitted] ConclusionsOverall, RUNX1-RUNX1T1 MRD levels on DNA and RNA were quite similar. The level of mRNA expression did not seem to change during follow-up when compared with the amount of DNA. MRD monitoring on genomic DNA is a useful method, but with sensitivity variations depending on the patient's breakpoint sequence and the efficiency of the RQ-PCR assay. DNA has potential advantages: it is more stable than RNA and a best substrate for collection, processing, transport and storage. Additionally, interpretation of the results is easier because it is closely related to the number of leukemic cells. However, this method greatly increases complexity, time of implementation, and cost of monitoring MRD, which limits its interest in routine practice. Disclosures:No relevant conflicts of interest to declare.

Intermittent Dosing Of Gleevec Is Efficacious In Cases Of Childhood Chronic Myeloid Leukemia With Poor Compliance

Chronic myeloid leukemia (CML) is rare in childhood (less than 5% of all childhood leukemias). The main characteristic is the Philadelphia chromosome (BCR-ABL1 positive) and the tyrosine kinase inhibitor imatinib mesylate (Gleevec) is the treatment of choice, with the target oral dose being 440 mg/m2/day asdetermined by the COG-P9973 and COG-ADVL0122 trials, while allogeneic stem cell transplantation is postponed until CML becomes refractory to the drug. We administer a treatment dose of 400mg/m2/day but we have observed high toxicity levels associated with prolonged treatment.We present a girl with CML, with persistent residual disease (MRD), even two years following diagnosis, and serious side effects (dry skin, significant hair loss, gastrointestinal discomfort and diarrhoea) that affected her quality of life. The patient was tested for polymorphisms in the tyrosine kinase and was found negative. Careful interviewing of the family revealed that the persistent MRD was due to poor compliance of the patient to the therapeutic regimen. The child was unhappy due to the side effects and refused to take her pills (Gleevec), hence the poor compliance. Therefore, taking into consideration the child's wellbeing and psychological welfare, it was decided that she would receive the drug on alternate months (one on/one off). Gleevec was discontinued when the patient completed two years of being MRD negative. The patient remains in complete molecular remission four years after the discontinuation of Gleevec.To date, there are few reports on childhood CML so most data come from studies in adults. Even though Gleevec is currently implemented as the primary treatment method in children, there are still doubts as to whether it can result in a permanent cure and of the potential complications of long-term use in the growth and development of these children. No specific guidelines have been set on the dosage and duration of treatment with Gleevec, especially for childhood CML patients facing a potentially lifelong treatment, who might also be faced with a wide range of unknown side effects. Psychological factors should also be taken into account and special attention should be given in avoiding adverse effects that interfere with the quality of life and the psychological welfare of this extremely fragile population.Overall, in our case, despite the persistent MRD, intermittent dosing of Gleevec proved to be an efficient method both in keeping toxicity levels to a minimum and in achieving complete and continuous remission. Persistent MRD levels in this case were due to the interrupted treatment regime, i.e. due to poor compliance, and not due to additional cytogenetic abnormalities that were resistant to Gleevec. Future clinical trials in children should investigate whether intermittent dosing of the drug produces fewer side effects during the course of treatment and whether it may present a more favourable option when considering the normal growth development of the children treated for CML. Disclosures:No relevant conflicts of interest to declare.

Comparative Assessment Of Minimal Residual Disease In Acute Myeloid Leukemia By Flow Cytometry and Molecular Genetics

