Immunoglobulin T-cell Receptor Research Articles

Flow cell sorting followed by PCR-based clonality testing may assist in questionable diagnosis and monitoring of acute lymphoblastic leukemia.

Multicolor flow cytometry (MFC) has highly reliable and flexible algorithms for diagnosis and monitoring of acute lymphoblastic leukemia (ALL). However, MFC analysis can be affected by poor sample quality or novel therapeutic options (e.g., targeted therapies and immunotherapy). Therefore, an additional confirmation of MFC data may be needed. We propose a simple approach for validation of MFC findings in ALL by sorting questionable cells and analyzing immunoglobulin/T-cell receptor (IG/TR) gene rearrangements via EuroClonality-based multiplex PCR. We obtained questionable MFC results for 38 biological samples from 37 patients. In total, 42 cell populations were isolated by flow cell sorting for downstream multiplex PCR. Most of the patients (n=29) had B-cell precursor ALL and were investigated for measurable residual disease (MRD); 79% of them received CD19-directed therapy (blinatumomab or CAR-T). We established the clonal nature of 40 cell populations (95.2%). By using this technique, we confirmed very low MRD levels (<0.01% MFC-MRD). We also applied it to several ambiguous findings for diagnostic samples, including those with mixed-phenotype acute leukemia, and the results obtained impacted the final diagnosis. We have demonstrated possibilities of a combined approach (cell sorting and PCR-based clonality assessment) to validate MFC findings in ALL. The technique is easy to implement in diagnostic and monitoring workflows, as it does not require isolation of a large number of cells and knowledge of individual clonal rearrangements. We believe it provides important information for further treatment.

Digital Droplet PCR for Minimal Residual Disease Assessment in Philadelphia-Positive Acute Lymphoblastic Leukemia Using IG/TR Genes and BCR/ABL1 as Markers. Preliminary Results of a Comparative Analysis

