Immunoglobulin Receptor Gene Research Articles

Integration of Minimal Residual Disease with Other Patient Risk Factors Identifies a Population with Very Poor Overall Survival in Pediatric ALL: Results from the UKALL 2003 Trial

Background:Overall survival (OS) for children with ALL treated on contemporary protocols is now > 90%. To further improve outcomes, stratification must direct intensive treatment to those patients with the highest risk of relapse. Whilst minimal residual disease (MRD) has emerged as the most powerful predictor of outcome, its use in isolation is insufficient to identify those patients with the greatest risk. To address this, we performed a detailed analysis of the entire patient cohort from the recently reported large multi-center randomized controlled trial, UKALL 2003. Integration of end of induction (EOI) MRD with other predictive factors - cytogenetics, age and white cell count (WCC) - allowed the identification of very high risk groups that are likely to benefit from early treatment intensification or novel therapies.Methods:The UKALL 2003 trial recruited 3126 patients aged 1-25 years with Philadelphia-negative ALL between Oct 1, 2003, and June 30, 2011. Treatment was initially stratified based on NCI risk and cytogenetic results. Subsequent treatment was directed by EOI MRD measured using EuroMRD approved real-time quantitative PCR for immunoglobulin and T-cell receptor gene rearrangements. Analysis was performed on 2666 patients with available MRD results and focused on the systematic integration of MRD results with other patient characteristics to identify subgroups with a very poor OS (<75% at 5 years). MRD was considered positive at a level ≥0.005% and low risk if <0.005% unless otherwise stated.Results:Both MRD and cytogenetic risk group were independently predictive of survival. In particular, patients with high risk (HR) genetic abnormalities - low hypodiploidy, near haploidy, KMT2A (MLL) rearranged, iAMP21 and t(17;19)(q23;p13) (n=100) had a poor outcome with 5 yr OS of 76.0%. Combining these factors showed that MRD could stratify the HR cytogenetic subgroup with a significant difference in survival between those with low risk MRD and positive MRD (5yr OS 93.0 vs. 63.1%, p=0.002, n=43 vs. 57). Crucially, this demonstrates that HR cytogenetics are only truly poor risk in the context of poor treatment response. Importantly, there were almost no survivors following relapse in the HR cytogenetic/MRD positive group, whilst 50% of those with low risk MRD could be salvaged after relapse. In addition, whilst t(1;19)(q23;p13)was not considered a HR cytogenetic factor in this trial, combination with positive EOI MRD identified an additional subgroup of 20 patients with a 5yr OS of only 74.7% (vs. 96.7% in MRD low risk patients, p=0.003).Whilst WCC at presentation was also predictive of outcome, this difference was significant only in those with B-ALL and not T-ALL. In B-ALL, WCC>100 x109/L was associated with significantly poorer survival (5yr OS 95.1 vs. 84.0%, p<0.0001) whilst high WCC in T-ALL had little effect on survival, even at WCC >200 x109/L (5yr OS 90.2 vs. 86.7%, p=0.283). Integration of MRD results allowed the identification of a very high risk group with B-ALL, WCC>100 and positive EOI MRD (n=80) which had a 5yr OS of only 71.3%. Further breakdown by cytogenetic risk group showed those patients with good risk cytogenetics (ETV6-RUNX1, high hyperdiploidy) (n=19) had a remarkably good survival despite WCC>100 (5yr OS 94%). In contrast, those with intermediate (n=34) or HR cytogenetics (n=15) had a very poor 5yr OS (65.7% and 60.0% respectively).Older age (≥10yr) was also associated with poorer outcomes compared to those <10yr (5yr OS 95.8 vs. 87.4%, p<0.0001). Integration with MRD results further separated these groups but did not identify a very high risk subgroup. However, combining older age with a higher EOI MRD cut-off of 1% successfully identified a subgroup (n=96) with a very poor 5yr OS of 51.3% (vs. 91.4% in those with MRD<1%, p<0.0001). Interestingly, this was independent of cytogenetic risk group.Conclusions:Through the integration of EOI MRD with other risk factors in a large pediatric ALL cohort we have identified a population of patients with a very poor OS. When combined, these criteria select only 6% of all patients, but capture 35% of all deaths suggesting current treatment is suboptimal in this group. This information is essential for the planning of future trials to ensure that treatment intensification and novel therapies are targeted at those patients at greatest risk, whilst avoiding unnecessary treatment in those patients that already experience good outcomes. DisclosuresNo relevant conflicts of interest to declare.

Comparison between qualitative and real-time polymerase chain reaction to evaluate minimal residual disease in children with acute lymphoblastic leukemia.

IntroductionMinimal residual disease is an important independent prognostic factor that can identify poor responders among patients with acute lymphoblastic leukemia.ObjectiveThe aim of this study was to analyze minimal residual disease using immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements by conventional polymerase chain reaction followed by homo-heteroduplex analysis and to compare this with real-time polymerase chain reaction at the end of the induction period in children with acute lymphoblastic leukemia.MethodsSeventy-four patients diagnosed with acute lymphoblastic leukemia were enrolled. Minimal residual disease was evaluated by qualitative polymerase chain reaction in 57 and by both tests in 44. The Kaplan–Meier and multivariate Cox methods and the log-rank test were used for statistical analysis.ResultsNine patients (15.8%) were positive for minimal residual disease by qualitative polymerase chain reaction and 11 (25%) by real-time polymerase chain reaction considering a cut-off point of 1 × 10−3 for precursor B-cell acute lymphoblastic leukemia and 1 × 10−2 for T-cell acute lymphoblastic leukemia. Using the qualitative method, the 3.5-year leukemia-free survival was significantly higher in children negative for minimal residual disease compared to those with positive results (84.1% ± 5.6% versus 41.7% ± 17.3%, respectively; p-value = 0.004). There was no significant association between leukemia-free survival and minimal residual disease by real-time polymerase chain reaction. Minimal residual disease by qualitative polymerase chain reaction was the only variable significantly correlated to leukemia-free survival.ConclusionGiven the difficulties in the implementation of minimal residual disease monitoring by real-time polymerase chain reaction in most treatment centers in Brazil, the qualitative polymerase chain reaction strategy may be a cost-effective alternative.