BackgroundMinimal residual disease (MRD) is quantified in patients (pts) with acute myeloid leukemia (AML) by both flow cytometry (FC) and molecular genetics (MG). Advantages und disadvantages for the respective approaches are under debate. AimTo assess the respective impact of FC and MG for MRD quantification in AML. Patients and methods420 bone marrow (BM) samples from 252 pts with AML undergoing intensive first-line therapy were analysed by FC and MG in parallel for MRD. FC quantified cells displaying aberrant immunophenotypes identified at diagnosis. Targets for MG included RUNX1-RUNXT1, CBFB-MYH11, EVI1 expression, MLL fusion genes, MLL-PTD, NPM1 mutation, NUP98-NSD1, and PML-RARA. MRD was expressed as log difference of disease level between diagnosis and MRD assessment (LDF for FC and LDM for MG). Four periods were analyzed separately: ≤day 60 after start of therapy, day 61-120, day 121-365, beyond 1 year. Non-MRD factors univariately related to EFS were age (p<0.001, HR 1.25 per decade), WBC count (p=0.010, HR 1.04 per 10 G/L), BM blasts by cytomorphology (p=0.017, HR 1.07 per 10%), and cytogenetics according to MRC [Grimwade et al. Blood 2010] (CG; p<0.001, HR 3.40 per step). Parameters related to OS were age (p=0.002, HR 1.27 per decade), WBC count (p=0.002, HR 1.06 per 10 G/L), BM blasts (p=0.023, HR 1.09 per 10%), and CG (p=0.001, HR 2.34 per step). ResultsLDF and LDM did not differ significantly for the first period (mean 2.94 vs 2.62), but for the other three periods LDF was significantly lower than LDM (3.04 vs 3.90, 3.20 vs 4.20, 3.05 vs 4.06; p<0.001 for each).In the first period both LDF (Cox, p=0.045, HR 0.80) and LDM (p=0.030, HR 0.84) significantly correlated with EFS. In a multivariate model LDM was the only independent parameter (p=0.028, HR 0.80). EFS in pts with LDM below the 25%ile (1.34) was significantly shorter than in the others (median 8.6 vs 16.3 months, p=0.003).In the second period LDM was significantly related to EFS (p=0.001, HR 0.74) while LDF was not. In multivariate analysis, LDM was the most important independent factor (p<0.001, HR 0.73) besides CG (p=0.017, HR 2.94) and age (p=0.030, HR 1.28). EFS in pts with LDM below the median (4.04) was significantly shorter than in the others (median 10.3 vs 26.5 months, p<0.001).In the third period both LDF (p=0.022, HR 0.60) and LDM (p<0.001, HR 0.64) were significantly related to EFS. Multivariate analysis again confirmed LDM as strongest parameter (p<0.001, HR 0.71) followed by CG (p<0.001, HR 3.48) and BM blasts at diagnosis (p=0.038, HR 1.10 per 10%). Separation of pts according to the respective median values resulted in significant differences in EFS, however, separation was more powerful by median LDM (4.57; 3-year-EFS 29% vs 67%, p<0.001) than by median LDF (3.27; 3-year-EFS 38% vs 59%, p=0.046).In the last period again both LDF (p=0.029, HR 0.66) and LDM (p<0.001, HR 0.60) were significantly related to EFS with only LDM being confirmed independent (p<0.001, HR 0.48) besides WBC count at diagnosis (p=0.005, HR 1.23 per 10 G/L). Separation of pts according to median LDM (4.51) again resulted in significant differences in EFS (3-year-EFS 42% vs 94%, p<0.001).For all four periods LDM was significantly related to OS (p=0.024, HR 0.79; p<0.001, HR 0.63; p=0.001, HR 0.69; p=0.020, HR 0.68; respectively) while LDF was not. Most importantly, all multivariate analyses of OS for the four periods revealed the respective LDM as the only independent parameter (p=0.012, HR 0.67; p<0.001, HR 0.65; p=0.024, HR 0.74; p=0.014, HR 0.57), except for the last period for which also WBC was independent (p=0.018, HR 1.23). Separation of pts according to the median LDM for the second period resulted in highly significant differences in OS (3-year-OS 32% vs. 75%, p<0.001).In 34 pts increases of MRD during repeated analyses until relapse were compared between FC and MG. Interestingly, mean increases were similar amounting to 0.276 and 0.281 logs per month, respectively. This was also true when considering disease subgroups with different relapse kinetics like those with NPM1 mutations (FC vs MG, 0.358 vs 0.362) and those with MLL-PTD (0.193 vs 0.206). However, the dynamic range of the lowest MRD level and the level at relapse clearly was higher for MG as compared to FC (4.12 vs 2.69, p<0.01). ConclusionMolecular genetics provides the more powerful tool to quantify MRD in AML as compared to flow cytometry mostly due to its higher sensitivity and its broader dynamic range. Disclosures:Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Rose:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Significance of minimal residual disease before myeloablative allogeneic hematopoietic cell transplantation for AML in first and second complete remission