Beldinanzi M, Della Starza I, contributed equally Background. The Philadelphia (Ph) chromosome is the most frequent genetic abnormality in adult acute lymphoblastic leukemia (ALL). BCR/ABL1 fusion transcript levels and clonal immunoglobulin/T-cell receptor (IG/TR) gene rearrangements are used as biomarkers for the study of minimal residual disease (MRD) in Ph+ ALL, with IG/TR used mainly in pediatric cohorts. There is evidence suggesting a poor concordance between these two molecular markers and there is a growing interest in evaluating MRD using a "double-hit strategy", especially in the adult setting where data are lacking. The real-time quantitative polymerase chain reaction (RQ-PCR) represents the current gold standard for MRD monitoring. It carries, however, some intrinsic limitations that digital droplet PCR (ddPCR), an innovative evolution of standard method, could overcome. Aim of this study. To carry out in adult Ph+ ALL patients a parallel MRD monitoring at early time points by both molecular targets - BCR/ABL1 gene fusion and clonal IG/TR gene rearrangements - by ddPCR. For BCR/ABL1 this was carried out by both RQ-PCR and ddPCR, in order to determine the concordance between the MRD values. Materials and Methods. The study comprised samples derived from 17 adults with newly diagnosed Ph+ ALL enrolled in the ongoing phase III GIMEMA LAL2820 clinical trial. Bone marrow (BM) samples were collected at the end of the induction phase (day +70) with ponatinib in the experimental arm or with a chemotherapy backbone plus imatinib in the control arm. At diagnosis, all patients underwent also a IG/TR gene rearrangements screening and MRD monitoring was performed by RQ-PCR and ddPCR for the quantification of BCR/ABL1 transcript levels and by ddPCR for IG/TR rearrangements, as described. MRD results were considered discordant in case of a difference greater than one log10 or in case of positivity/negativity. Results. The comparison between MRD values obtained in all samples by RQ-PCR and ddPCR using BCR/ABL1 mRNA level as biomarker showed an excellent correlation degree (R2=0.99). At variance, by IG/TR screening, it was possible to identify a clonal rearrangement only in 11/17 cases (65%), while 6/17 (35%) had no marker (NM) (3 p190, 3 p210; 2 IKZF1plus, 2 IKZF1 loss only and 2 IKZF1 WT). Moreover, of the 11 patients with a clonal rearrangement, in 3 (27%) (3 p190; 1 IKZF1plus, 2 IKZF1 WT) it was not possible to design a sensitive allele-specific oligonucleotide (ASO) for MRD monitoring. Overall, 8 samples were evaluated using both molecular markers by ddPCR, showing a poor correlation (R2=-0.21) and a low concordance rate. Indeed, 3/8 (37.5%) samples were concordant by both targets, either having highly positive (2/3) or negative (1/3) MRD values, while 5/8 (62.5%) were discordant; among the discordant cases, 1/5 was MRD-positive by both targets but outside the concordance range, while in 4/5 IG/TR MRD monitoring was negative, while BCR/ABL1 MRD monitoring was positive (range 10-3-10-1). Finally, in the 9 patients who failed IG/TR MRD monitoring, the BCR/ABL1 RQ-PCR analysis revealed a quantifiable MRD positivity in 6 cases (ranged 10-1-10-3), 2 proved "positive non-quantifiable" (PNQ) and 1 was MRD-negative (Figure 1). Conclusions. In adult Ph+ ALL, ddPCR with BCR/ABL1 as marker proved to be a sensitive and reliable tool for MRD monitoring at least comparable to RQ-PCR, showing a very high concordance rate. At variance, at least at early time points, the degree of concordance between BCR/ABL1 fusion transcript levels and IG/TR clonal rearrangements appeared suboptimal. Although IG/TR gene rearrangements are the most widely used biomarker for MRD monitoring in pediatric patients, the rate of failure in IG/TR marker identification in the adult Ph+ ALL setting is higher than in other subtypes. These findings suggest that the BCR/ABL1 fusion transcript remains the main sensitive target in adult Ph+ ALL and lead to hypothesize that in in this setting IG/TR clonal rearrangements represent a surrogate marker. Cohort expansion is warranted and clinical correlations are ongoing. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Minimal residual disease comparison between Ig/TCR PCR versus NGS assays in children with Philadelphia chromosome-positive acute lymphoblastic leukemia: A report from the COG AALL1631 study.

10023 Background: Minimal residual disease (MRD) assessment by immunoglobulin/T-cell receptor (Ig/TCR) polymerase chain reaction (PCR) is currently being used in the international pediatric Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) trial EsPhALL2017/AALL1631 for risk stratification. MRD concordance has previously been demonstrated between Ig/TCR PCR and flow cytometry in Ph+ALL. We sought to assess concordance of MRD assessment between conventional Ig/TCR PCR and next-generation sequencing (NGS) assays. Methods: MRD was assessed in all pts on AALL1631 by Ig/TCR PCR at end-induction IB; those with MRD &lt;5x10-4 were classified as standard-risk (SR) and randomized to treatment with imatinib and one of two chemotherapy regimens without hematopoietic stem cell transplant (HSCT), whereas pts with end-induction 1B MRD ≥ 5x10-4 were considered high-risk (HR) and assigned to HSCT after consolidation chemotherapy. Residual diagnostic and end-induction IB samples from consenting pts were assessed for NGS MRD by the clonoSEQ assay (Adaptive Biotechnologies) in blinded fashion and subsequently compared to Ig/TCR MRD to determine concordance as related to MRD-based HSCT recommendations ( ie, MRD ≥ 5x10-4 consistent with HR group assignment). MRD values were calculated using the kappa statistic for agreement above chance. Results: Sixty-seven pts had matched samples available for MRD assessment at end-induction 1B by both Ig/TCR PCR and NGS (Table). NGS MRD was evaluable for all 67 pts and stratified as 62 SR (&lt;5x10-4) and 5 HR (≥5x10-4). In contrast, Ig/TCR PCR results were inevaluable for 3 pts (unsatisfactory sample quality) and indeterminate (positive, but not quantifiable) in 4 pts. Of the remaining 60 pts, 55 met SR and 5 HR criteria using Ig/TCR PCR. There was only 1 discordant case between the two methods for MRD-based HSCT recommendation among these 60 pts with a kappa statistic for agreement above chance of 0.88. Conclusions: NGS and Ig/TCR PCR assays were highly concordant in MRD assessment for risk stratification at a threshold of 5x10-4 in pediatric pts with Ph+ALL enrolled on AALL1631. Of note, the NGS assay yielded MRD results amenable for risk stratification in 100% pts compared to 89.6% for the Ig/TCR PCR methodology. These data support the use of NGS MRD testing for risk stratification in pediatric Ph+ALL.[Table: see text]