Diagnostic value of endoscopic ultrasonography combined with detection of immunoglobulin and T-cell receptor gene rearrangements in primary gastrointestinal lymphoma

Objective To evaluate the diagnostic value of endoscopic ultrasonography (EUS) combined with detection of immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements in primary gastrointestinal lymphoma (PGIL). Methods From 12nd January, 2012 to 23rd May, 2014, the clinical data of 24 patients with suspicious PGIL under endoscopy (regular biopsy negative and without treatment) and underwent further EUS examination was retrospectively analyzed. All patients received EUS-guided biopsy or EUS-guided fine needle aspiration (FNA) and the tissue specimens were detected for Ig and TCR gene rearrangements. Considering biopsy result, surgical pathological diagnosis and follow-up result as gold standard, the clinical significance of sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy of EUS combined with Ig/TCR gene rearrangements in PGIL were explored. Results Among 24 patients, 19 patients were finally diagnosed as PGIL, which were all non-Hodgkin's lymphoma (NHL); monoclonal gene rearrangement was found in 13 cases of the 19 cases. The left five cases were not lymphoma lesions (three cases of gastritis, one case of leather stomach and one case of malignant melanoma) with no monoclonal gene rearrangement. In cases diagnosed as PGIL, 14 were B cell NHL, which included eight cases of muscosa-associated lymphoid tissue (MALT) lymphoma and six cases of diffuse large B cell lymphoma. Of which, immunoglobulin heavy chain (IgH)/immunoglobulin kappa (IgK) gene rearrangement was found in 11 cases. A total of five cases were T cell NHL including one case of anaplastic large cell lymphoma, one case of NK/T cell lymphoma and three cases of enteropathy-associated T-cell lymphomas. TCR gene rearrangement was found in two cases. Because theoretically, there was no gene rearrangement in natural killer (NK)/T cell lymphoma, so NK/T cell lymphoma was excluded from statistical analysis. The sensitivity, specificity, PPV, NPV and accuracy of EUS combined with Ig/TCR gene rearrangements detection in PGIL were 72.2%, 100.0%, 100.0%, 50.0% and 78.3%, respectively. Conclusion The detection of monoclonal gene rearrangement in tissues from EUS-guided biopsy or EUS-guided FNA had better diagnostic value in PGIL, which improved the objectivity and accuracy of lymphoma diagnosis. Key words: Gastrointestinal neoplasms; Lymphoma; Gene rearrangement; Endoscopy

Killer immunoglobulin receptor genes and their HLA-C ligand are associated with Type 1 diabetes in an Eastern Indian population.

Killer immunoglobulin-like receptors (KIRs) and their interaction with HLA class I ligands have been shown to be associated with Type 1 diabetes mellitus. The aim of our study was to investigate the influence of KIR genes and their HLA-C ligands for susceptibility to Type 1 diabetes in patients from Eastern India. A total of 135 patients with Type 1 diabetes and 98 healthy subjects from Eastern India were typed for KIR genes and HLA-C ligands using PCR-based genotyping. The frequencies of these genes were compared between patients and controls. Comparison of KIR genes between Type 1 diabetes patients and healthy subjects revealed significantly different frequencies of KIRs 2DL2 and 2DS4. The presence of HLA-C1 was negatively associated with disease. The presence of both HLA-C1 and -C2 showed a negative association with Type 1 diabetes, whereas the absence of C1 and presence of C2 was positively associated with disease. Stratification analysis of HLA-C ligands and KIRs showed significant associations between Type 1 diabetes and 2DL2+/C1-, 2DL2-/C1+, 2DL3+/C1+, 2DL3+/C1- and 2DS2+/C1-. Our results suggest that the interaction of KIRs with HLA-C ligands are significant and certain combinations contribute to susceptibility to and protection against Type 1 diabetes.

278. Successful Correction of Rag1 Severe Combined Immunedefciency at Clinically Relevant Vector Copy Numbers

RAG1 -SCID patients lack B- and T lymphocytes due to the inability to rearrange immunoglobulin (Ig) and T-cell receptor (TCR) genes. Gene therapy is a valid treatment alternative for RAG1-SCID patients lacking an appropriate bone marrow donor. However, developing such therapy for RAG1 has proven difficult. As proof-of-principle, we have previously shown with an SFFV promoter element that sufficient RAG1 expression could be achieved for correction at clinically acceptable vector copy numbers (VCN).In our current study, we tested clinically relevant lentiviral SIN vectors with different internal promoter elements (UCOE, PGK, MND, and UCOE-MND in tandem) to deliver human RAG1 sequences. In vitro assessment of promoter strength was done on transduced mouse lineage-depleted cells. The highest RAG1 expression per VCN was reached with the MND and the UCOE-MND containing vectors, followed by UCOE. The PGK displayed the lowest promoter strength, 3 to 10 times weaker than the other elements.Next, we used Rag1-/- mice as a preclinical model for RAG-SCID to assess the capacity of the various vectors to correct the SCID phenotype at very low to moderate VCN (0.01-0.2), where the VCN positively correlating with the RAG1 expression levels. All gene therapy-treated mice reconstituted their peripheral T cell compartment, however in animals that received a transplant with low RAG1 expression (n=9) this effect was limited to peripheral blood. When assessing phenotypic data from thymus 17 weeks after transplantation, only mice that received a transplant with high RAG1 expression, showed a thymocyte subset composition comparable to mice receiving wild type cells. This included a high percentage and number of double positive (DP) cells, assuring an ongoing supply of peripheral T cells. Mice receiving a transplant with low RAG1 expression levels, were mostly lacking the DP population.Similarly, when looking at developing B cells in the bone marrow, animals that were transplanted with cells with low RAG1 expression did not have a substantial number of surface IgM+ cells. This in contrast to animals that received cells with high RAG1 expression levels in which developing B cell subsets were readily detectable. These findings were paralleled in the peripheral blood, where the fraction of B220+ cells positively correlated with RAG1 expression in the transplant material.In the low RAG1 expression groups, we found that a number of mice developed skin rashes, while animals in the high RAG1 expression group as well as animals that received wild type cells or uncorrected Rag1-/- cells did not display any health problems. We are currently determining the TCR repertoire, since a limited repertoire could point to autoreactive T cells causing the rashes.To conclude, the level of B- and T cell development in primary lymphoid organs is directly correlated to the RAG1 expression levels in the transplant and we were able to obtain sufficient RAG1 expression for full T and B cell reconstitution even at a moderate, but clinically relevant, VCN.