AbstractMinimal residual disease (MRD) before myeloablative hematopoietic cell transplantation (HCT) is associated with adverse outcome in acute myeloid leukemia (AML) in first complete remission (CR1). To compare this association with that for patients in second complete remission (CR2) and to examine the quantitative impact of MRD, we studied 253 consecutive patients receiving myeloablative HCT for AML in CR1 (n = 183) or CR2 (n = 70) who had pre-HCT marrow aspirates analyzed by 10-color flow cytometry. Three-year estimates of overall survival were 73% (64%-79%) and 32% (17%-48%) for MRDneg and MRDpos CR1 patients, respectively, and 73% (57%-83%) and 44% (21%-65%) for MRDneg and MRDpos CR2 patients, respectively. Similar estimates of relapse were 21% (14%-28%) and 58% (41%-72%) for MRDneg and MRDpos CR1 patients, respectively, and 19% (9%-31%) and 68% (41%-85%) for MRDneg and MRDpos CR2 patients, respectively. Among the MRDpos patients, there was no statistically significant evidence that increasing levels of MRD were associated with increasing risks of relapse and death. After multivariable adjustment, risks of death and relapse were 2.61 times and 4.90 times higher for MRDpos patients (P < .001). Together, our findings indicate that the negative impact of pre-HCT MRD is similar for AML in CR1 and CR2 with even minute levels (≤0.1%) as being associated with adverse outcome.

Minimal Residual Disease-Based Risk Stratification in Chinese Childhood Acute Lymphoblastic Leukemia by Flow Cytometry and Plasma DNA Quantitative Polymerase Chain Reaction

Minimal residual disease, or MRD, is an important prognostic indicator in childhood acute lymphoblastic leukemia. In ALL-IC-BFM 2002 study, we employed a standardized method of flow cytometry MRD monitoring for multiple centers internationally using uniformed gating, and determined the relevant MRD-based risk stratification strategies in our local patient cohort. We also evaluated a novel method of PCR MRD quantitation using peripheral blood plasma. For the bone marrow flow MRD study, patients could be stratified into 3 risk groups according to MRD level using a single time-point at day-15 (Model I) (I-A: <0.1%, I-B: 0.1–10%, I-C: >10%), or using two time-points at day-15 and day-33 (Model II) (II-A: day-15<10% and day-33<0.01%, II-B: day-15≥10% or day-33≥0.01% but not both, II-C: day-15≥10% and day-33≥0.01%), which showed significantly superior prediction of relapse (p = .00047 and <0.0001 respectively). Importantly, patients with good outcome (frequency: 56.0%, event-free survival: 90.1%) could be more accurately predicted by Model II. In peripheral blood plasma PCR MRD investigation, patients with day-15-MRD≥10−4 were at a significantly higher risk of relapse (p = 0.0117). By multivariate analysis, MRD results from both methods could independently predict patients’ prognosis, with 20–35-fold increase in risk of relapse for flow MRD I-C and II-C respectively, and 5.8-fold for patients having plasma MRD of ≥10−4. We confirmed that MRD detection by flow cytometry is useful for prognostic evaluation in our Chinese cohort of childhood ALL after treatment. Moreover, peripheral blood plasma DNA MRD can be an alternative where bone marrow specimen is unavailable and as a less invasive method, which allows close monitoring.

Pediatrik B Hücreli Akut Lenfoblastik Lösemide Minimal Kalıt Hastalığının Akan Hücre Ölçer ile Saptanması

Objective: Improvements in the treatment of childhood acute lymphoblastic leukemia (ALL) provide a complete remission in many patients. Several study groups indicated that determining submicroscopic levels of leukemia cells in the bone marrow (minimal residual disease-MRD) on day 15 is affects the prognosis. Flow cytometry (FCM) is a fast and cheap approach when compared to other molecular methods. In this study, day 15 bone marrow MRD levels and demographic profile in B-ALL patients assessed in our laboratory between December 2010 and August 2011 were discussed. Material and Methods: Bone marrow samples obtained on treatment day 0 and 15 from patients diagnosed with ALL (n=45) and the expressions of CD10, CD11a, CD19, CD20, CD34, CD38, CD45 and CD58 have been determined using FACSCalibur according to the Associazione Italiana Ematologia Oncologia Pediatrica- Berlin Frankfurt Münster (AIEOP-BFM) protocol and evaluated by CELLQuest-Pro software. After determination of the number of nucleated cells with Syto16, the percentages of leukemic cells (blast-MRD percentage) detected in the nucleated CD19+ B cell population were classified as flow low (FLR), flow medium (FMR) and flow high (FHR) risk. Results: Six of the 45 cases (10 females, 35 males, 6.17±3.90 years) in our survey were MRD negative and 39 were positive. Eleven cases were determined as FLR (24.4%), 26 as FMR (57.8%) and 8 as FHR (17.8%) according to MRD risk. Conclusion: All of the evaluations were approved by an AIEOP-BFM partner (100%). Since 30 August 2011, our institute has become the first center in Turkey to evaluate its own cases with the qualifications of our AIEOP-BFM partner (Vienna). In the future, we plan to investigate correlation of MRD-FCM results with prognosis and relapse, and to compare these findings with the polymerase chain reaction results.