Measurable residual disease analysis in paediatric acute lymphoblastic leukaemia patients with ABL-class fusions

BackgroundABL-class fusions including NUP214-ABL1 and EBF1-PDGFRB occur in high risk acute lymphoblastic leukaemia (ALL) with gene expression patterns similar to BCR-ABL-positive ALL. Our aim was to evaluate new DNA-based measurable residual disease (MRD) tests detecting these fusions and IKZF1-deletions in comparison with conventional immunoglobulin/T-cell receptor (Ig/TCR) markers.MethodsPrecise genomic breakpoints were defined from targeted or whole genome next generation sequencing for ABL-fusions and BCR-ABL1. Quantitative PCR assays were designed and used to re-measure MRD in remission bone marrow samples previously tested using Ig/TCR markers. All MRD testing complied with EuroMRD guidelines.ResultsABL-class patients had 46% 5year event-free survival and 79% 5year overall survival. All had sensitive fusion tests giving high concordance between Ig/TCR and ABL-class fusion results (21 patients, n = 257 samples, r2 = 0.9786, P < 0.0001) and Ig/TCR and IKZF1-deletion results (9 patients, n = 143 samples, r2 = 0.9661, P < 0.0001). In contrast, in BCR-ABL1 patients, Ig/TCR and BCR-ABL1 tests were discordant in 32% (40 patients, n = 346 samples, r2 = 0.4703, P < 0.0001) and IKZF1-deletion results were closer to Ig/TCR (25 patients, n = 176, r2 = 0.8631, P < 0.0001).ConclusionsMRD monitoring based on patient-specific assays detecting gene fusions or recurrent assays for IKZF1-deletions is feasible and provides good alternatives to Ig/TCR tests to monitor MRD in ABL-class ALL.

Intensification of Early-Phase Therapy to Diminish the Prognostic Effect of Myeloid Antigen Expression in Infants with KMT2A-Rearranged Acute Lymphoblastic Leukemia: A Report from the JPLSG MLL-10 Trial