Circulating tumour DNA and CT monitoring in patients with untreated diffuse large B-cell lymphoma: a correlative biomarker study

Diffuse large-B-cell lymphoma is curable, but when treatment fails, outcome is poor. Although imaging can help to identify patients at risk of treatment failure, they are often imprecise, and radiation exposure is a potential health risk. We aimed to assess whether circulating tumour DNA encoding the clonal immunoglobulin gene sequence could be detected in the serum of patients with diffuse large-B-cell lymphoma and used to predict clinical disease recurrence after frontline treatment. We used next-generation DNA sequencing to retrospectively analyse cell-free circulating tumour DNA in patients assigned to one of three treatment protocols between May 8, 1993, and June 6, 2013. Eligible patients had diffuse large-B-cell lymphoma, no evidence of indolent lymphoma, and were previously untreated. We obtained serial serum samples and concurrent CT scans at specified times during most treatment cycles and up to 5 years of follow-up. VDJ gene segments of the rearranged immunoglobulin receptor genes were amplified and sequenced from pretreatment specimens and serum circulating tumour DNA encoding the VDJ rearrangements was quantitated. Tumour clonotypes were identified in pretreatment specimens from 126 patients who were followed up for a median of 11 years (IQR 6·8-14·2). Interim monitoring of circulating tumour DNA at the end of two treatment cycles in 108 patients showed a 5-year time to progression of 41·7% (95% CI 22·2-60·1) in patients with detectable circulating tumour DNA and 80·2% (69·6-87·3) in those without detectable circulating tumour DNA (p<0·0001). Detectable interim circulating tumour DNA had a positive predictive value of 62·5% (95% CI 40·6-81·2) and a negative predictive value of 79·8% (69·6-87·8). Surveillance monitoring of circulating tumour DNA was done in 107 patients who achieved complete remission. A Cox proportional hazards model showed that the hazard ratio for clinical disease progression was 228 (95% CI 51-1022) for patients who developed detectable circulating tumour DNA during surveillance compared with patients with undetectable circulating tumour DNA (p<0·0001). Surveillance circulating tumour DNA had a positive predictive value of 88·2% (95% CI 63·6-98·5) and a negative predictive value of 97·8% (92·2-99·7) and identified risk of recurrence at a median of 3·5 months (range 0-200) before evidence of clinical disease. Surveillance circulating tumour DNA identifies patients at risk of recurrence before clinical evidence of disease in most patients and results in a reduced disease burden at relapse. Interim circulating tumour DNA is a promising biomarker to identify patients at high risk of treatment failure. National Cancer Institute and Adaptive Biotechnologies.

Expression of TCR-Vβ peptides by murine bone marrow cells does not identify T-cell progenitors.

Germline transcription has been described for both immunoglobulin and T-cell receptor (TCR) genes, raising questions of their functional significance during haematopoiesis. Previously, an immature murine T-cell line was shown to bind antibody to TCR-Vβ8.2 in absence of anti-Cβ antibody binding, and an equivalent cell subset was also identified in the mesenteric lymph node. Here, we investigate whether germline transcription and cell surface Vβ8.2 expression could therefore represent a potential marker of T-cell progenitors. Cells with the TCR phenotype of Vβ8.2+Cβ− are found in several lymphoid sites, and among the lineage-negative (Lin−) fraction of hematopoietic progenitors in bone marrow (BM). Cell surface marker analysis of these cells identified subsets reflecting common lymphoid progenitors, common myeloid progenitors and multipotential progenitors. To assess whether the Lin−Vβ8.2+Cβ− BM subset contains hematopoietic progenitors, cells were sorted and adoptively transferred into sub-lethally irradiated recipients. No T-cell or myeloid progeny were detected following introduction of cells via the intrathymic or intravenous routes. However, B-cell development was detected in spleen. This pattern of restricted in vivo reconstitution disputes Lin−Vβ8.2+Cβ− BM cells as committed T-cell progenitors, but raises the possibility of progenitors with potential for B-cell development.

Polyclonal, newly derived T cells with low expression of inhibitory molecule PD-1 in tonsils define the phenotype of lymphocytes in children with Periodic Fever, Aphtous Stomatitis, Pharyngitis and Adenitis (PFAPA) syndrome

PurposePFAPA syndrome is a benign, recurrent inflammatory disease of childhood. Tonsillectomy is one of the therapeutic options with a yet unexplained biological mechanism. We tested whether specific lymphocyte subsets recruited from blood to human tonsils participate in PFAPA pathogenesis. MethodsPaired tonsils/peripheral blood (PB) samples were investigated (a) from children with PFAPA that successfully resolved after tonsillectomy (n=10) (b) from children with obstructive sleep apnoea syndrome as controls (n=10). The lymphocyte profiles were analysed using 8-colour flow cytometry, immunoglobulin (IGH) and T-cell receptor (TCR) gene rearrangements via PCR and next generation sequencing; a TREC/KREC analysis was performed using qPCR. ResultsThe PFAPA tonsils in the asymptomatic phase had a lower percentage of B-lymphocytes than controls; T-lymphocyte counts were significantly higher in PB. The percentages of cytotoxic CD8pos T-lymphocytes were approximately 2-fold higher in PFAPA tonsils; the transitional B cells and naïve stages of both the CD4pos and CD8pos T-lymphocytes with a low expression of PD-1 molecule and high numbers of TREC were also increased. With the exception of elevated plasmablasts, no other differences were significant in PB. The expression levels of CXCL10, CXCL9 and CCL19 genes were significantly higher in PFAPA tonsils. The IGH/TCR pattern showed no clonal/oligoclonal expansion. DNA from the Epstein-Barr virus, Human Herpervirus-6 or adenovirus was detected in 7 of 10 PFAPA tonsils but also in 7 of 9 controls. ConclusionsOur findings suggest that the uninhibited, polyclonal response of newly derived lymphocytes participate in the pathogenesis of PFAPA. Because most of the observed changes were restricted to tonsils and were not present in PB, they partly explain the therapeutic success of tonsillectomy in PFAPA syndrome.

Immunomodulatory Effects of theAgaricus blazeiMurrill-Based Mushroom Extract AndoSan in Patients with Multiple Myeloma Undergoing High Dose Chemotherapy and Autologous Stem Cell Transplantation: A Randomized, Double Blinded Clinical Study

Forty patients with multiple myeloma scheduled to undergo high dose chemotherapy with autologous stem cell support were randomized in a double blinded fashion to receive adjuvant treatment with the mushroom extract AndoSan, containing 82% of Agaricus blazei Murrill (19 patients) or placebo (21 patients). Intake of the study product started on the day of stem cell mobilizing chemotherapy and continued until the end of aplasia after high dose chemotherapy, a period of about seven weeks. Thirty-three patients were evaluable for all study endpoints, while all 40 included patients were evaluable for survival endpoints. In the leukapheresis product harvested after stem cell mobilisation, increased percentages of Treg cells and plasmacytoid dendritic cells were found in patients receiving AndoSan. Also, in this group, a significant increase of serum levels of IL-1ra, IL-5, and IL-7 at the end of treatment was found. Whole genome microarray showed increased expression of immunoglobulin genes, Killer Immunoglobulin Receptor (KIR) genes, and HLA genes in the Agaricus group. Furthermore, AndoSan displayed a concentration dependent antiproliferative effect on mouse myeloma cells in vitro. There were no statistically significant differences in treatment response, overall survival, and time to new treatment. The study was registered with Clinicaltrials.gov NCT00970021.

Long-Range Control of V(D)J Recombination & Allelic Exclusion: Modeling Views.