Prognostic Impact of the MRD Status After Induction Treatment with Rituximab Plus Fludarabine, Cyclophosphamide and Mitoxantrone (R-FCM) in Patients with Chronic Lymphocytic Leukemia Receiving Rituximab Maintenance Therapy

AbstractAbstract 3930Chemoimmunotherapy combination regimens achieve high rates of negative minimal residual disease responses in CLL, which has been correlated with prolonged PFS and OS. In the present study, we addressed the prognostic value of MRD levels obtained after rituximab, fludarabine, cyclophosphamide, and mitoxantrone (R-FCM) induction treatment in the response duration of patients with CLL included in the GELLC-1 trial and receiving maintenance rituximab treatment (Bosch et al. J Clin Oncol 27:4578–4584, 2009). Patients achieving CR or PR after R-FCM induction received rituximab maintenance consisting of rituximab 375 mg/m2 every three months for two years (up to 8 cycles). MRD was evaluated by four-color flow cytometry assays giving a sensitivity < 10−4 in paired peripheral blood (PB) and bone marrow (BM) samples three months after R-FCM induction therapy, every 6 months during rituximab maintenance, and at the final restaging 3 months after conclusion of treatment. Sixty-seven patients (median age 60 years, 70% male) received a median of 8 cycles of rituximab maintenance (range, 1 to 8), 76% of them completing the entire planned treatment. After R-FCM induction, MRD was considered negative in 45/59 patients (76%) in PB and in 35/63 patients (55%) in BM. Of note, these patients with negative MRD in PB had longer PFS in comparison to those with detectable MRD (at 4 years, 88%, [95%IC 98%-78%] vs 27%, [95%IC 51%-3%] respectively; p<0.01) (Figure 1). MRD negativity in BM showed a trend for a prolonged PFS (p=0.056). When MRD levels in BM after R-FCM induction where categorized, we observed that PFS was similar between the MRD negative (<10−4; n=35) and intermediate (>10−4 to <10−2; n=20) subgroups, whereas it was significantly shorter in patients showing high (>10−2, n=8) MRD levels (PFS at 4 years, 84%, 74%, and 25%, respectively, p<0.01). MRD levels after RFCM induction were compared between PB and BM paired samples. Whereas 12/57 patients (21%) that were MRD negative in PB resulted positive in BM, all patients with negative MRD in BM also had negative MRD in PB. Patients with discrepancies (negative in PB but positive in BM) in their MRD status presented a similar PFS than those with negative MRD in BM (4 year PFS, 85% vs. 90%, P=NS). The impact of MRD levels in PB achieved after R-FCM induction on PFS was also analyzed. MRD status proved to be a superior predictor for PFS than clinical response (Figure 2). In addition, when different prognostic variables (lymphocyte doubling time [LDT, cutoff 12 months], ZAP-70, serum ß2microglobulin and LDH, cytogenetic abnormalities, and MRD levels) were analyzed as predictors for PFS, only MRD status in PB along with LDT remained significantly predictive. After rituximab maintenance, 40.6% of patients achieved a MRD-negative CR, 40.6% a MRD+ CR, 5% a PR, and 14% failed to treatment. Median time to conversion from negative to positive MRD was 45.4 months, significantly longer than that observed in patients treated with FCM only (45.4 vs. 16.4 months; p=0.011) (Bosch et al. Clin Can Res 14:155–161,2008). Moreover, 3 patients that achieved MRD negative in PB but remained MRD positive in BM after the initial R-FCM treatment, became MRD-negative in BM upon rituximab maintenance. Patients that remained MRD negative in PB at the end of rituximab maintenance treatment had an excellent outcome with a PFS of 93% at 4 years in comparison to 68% in patients with MRD positive status (p=0.016). In conclusion, in patients receiving rituximab maintenance MRD levels obtained after R-FCM induction correlated with PFS duration, this correlation being independent of the clinical response attained. The sensitivity of the detection of MRD in these patients was higher in BM than in PB. Finally, maintenance treatment with rituximab seems to prolong the PFS of patients with MRD positive status, minimizing the negative impact of low levels of MRD after induction therapy. [Display omitted] [Display omitted] Disclosures:Off Label Use: Rituximab is currently not approved as maintenance therapy for patients with chronic lymphocytic leukemia. Bosch:Hoffman La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.