Background KMT2A-rearranged acute lymphoblastic leukemia (KMT2A-r ALL) is a rare and dismal disease in infants. Despite restriction of the indication of allogeneic hematopoietic stem cell transplantation to the high-risk group (patients aged <180 days with KMT2A-r ALL or central nervous system involvement), adoption of an Interfant-06-based induction therapy with stricter age-related dosing followed by COG AALL0631-based post-remission chemotherapy with additional administration of high-dose cytarabine in the early intensification phase led to rapid clearance of minimal residual disease (MRD) in the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) MLL-10 trial. The MLL-10 trial demonstrated an improved outcome of 66.2% in 3-year event-free survival (EFS) among infants with KMT2A-r ALL (Tomizawa D. Blood 2021). As the Interfant-06 study showed an association between the expression levels of myeloid markers (MM) and poor MRD clearance at end of induction (EOI) and a high relapse rate (Stutterheim J, J Clin Oncol.2021), we analyzed the significance of MM expression in the MLL-10 cohort and its association with prognosis.MethodsWe analyzed and compared the MM expression (defined as at least one positive marker [with a positive blast subset ≥ 10%] among CD117, CD13, CD33, and CD65/CD15) by using flow cytometry (FCM) in infants with KMT2A-r ALL who were registered in the JPLSG MLL-10 trial. We also compared the results of immunoglobulin/T-cell receptor (Ig/TCR) gene-based polymerase chain reaction (PCR)-MRD analyses or 4-color FCM-MRD assay between the MM-positive and MM-negative groups at EOI and end of early consolidation. The Ig/TCR-MRD results were classified as negative if <5 × 10 −4 and positive if ≥5 × 10 −4, while the FCM-MRD results were classified as negative if <0.01% and positive if ≥0.01%. We prioritized PCR-MRD and used FCM-MRD when we could not make a primer for PCR-MRD. The presence of MRD was not used as a basis for choosing the appropriate therapy.Results and DiscussionAmong the patients with KMT2A-rALL, 74 were included in this study, excluding one who was not evaluated with FCM at diagnosis. Of these patients, 42 were MM-positive and 32 were MM-negative. The 3-year EFS rates of the MM-positive and MM-negative patients were 62.3% (95% confidence interval [CI], 45.5-75.3) and 70.0% (95% CI, 50.3-83.1), respectively (p = 0.61). Their 3-year overall survival rates were 80.6% (95% CI, 65.0-89.8) and 87.5% (95% CI, 70.0-95.1), respectively (p = 0.74). The numbers of MM-positive and MM-negative patients according to age group are summarized in Table 1, and the difference in age distribution between the two groups was not significant. The MRD statuses of the patients at EOI in the two groups are also summarized in Table 1. No significant difference in MRD clearance was found between the MM-positive and MM-negative groups.ConclusionIn this study, we found no significant difference in survival rate between the MM-positive and MM-negative groups. The MM expression was not a prognostic marker in the infants with KMT2A-r ALL in the MLL-10 cohort. We believe that rapid MRD clearance in the early phase of treatment with enhanced chemotherapy would have the greatest contribution to the improvement of prognosis. In this study, the MM-positive patients from the MLL-10 cohort might have benefited from early-phase treatment intensification in terms of MRD clearance. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Analytical Quality Controls for ddPCR Detection of Minimal Residual Disease in Acute Lymphoblastic Leukemia

Abstract Background Droplet digital PCR (ddPCR) is a promising technique for absolute quantification of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL), but there is no comprehensive quality assurance program to enable its application in clinical laboratories. Current guidelines for real-time quantitative PCR (qPCR) assays targeting immunoglobulin/T-cell receptor (Ig/TCR) gene rearrangements needed adaptation for ddPCR to cover droplet generation, intraassay variation, and interassay variation in the absence of standard curves. Methods Six qPCR MRD assays for Ig/TCR gene rearrangements and a standard albumin control gene assay were migrated to a ddPCR platform and used to test 82 remission samples from 6 patients with ALL. Three analytical quality controls (QC) were developed and evaluated for ddPCR MRD detection. Results Analytical QC for droplet number generation (DN-QC), for albumin ddPCR assay performance (Alb-QC) and for patient-specific marker assay performance (PS-QC) were established with pass/fail limits and corresponding QC rules. Compared to established qPCRs, the ddPCR assays had comparable sensitivity and quantitative range. Overall, there was close agreement (91%) of MRD results between qPCR and ddPCR (κ = 0.86, P &amp;lt; 0.0001) and stronger concordance in 32 quantifiable samples (R2 = 0.97, P &amp;lt; 0.0001). Conclusions The use of this newly developed quality control system for ddPCR MRD testing avoids the need to repeat standard curves and provides reliable results comparable to standardized qPCR methods for MRD detection in ALL.

PCR Technology to Identify Minimal Residual Disease.