Allelic exclusion of immunoglobulin (Ig) and T-cell receptor (TCR) genes ensures the development of B and T lymphocytes operating under the mode of clonal selection. This phenomenon associates asynchronous V(D)J recombination events at Ig or TCR alleles and inhibitory feedback control. Despite years of intense research, however, the mechanisms that sustain asymmetric choice in random Ig/TCR dual allele usage and the production of Ig/TCR monoallelic expressing B and T lymphocytes remain unclear and open for debate. In this chapter, we first recapitulate the biological evidence that almost from the start appeared to link V(D)J recombination and allelic exclusion. We review the theoretical models previously proposed to explain this connection. Finally, we introduce our own mathematical modeling views based on how the developmental dynamics of individual lymphoid cells combine to sustain allelic exclusion.

BLAST: A Confirmatory, Single-Arm, Phase 2 Study of Blinatumomab, a Bispecific T-Cell Engager (BiTE®) Antibody Construct, in Patients with Minimal Residual Disease B-Precursor Acute Lymphoblastic Leukemia (ALL)

▪Introduction: In ALL, minimal residual disease (MRD) is defined as the detection of leukemic cells in bone marrow by PCR or flow cytometry in the presence of hematological complete remission (CR). Patients with persistent/recurrent MRD after induction therapy have a higher risk of relapse than those with no detectable MRD. Effective treatment of patients with MRD aims to avoid hematologic relapse, reduce MRD load, and provide a bridge to subsequent HSCT.Blinatumomab, an investigational BiTE® antibody construct, redirects CD3+ T cells to CD19+ target cells, resulting in serial lysis of CD19+ B cells. In a phase 2 study of blinatumomab in 21 patients with MRD+ ALL in first-line treatment, 80% of evaluable patients achieved a complete MRD response. BLAST, a confirmatory single-arm, phase 2 study evaluated efficacy, safety, and tolerability of blinatumomab in patients with MRD+ ALL in a larger population.Methods: Adults (≥18 years) with B-precursor ALL in hematologic CR (<5% blasts in bone marrow) after ≥3 intensive chemotherapy treatments and with MRD ≥10-3 were eligible. Patients with current CNS pathology or extramedullary disease, previous allogeneic HSCT, or Ph+ ALL eligible for treatment with tyrosine kinase inhibitors were excluded. Blinatumomab 15 µg/m²/day was given by continuous IV infusion for 4 weeks, followed by a 2-week treatment-free period (1 cycle). Responders could receive ≤4 cycles of treatment or undergo HSCT after ≥1 cycle. Patients with hematologic relapse discontinued treatment. Rate of complete MRD response (absence of MRD confirmed with a sensitivity of at least 10-4 after 1 cycle) was the primary endpoint. MRD was assessed by allele-specific quantitative real-time PCR for clonal rearrangements of immunoglobulin or T-cell receptor genes by a central laboratory. Overall survival (OS), relapse-free survival (RFS), duration of complete MRD response, and incidence and severity of adverse events (AEs) were secondary endpoints. Per protocol, OS and RFS will be analyzed after 18 months minimum follow-up.Results: 116 patients enrolled and received blinatumomab. Median (range) age was 45 (18–76) years; 15 (13%) patients were ≥65 years of age. 35% of the patients were treated in second or later remission (Table). As of February 2014, 106 patients had ended treatment: 74 had completed treatment (4 cycles or 1 cycle followed by HSCT) and 32 had discontinued treatment due to AEs, disease relapse, or investigators’ decision; 79 patients were still alive and being followed. Three patients were excluded from the efficacy analysis: one had no central lab assay and two had assays with a sensitivity of 5×10-4. Eighty-eight patients (78% [95% CI, 69%–85%]) had a complete MRD response after 1 cycle of treatment. The lower CI bound exceeded 44% (the null hypothesis response rate), confirming that the study met its primary objective. Two additional patients had a complete MRD response after >1 cycle of blinatumomab; across all cycles the complete MRD response rate was 80%. The rate of complete MRD response did not differ significantly across baseline age, sex, line of treatment, and MRD burden categories (Table). All patients experienced ≥1 AE. AEs occurring in ≥20% of patients included pyrexia (88%), headache (38%), tremor (29%), chills (25%), fatigue (24%), nausea (22%) and vomiting (22%). Serious AEs (SAEs) occurred in 60% of patients; 59% and 27% of patients had grade ≥3 and grade ≥4 AEs, respectively. SAEs occurring in ≥5% of patients were pyrexia (15%), tremor (7%), aphasia (5%), encephalopathy (5%) and overdose (5%). Two fatal AEs occurred on treatment: subdural hemorrhage and atypical pneumonia (the latter was deemed treatment-related).Conclusion: This is the largest prospective trial with an experimental compound in MRD+ ALL.Blinatumomab treatment resulted in complete MRD response across multiple patient demographics including patients in second-line treatment and those with high MRD burden. With a complete MRD response rate of 78%, the study met its primary objective. Among patients with a complete MRD response, 98% had a response within the first treatment cycle. In patients with MRD+ ALL following intensive therapy, rapid MRD response induced by blinatumomab has the potential to improve patient outcomes. [Display omitted] DisclosuresGoekbuget:Amgen Inc.: Consultancy, Honoraria, Research Funding. Off Label Use: This presentation will discuss the off-label use of blinatumomab, as this agent is not approved for use by the FDA, EMA or any other regulatory authorities.. Dombret:Amgen Inc.: Honoraria, Research Funding. Bonifacio:Amgen Inc.: Consultancy. Graux:Amgen Inc.: Honoraria, Membership on an entity’s Board of Directors or advisory committees. Buss:Amgen Inc.: Reimbursement for participation in clinical studies, Reimbursement for participation in clinical studies Other; BMS: Travel support, Travel support Other; Novartis: Travel support Other; Pfizer: Reimbursements for participation in clinical studies, Reimbursements for participation in clinical studies Other. Bruggemann:Amgen Inc.: Consultancy, Research Funding. Stieglmaier:Amgen Inc.: Equity Ownership; Amgen Research (Munich) GmbH: Employment. Wessels:Amgen Inc.: Equity Ownership; Amgen Research (Munich) GmbH: Employment. Haddad:Amgen Ltd.: Employment. Zugmaier:Amgen Research (Munich) GmbH: Employment; Amgen Inc.: Equity Ownership. Nagorsen:Amgen Inc.: Employment, Equity Ownership, Related to blinatumomab Patents & Royalties. Bargou:Amgen Inc.: Consultancy, Honoraria.

Comparison of MRD Monitoring By IG/TR Rearrangement and BCR/ABL1 Transcript in Ph+ Pediatric Acute Lymphoblastic Leukemia Patients Treated with Imatinib in the Esphall Study

Background. In the European Study for Pediatric Ph+ ALL (EsPhALL), a total of 128 patients aged 1 to 18 years diagnosed with ALL and evidence of t(9;22)(q34;q11) received post-Induction Imatinib on top of BFM high risk chemotherapy backbone, either in Good Risk (GR) (after randomization, n=58) or in Poor Risk (PR) (n=70), as defined according to early response to the National induction treatment. Although not used for treatment decision, MRD was prospectively measured at relevant time-points (TPs) by real-time quantitative PCR analysis of both rearranged immunoglobulin (IG) and T-cell receptor (TR) genes and BCR/ABL1 (B/A) transcript. Previous studies reported a different levels of concordance of the two methods, which could underline biological differences and might have implication in the clinical practice.Aim. To define the concordance of MRD measured at DNA (IG/TR) and RNA (B/A) levels in a large clinically controlled study by applying consensus methods.Methods. MRD was analyzed in national reference laboratories by real-time quantitative PCR of rearranged IG/TR genes as well as B/A fusion transcript, on subsets of study patients with available material at different TPs during therapy: 50/128, 39% at TP1 (end of IA), 56/128, 44% at TP2 (end of IB), 40/128, 31% at TP3 (end of HR1 Block), 37/128, 29% at TP4 (end of HR2 Block) and 13/128, 10% at TP5 (end of HR3 Block). Overall, 99/128 (77%) patients had at least one MRD measurement within TP5, and IG/TR versus B/A comparison was performed in 196 samples. Common guidelines developed within the EuroMRD consortium were applied for performing both methods and interpreting MRD results. Results were considered discordant in case of at least 1 log difference if both positive; similarly, in case of negative result by one of the two methods, discordance was called if the difference between the positive result and the sensitivity of the other method was more than 1 log.Results. Overall, the two methods were concordant in 70% of tests (n=137) (see figure), with the lowest concordance at TP3 (65%) and the highest at TP5 (77%). The vast majority of the 59 discordances reflected B/A results higher than IG/TR (47, 80%). IG/TR results tend to reduce earlier than B/A, as indicated by persistent positivity by B/A only at later TPs. Indeed, at TP1 (before Imatinib administration) 3 cases (6% of analyzed) were negative by IG/TR and positive by B/A, and 2 vice versa (4%). At later TPs, no more cases were found negative by B/A and positive by IG/TR, while 6/56 cases (11%) were B/A+ and IG/TR- at TP2, 7/40 (17%) at TP3, 6/37 (16%) at TP4, and 2/13 (15%) at TP5. Out of the 3 cases already IG/TR- and B/A+ at TP1, 2 were evaluated by 2 IG/TR markers and were persistently positive by B/A at subsequent TPs (7.3E-02 to 1.1E-01 at TP4); however, all 3 are alive in CCR, 2 after HSCT (1 GR and 1 PR) and 1 (GR) after Imatinib + chemo only. On the other hand, of the 6 cases that were IG/TR- but B/A+ at TP4, 3 were found negative by IG/TR already at TP1 and are described above. The remaining 3 progressively reduced tumor load by IG/TR and were all negative at TP3. One was GR and was alive in CCR after HSCT, while 2 were PR, one relapsed and one died in CCR, both after HSCT.Relapses in PR cases discordant at TP2 (2/10, 20%) (after the first exposure to Imatinib) were less frequent than in concordant cases (8/23, 35%), but were higher for discordant at TP3 (6/9, 67% vs 2/12, 17%) and were not different at TP4 (2/6, 33% vs 4/15, 27%) (too few data at TP5).If MRD would be used as a criteria for eligibility to HSCT (MRD≥5x10-4 at TP2 and if MRD<5x10-4 at TP2, MRD positive at any level at TP5), eligibility would differ according to the method, with 37/62 patients eligible by IG/TR (60%; no call possible for 14 cases, 18%) versus 46/57 eligible by B/A (81%; no call for 7 cases, 11%).Conclusions. The overall concordance of MRD monitoring by IG/TR or by B/A in pediatric patients treated with Imatinib is quite high. However, a relatively high proportion of cases are discordant at different TPs. Discordance (mainly B/A higher than IG/TR) does not necessarily imply a different outcome, even considering that most EsPhALL patients underwent HSCT. Indeed, these differences might reflect biological mechanisms that need to be experimentally approached. It must be tightly monitored (by multiple methods) whether a reduced eligibility to HSCT in the future protocols would result in a higher incidence of relapses. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Extensive Molecular Analysis Strongly Improves the Distinction Between AML and ALL in Adult Acute Leukemias of Ambiguous Lineage