Although new techniques (i.e., droplet digital-PCR, next-generation sequencing, advanced flow cytometry) are being developed, DNA-based allele-specific real-time quantitative (RQ)-PCR is still the gold standard for sensitive and accurate immunoglobulin/T cell receptor (IG/TR)-based minimal residual disease (MRD) monitoring, allowing the detection of up to 1 leukemic cell in 100,000 normal lymphoid cells. We herewith describe the standard PCR procedure which has been developed and standardized (with minor modification in single labs) through the last 20years of activity of the EuroMRD Consortium, a volunteer activity of expert laboratories that is continuously providing education, standardization, quality control rounds, and guidelines for interpretation of RQ-PCR data.

Capture-based Next-Generation Sequencing Improves the Identification of Immunoglobulin/T-Cell Receptor Clonal Markers and Gene Mutations in Adult Acute Lymphoblastic Leukemia Patients Lacking Molecular Probes.

The monitoring of minimal residual disease (MRD) in Philadelphia-negative acute lymphoblastic leukemia (ALL) requires the identification at diagnosis of immunoglobulin/T-cell receptor (Ig/TCR) rearrangements as clonality markers. Aiming to simplify and possibly improve the patients’ initial screening, we designed a capture-based next-generation sequencing (NGS) panel combining the Ig/TCR rearrangement detection with the profiling of relevant leukemia-related genes. The validation of the assay on well-characterized samples allowed us to identify all the known Ig/TCR rearrangements as well as additional clonalities, including rare rearrangements characterized by uncommon combinations of variable, diversity, and joining (V-D-J) gene segments, oligoclonal rearrangements, and low represented clones. Upon validation, the capture NGS approach allowed us to identify Ig/TCR clonal markers in 87% of a retrospective cohort (MRD-unknown within the Northern Italy Leukemia Group (NILG)-ALL 09/00 clinical trial) and in 83% of newly-diagnosed ALL cases in which conventional method failed, thus proving its prospective applicability. Finally, we identified gene variants in 94.7% of patients analyzed for mutational status with the same implemented capture assay. The prospective application of this technology could simplify clonality assessment and improve standard assay development for leukemia monitoring, as well as provide information about the mutational status of selected leukemia-related genes, potentially representing new prognostic elements, MRD markers, and targets for specific therapies.

High sensitivity and clonal stability of the genomic fusion as single marker for response monitoring in ETV6-RUNX1-positive acute lymphoblastic leukemia.

Assessment of minimal residual disease (MRD) is an integral component for response monitoring and treatment stratification in acute lymphoblastic leukemia (ALL). We aimed to evaluate the genomic ETV6-RUNX1 fusion sites as a single marker for MRD quantification. In a representative, uniformly treated cohort of pediatric relapsed ALL patients (n=52), ETV6-RUNX1 fusion sites were compared to the current gold standard, immunoglobulin/T-cell receptor (Ig/TCR) gene rearrangements. Primer/probe sets designed to ETV6-RUNX1 fusions achieved significantly more frequent a sensitivity and a quantitative range of at least 10-4 compared to the gold standard with 100% and 73%versus 76% and 47%, respectively. The breakpoint sequence was identical at diagnosis and relapse in all tested cases. There was a high degree of concordance between quantitative MRD results assessed using ETV6-RUNX1 and the highest Ig/TCR marker (Spearman's 0.899, P<.01) with differences>½ log-step in only 6% of patients. A high proportion of ETV6-RUNX1-positive ALL relapses (40%) in our cohort showed a poor response to induction treatment at relapse, and therefore had an indication for hematopoietic stem cell transplantation, demonstrating the need of accurate identification of this subgroup. ETV6-RUNX1 fusion sites are highly sensitive and reliable MRD markers. Our data confirm that they are unaffected by clonal evolution and selection during front-line and second-line chemotherapy in contrast to Ig/TCR rearrangements, which require several markers per patient to compensate for the observed loss of target clones. In future studies, the genomic ETV6-RUNX1 fusion can be used as single MRD marker.

Quantitative monitoring of minimal residual disease in childhood acute lymphoblastic leukemia using TEL-AML1 fusion transcript as a marker.