BackgroundIn 1995 the European Group for the Immunological Classification of Leukemias (EGIL) presented guidelines to classify bilineage and biphenotypic acute leukemias (BAL). Subsequently, the WHO in 2008 defined mixed phenotype acute leukemias (MPAL), as leukemias coexpressing antigens of both lymphoid and myeloid lineage, and leukemias with distinct blast populations of more than one lineage. However, a classifying diagnosis towards myeloid or lymphoid leukemia based on an extensive molecular-biological characterization could provide much better distinction, which could be useful for selecting AML or ALL like treatment approaches. We investigated whether extensive molecular analysis might improve a diagnosis of BAL/MPAL into predominant AML or ALL.Patients and methodsFor this study 25 adults, diagnosed between 2000 and 2009 with BAL/MPAL were identified. Flowcytometry and cytogenetics were performed at diagnosis. In addition, molecular analysis was performed to identify BCR-ABL, MLL, AML1-ETO, CBF-MYH11, SIL-TAL1, SET-NUP, NUP-ABL fusion transcripts, FLT3-ITD, CEBPA, NPM1, NOTCH1 mutations and IKZF1, CDKN2A, CDKN2B deletions. Furthermore, multiplex PCR assays to detect immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements were performed. Gene expression profiles (GEP) were generated, and results were compared to an AML/ALL classifier profile. A classifying diagnosis of either ALL or AML was made if at least two of the 5 assays applied, were highly suggestive for a specific lineage without explicit contradicting results of the remaining assays.ResultsTwenty-four of the 25 BAL/MPAL cases could be diagnosed into predominant AML or ALL by extensive molecular analysis (Table 1). In 11 cases the WHO2008 criteria classified BAL cases as AML or ALL, and extensive molecular analysis also indicated the same lineage. Adding standard cytogenetic and molecular analysis improved the WHO classification in another 4 patients. Remarkably, extensive molecular analysis clearly showed preference for either AML or ALL in all of the MPAL cases. As a result, we were able to improve a diagnosis in all MPAL cases. Only 1 of the 25 patients remained unclassified. Statistical analysis did not show significant correlation between the different assays. Twelve patients received AML-based chemotherapy, including 3 patients ultimately classified as ALL. Moreover, among 12 patients who received ALL type therapy, 1 was retrospectively classified as AML. In general, outcome appeared very poor with a 3 year overall survival of 32% (Kaplan-Meier estimate, 95%CI: 14%-51%).ConclusionsThis study suggests that a differential diagnosis of AML versus ALL is partly improved by applying the WHO2008 criteria instead of the EGIL classification. However, extensive molecular analysis, including GEP and Ig/TCR, more strongly enabled the identification of the lineage of origin, ultimately resulting in a classifying diagnosis in 24 out of 25 patients. So far no assay appeared as golden standard but different complementary techniques showed additive value. Collectively these results strongly argue for advanced, centralized diagnostic procedures in patients with acute leukemia of ambiguous lineage, which may subsequently enable the development of specific therapeutic approaches in these poor-risk patients.Table 1Patient characteristicsAge (year)EGILWHO 2008Molecular aberrationsGEP probabilityIg/TCRClassificationAMLALL51TMMPAL*0,0001,000POSALL52TMMPALFLT3-ITD0,9700,030NEGAML20BMMPALMLL-AF4 CEBPA0,0001,000POSALL18BMMPALIKZF1 CDKN2A-2B0,0001,000POSALL49BMMPALBCR-ABL IKZF1NDNDPOSALL57BMMPALCDKN2A0,0001,000POSALL40BMMPALCDKN2A-2BNDNDPOSALL23TMPALCDKN2ANDNDPOSALL24BMPAL*0,0001,000POSALL65BMPALBCR-ABL IKZF1 CDKN2ANDNDPOSALL58BMAML*1,0000,000NEGAML52BMAMLNPM1NDNDNEGAML59BMAMLFLT3-ITD NPM1NDNDNEGAML41BMAMLFLT3-ITD CEBPA NPM1NDNDPOSAML69BMAMLMLLNDNDNEGAML18BMAMLCBF-MYH11NDNDPOSAML71BM#FLT3-ITD CDKN2A-2B0,7090,291NEGAML67BM#*0,0030,997POSALL15BMALLIKZF1 CDKN2A-2BNDNDPOSALL23BM#*0,9980,002POS?32BTM#*0,9380,062POSAML27BTMALLIKZF10,0010,999POSALL69BTMALLCDKN2A-2B0,0980,902NEGALL16BTMALLCDKN2A-2BNDNDPOSALL54TMALLNOTCH10,1480,852NEGALLAbbreviations: ND, not determined.* No abnormalities# Unclear classification according to WHO2008 DisclosuresNo relevant conflicts of interest to declare.

FLT3-ITD As a Target for Minimal Residual Disease Monitoring in Early T-Cell Precursor (ETP) Acute Lymphoblastic Leukemia

Background. The 'early T-cell precursor’ (ETP) subtype of T-ALL comprises up to 15% of T-ALL and has been reported to be associated with high risk of relapse. In addition to properties of T cell development, gene expression profile and immunophenotype of ETP-ALL show stem cell and early myeloid features. Consistently, this leukemia subgroup shows lower frequencies of prototypical T-ALL lesions and a higher prevalence of mutations typically associated with AML, including RAS and FLT3 mutations. In particular, FLT3-ITD was identified in up to 35% of adult ETP-ALL but data on its prevalence in pediatric ETP-ALL are lacking.In agreement with its stem-cell signature, ETPs frequently lack Immunoglobulin (IG) and T-cell receptor (TR) gene rearrangements, the most used and sensitive targets for MRD monitoring. As a consequence, alternative markers are required to extend the application of molecular MRD to most ETP-ALL patients.Aim. We explored the prevalence of FLT3-ITD mutation in a large series of pediatric ETP-ALL enrolled in two consecutive protocols of the Italian Association of Pediatric Hematology and Oncology (AIEOP), and we evaluated the potential use of FLT3-ITD as an alternative DNA marker for MRD monitoring.Methods. Out of 439 T-ALL patients enrolled in Italy into the AIEOP-BFM ALL2000 and AIEOP ALLR2006 consecutive protocols, 145/168 Early-T ALL (TI/II) patients were screened for FLT3-ITD occurrence. Among Early-T patients, 34 were defined as ETP according to immunophenotype, and 31 of them were screened for FLT3-ITD. Twenty-two ETP patients enrolled in Italy into the ongoing AIEOP-BFM ALL2009 were also screened, together with T-ALL cases without IG/TR molecular markers only for the technical validation of the method. PCR screening and RQ-PCR for FLT3-ITD were performed as previously reported (Nakao, Leukemia 1996;10:1911; Beretta, Leukemia 2004;18:1441). Parallel MRD analysis for IG/TR on the same samples, and flow cytometry-MRD were performed by standard procedures. EuroMRD guidelines were applied for performance and interpretation of RQ-PCR.Results. Among ALL2000/R2006 and ALL2009 ETP cases, 4/31 (12.9%) and 3/22 (13.6%) were FLT3-ITD positive, respectively; 5/7 were PPR, and all 7 were stratified as high risk. For ALL2000/R2006 patients, IG/TR MRD monitoring was feasible in 2 cases, and both were MRD-HR; 3/4 cases are alive in CCR, and one died after HSCT. Overall, the FLT3-ITD marker was detected in 12 T-ALL cases; only 4 of them had valuable IG/TR markers, while 8/12 (66%) did not present a suitable IG/TR MRD marker. FLT3-ITD MRD monitoring was performed on 11/12 FLT3-ITD positive T-ALL cases. Mean length of the ITD was 44 nucleotides (nts) (range 24-71), with a mean of 7 randomly inserted nts (range 1-26). Standard curves performed by 10-fold dilutions in DNA from PB Healthy Donor, showed a quantitative range of at least 5.0E-04 in all cases and 1.0E-04 in 5/11. Sensitivity of the assay was at least 1.0E-04 in all tested cases, and 1.0E-05 in 7/11.A comparison between IG/TR and FLT3-ITD was feasible in 3 out of 4 cases (1 is ongoing); all 3 cases were monitored by 2 IG/TR markers. At day15 and day33 of the Induction therapy, when MRD was very high (10-1 to 10-3 range), the IG/TR and FLT3-ITD were fully comparable, with less than 2 times difference. At day78 (after IB Induction block) 1 case was fully negative for both markers, 1 was slightly positive by FLT3-ITD (although not quantifiable and at the limit of the sensitivity) and negative for both IG/TR. The latter case was highly positive for both IG/TR (5.0E-03) but low positive (<1.0E-04) by FLT3-ITD. In this case, the IG/TR and FLT3-ITD were concordant and finally both were negative at subsequent time points.Conclusions. This is the first report on FLT3-ITD prevalence in a consecutive series of children with ETP-ALL, which resulted to be 14.8% (4/31), a value lower than that reported in adult ETP-ALL. The limited number of cases does not allow to draw conclusions on the prognostic impact of FLT3-ITD in ETP-ALL, although 3 out of 4 patients are alive in CCR. Although available in a limited subset, FLT3-ITD can be used as a marker for sensitive molecular MRD monitoring in ETP-ALL, when IG/TR markers are not available (about 2/3 of cases). The results of MRD monitoring in a limited set of cases suggests that ETP patients might respond well to IB Induction therapy. As already known for AML, caution for false negative results is required when only FLT3-ITD is monitored. DisclosuresNo relevant conflicts of interest to declare.

Next Generation Amplicon Sequencing of Immunoglobulin Heavy Chain Gene Rearrangaments for Minimal Residual Disease (MRD) Stratification in Childhood Acute Lymphoblastic Leukemia (ALL): A Comparison with Classical qPCR-Based Technique

▪IntroductionMRD is an important predictor of outcome in childhood ALL. Since 2000, MRD detected by quantitative PCR (qPCR) for immunoglobulin and T-cell receptor gene rearrangements with a minimal sensitivity of 1E-04 has been used for risk group stratification in pediatric BFM trials. Next generation sequencing (NGS) permits rapid parallel sequencing of large numbers of DNA segments. It can overcome most of the limitations of qPCR: it allows highly specific molecular detection of MRD without laborious optimization of patient-specific assays. Moreover it enables not only monitoring of malignant clone but also shows the picture of entire immune background.AimsTo develop an assay for immunoglobulin heavy chain (IgH) rearrangements detection on Ion Torrent PGM/Ion Proton platforms and compare the MRD levels with qPCR at BFM stratification timepoints.MethodsTwo round PCR was used for library preparation. Libraries were created from 450ng (equivalent of 70,000 DNA copies) of bone marrow DNA and 50ng of Human Genomic DNA (Roche). In the first round of PCR rearranged IgH genes were amplified using IGH FR3 BIOMED-2 primers (van Dongen, Leukemia 2003). In the second round the sequencing adapters and multiplex identifiers were added. Sequencing was performed on Ion Torrent PGM/Ion Proton sequencers using a 200bp chemistry. We developed a bioinformatics algorithm for detection of reads with known clonal V-D-J rearrangements within the resulting fastq files.For validation of the assay we sequenced 1E-1 to 1E-5 dilutions of diagnostic samples from 2 patients in multiplicates. The results show that the assay gives reproducible quantitative results up to 1E-4 dilution.ResultsWe sequenced 183 samples from 67 patients (52×day 15, 65×day 33, 66×day 78) with childhood ALL treated according to AIEOP-BFM ALL 2000 protocol with the median coverage 587,406 reads per sample. Eighty-three (45.4%) samples were negative by both methods. Fifteen (8.2%) samples were positive by NGS and negative by qPCR and 14 (7.7%) samples were positive by qPCR and negative by NGS. All the discordant samples had MRD levels below the sensitivity of both methods. The overall correlation of all double positive and double negative samples was very good (R2=0.93). If risk group stratification based on NGS results would be performed, 8 patients would be classified as intermediate risk (IR) instead of standard risk (SR) (one of whom relapsed) and 8 patients as SR instead of IR. One patient would be relocated from IR to slow early responders (SER) group, and two patients from SER to IR (one of them relapsed). One patient who relapsed would be classified as high risk (HR) instead of SER. All 5 patients who were MRD negative at d15 by NGS remained MRD negative in later timepoints and none of them relapsed.DiscussionWe present a cost-effective and widely adoptable NGS-based method that provides clinically relevant results in childhood ALL. NGS has a great potential to reduce the laboriousness associated with patient-specific qPCR analysis and to speed up the process of MRD detection. The sensitivity of both methods is comparable when ~500ng of DNA is used. The majority of the differences were in the samples with MRD levels below 1E-4 and most of treatment stratification changes occurred between SR/IR. However, the different stratification mostly concerned patients who did not relapse. The sensitivity of NGS could be improved if more DNA was analyzed. However, the benefit of increased MRD sensitivity is questionable due to possible overtreatment of patients with very low MRD loads after induction treatment. “Online” identification of d15 MRD negative patients reported as having an excellent prognosis previously is possible only by NGS, because optimization of patient-specific qPCR takes several weeks.The addition of 10% polyclonal DNA seems to solve the problem of MRD overestimation by NGS in samples with B-cell aplasia. At present, the main drawback of the Ig/TCR-exploring NGS methods is lack of standardization both in the experimental setting and in data analysis. Therefore, recently a European network, the EuroClonality NGS Consortium, has been formed to standardize the whole workflow of analytics, pre-analytics and bioinformatics not only for MRD quantification but also for clonality assessment in lymphoid neoplasms and for repertoire analysis.Supported by IGA NT14343, IGA NT12397, IGA NT13462-4 and GAUK 394214. DisclosuresNo relevant conflicts of interest to declare.

Clonal origins of ETV6-RUNX1⁺ acute lymphoblastic leukemia: studies in monozygotic twins.

Studies on twins with concordant acute lymphoblastic leukemia (ALL) have revealed that ETV6-RUNX1 gene fusion is a common, prenatal genetic event with other driver aberrations occurring subclonally and probably postnatally. The fetal cell type that is transformed by ETV6-RUNX1 is not identified by such studies or by the analysis of early B-cell lineage phenotype of derived progeny. Ongoing, clonal immunoglobulin (IG) and cross-lineage T-cell receptor (TCR) gene rearrangements are features of B-cell precursor leukemia and commence at the pro-B-cell stage of normal B-cell lineage development. We reasoned that shared clonal rearrangements of IG or TCR genes by concordant ALL in twins would be informative about the fetal cell type in which clonal advantage is elicited by ETV6-RUNX1. Five pairs of twins were analyzed for all varieties of IG and TCR gene rearrangements. All pairs showed identical incomplete or complete variable-diversity-joining junctions coupled with substantial, subclonal and divergent rearrangements. This pattern was endorsed by single-cell genetic scrutiny in one twin pair. Our data suggest that the pre-leukemic initiating function of ETV6-RUNX1 fusion is associated with clonal expansion early in the fetal B-cell lineage.

Monoclonality and cytogenetic abnormalities in hyaline vascular Castleman disease

Hyaline vascular Castleman disease is traditionally regarded as a reactive hyperplastic process. Occasional cases, however, have been reported with cytogenetic anomalies bringing this concept into question. In this study, we used conventional and methylation-specific polymerase chain reaction methods to assess the human androgen receptor α (HUMARA) gene in 29 female patients with hyaline vascular Castleman disease and compared the results with three cases of plasma cell Castleman disease and 20 cases of age-matched lymphoid hyperplasia. We also assessed for immunoglobulin gene and T-cell receptor gene rearrangements, and conventional cytogenetic analysis was performed in three cases of hyaline vascular Castleman disease. In cases with informative results, conventional and methylation-specific human androgen receptor α gene analyses yielded a monoclonal pattern in 10 of 19 (53%) and 17 of 23 (74%) cases of hyaline vascular Castleman disease, respectively. A monoclonal pattern was also detected in three cases of plasma cell Castleman disease but not in cases of lymphoid hyperplasia. The frequency of monoclonality was higher for lesions >5 cm in size (100%) and for the stromal-rich variant (91%). Cytogenetic abnormalities in stromal cells were revealed in two cases of hyaline vascular Castleman disease and no cases showed monoclonal immunoglobulin or T-cell receptor gene rearrangements. Follow-up data showed persistent disease in 4 of 23 (17%) patients. We conclude that hyaline vascular Castleman disease is often a monoclonal proliferation, most likely of lymph node stromal cells.

Pediatric posttransplant relapsed/refractory B-precursor acute lymphoblastic leukemia shows durable remission by therapy with the T-cell engaging bispecific antibody blinatumomab.

We report on posttransplant relapsed pediatric patients with B-precursor acute lymphoblastic leukemia with no further standard of care therapy who were treated with the T-cell engaging CD19/CD3-bispecific single-chain antibody construct blinatumomab on a compassionate use basis. Blast load was assessed prior to, during and after blinatumomab cycle using flow cytometry to detect minimal residual disease, quantitative polymerase chain reaction for rearrangements of the immunoglobulin or T-cell receptor genes, and bcr/abl mutation detection in one patient with Philadelphia chromosome-positive acute lymphoblastic leukemia. Blinatumomab was administered as a 4-week continuous intravenous infusion at a dosage of 5 or 15 μg/m(2)/day. Nine patients received a total of 18 cycles. Four patients achieved complete remission after the first cycle of treatment; 2 patients showed a complete remission from the second cycle after previous reduction of blast load by chemotherapy. Three patients did not respond, of whom one patient proceeded to a second cycle without additional chemotherapy and again did not respond. Four patients were successfully retransplanted in molecular remission from haploidentical donors. After a median follow up of 398 days, the probability of hematologic event-free survival is 30%. Major toxicities were grade 3 seizures in one patient and grade 3 cytokine release syndrome in 2 patients. Blinatumomab can induce molecular remission in pediatric patients with posttransplant relapsed B-precursor acute lymphoblastic leukemia and facilitate subsequent allogeneic hematopoietic stem cell transplantation from haploidentical donor with subsequent long-term leukemia-free survival.

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.

Comparison Of Deep Sequencing and Allele-Specific Oligonucleotide PCR Methods For MRD Quantitation In Acute Lymphoblastic Leukemia and Mantle Cell Lymphoma: CALGB 10403 and CALGB 59909 (Alliance)

BackgroundThe assessment of minimal residual disease (MRD) is a key component of prognosis and monitoring in acute lymphoblastic leukemia (ALL) and mantle cell lymphoma (MCL). Allele-specific oligonucleotide (ASO)-PCR can be used to assess MRD; however, this technique requires preparation of clonotype-specific primers for each patient, which is laborious and time-consuming. We demonstrated the utility of sequencing-based MRD assessment in ALL (Faham et al., Blood 2012). This quantitative approach relies on amplification and sequencing of immunoglobulin and T-cell receptor gene segments using consensus primers and can address some of the limitations associated with traditional MRD detection. Here we compared the ability of the sequencing and ASO-PCR methods to identify clonal cancer gene rearrangements at diagnosis and evaluated the concordance of MRD detection in bone marrow (BM) or blood samples from 37 patients (pts) with ALL and 22 pts with MCL entered onto prospective CALGB treatment trials, 10403 and 59909 (Alliance), respectively . MethodsUsing the quantitative ASO-PCR and sequencing assays, we analyzed diagnostic blood and BM samples from ALL pts for clonal rearrangements of immunoglobulin (IGH-VDJ, IGH-DJ, IGK) and T cell receptor (TRB, TRD, TRG) genes. We then assessed MRD at the IGH and/or TRG locus in 84 follow-up samples from ALL pts. Similarly, we analyzed samples from 22 MCL pts for immunoglobulin (IGH-VDJ, IGH-DJ, IGK) clonal gene rearrangements and measured MRD at the IGH and/or IGK locus in 114 follow-up samples. Sensitivity of the ASO-PCR vs sequencing was 1 X 10 4-5 vs 1 X 106, respectively. Concordance between sequencing and ASO-PCR MRD assessment on serial samples collected during and post-treatment was evaluated across all pts and within disease groups using concordance correlation coefficients for repeated measures (CCC-RM). Concordance in identification of detectable vs. undetectable MRD by both methods was also evaluated using Kappa statistics. ResultsUsing the sequencing platform, high frequency clonal rearrangements were observed in at least two receptors in 97% and 95% of pts with ALL and MCL, respectively. Selected ALL samples were known to have IGH-VDJ or TRG clonal rearrangements by ASO-PCR; however, sequencing revealed additional clonal rearrangements in 36/37 (97%) pts with ALL. Good concordance was observed with identification of MRD positive vs. negative between the methods (K=0.62; p<0.0001), where better agreement was observed within the ALL (K=0.63) than within the MCL (K=0.59) cohorts. Across both diseases, the sequencing method proved a more sensitive measure and CCC-RM was 0.82, reflecting the fact that 28 pts with undetectable MRD by ASO-PCR methods had low detectable MRD measures by the sequencing approach. The sequencing platform also provided new insights into kinetics of relapse. In one ALL case (Fig 1), we observed three high frequency IGH-VDJ clones, two of which are related based on VDJ replacement model. Dramatic reduction in frequency of one clone (clone 3, Fig 1) was observed following initial induction chemotherapy in both BM and blood. Only this single clone was monitored using ASO-PCR, and the patient appeared to be in molecular remission. However, the other two high frequency clones were chemo-resistant, and sequencing MRD monitoring revealed no reduction in clone frequency in either the BM or blood. The patient experienced early relapse at day 56, which may have resulted from the expansion of a clone carrying one or both of the clonal sequences that was not monitored by ASO-PCR. Thus, the sequencing method can be used to monitor response to treatment at the individual clone level. [Display omitted] ConclusionsThis study demonstrates concordance between identification of detectable MRD in ALL and MCL by sequencing and ASO-PCR Methods. The sequencing approach offers improvements over ASO-PCR including the ability to monitor multiple clonal sequences, faster turnaround time (results in 1 week), and greater sensitivity. The clinical significance of this greater sensitivity remains to be tested prospectively and must be correlated with clinical results. Nevertheless, the sequencing method represents an alternative approach for clinical MRD monitoring which could fundamentally improve the ability to monitor disease progression and recurrence in patients with lymphoid malignancies. Disclosures:Carlton: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.