ABSTRACTImportanceBy demonstrating with TEL‐AML1, this study indicated that mRNAs transcribed from fusion genes are ideal targets for minimal residual disease (MRD) monitoring in childhood acute lymphoblastic leukemia, and that different thresholds are needed to apply them into the risk stratification.Objective TEL‐AML1 expression was measured at three time points to 1) determine cut‐off values for predicting acute lymphoblastic leukemia (ALL) relapse; 2) investigate the prognostic value of this method and how well the results at these time points correlated; 3) determine the correlation between MRD levels assessed using this marker and that determined by immunoglobulin/T‐cell receptor (Ig/TCR) rearrangement detection.Methods TEL‐ AML1 expression in 62 children with ALL was quantitated by real‐time quantitative PCR at day 15, day 33, and month 3. The relationship between patient outcome and TEL‐AML1 level was analyzed at each time point. The correlation between the MRD levels determined by TEL‐AML1 or Ig/TCR rearrangements was also analyzed.ResultsFor day 33, 6.68 TEL‐AML1 copies/104 ABL copies was determined to be the best cut‐off value. Higher levels were correlated with relapse (P = 0.001). For day 15 and month 3, the best cut‐off values were 336.5 and 0.85 copies/104 ABL copies respectively; patients with higher expression levels had lower RFSs (day 15: P = 0.027; month 3: P = 0.023). For days 15 and 33, MRD levels assessed using TEL‐AML1 or Ig/TCR rearrangements were strongly correlated [Spearman rank correlation coefficient (ρ) = 0.729 (day 15), 0.719 (day 33); P < 0.001 (both)], and both methods were equally effective at predicting relapse. At month 3, there was moderate correlation between the results derived from the two markers (ρ = 0.418, P = 0.003); however, receiver operating characteristic curve analysis showed that TEL‐AML1 was a better prognostic marker.Interpretation TEL‐AML1 is an effective marker for MRD assessment and relapse prediction in children with ALL.

Switching Towards Monocytic Lineage and Discordancy between Flow Cytometric and PCR Minimal Residual Disease Results Is a Hallmark Feature of DUX4 Rearranged B-Cell Precursor Acute Lymphoblastic Leukemia

Introduction:Recently we described a subgroup of pediatric patients with B cell precursor acute lymphoblastic leukemia (BCP ALL) with switching from B to monocytic lineage in early phase of the therapy (Slamova et al., 2014). In a limited cohort of patients with switching ALL (swALL), we observed inferior response to treatment with discrepancy of minimal residual disease level (MRD) assessed by flow cytometry (FC) and quantitative polymerase chain reaction (qPCR) of Immunoglobulin-T cell receptor (Ig-TCR) rearrangements. In current Berlin-Frankfurt-Münster (BFM) treatment protocols, FC MRD value at day 15 (d15) and PCR MRD value at day 33 (d33) and week 12 (w12) are used for stratification. Using an extended cohort of patients with available RNA sequencing data (cohort mainly focused on B other cases or swALLs) we aimed to answer following questions:What is the genetic background of swALL? What is the frequency among swALLs of the recently described DUX4 rearranged subgroup?How do B cell oriented FC and PCR MRD correlate in standard protocol timepoints, i.e. day 8 (d8) (peripheral blood, PB) d15, d33 and w12 (bone marrow, BM) of treatment?What is the characteristic MRD response to treatment in swALL?Results:We performed RNA sequencing in 177 patients (median age 6.1 years, range 0-18) treated by several treatment protocols (ALL BFM 95 n=5, ALL IC BFM 2002 n=14, ALL AIEOP BFM 2000 n=17, ALL AIEOP BFM 2009 n=135, Interfant n=3, ALL IC/Interfant n=2, EsPhALL n=1). In 68 patients we observed switching phenomenon by appearance of B/monocytoid population coexpressing B lineage (CD19, CD34) and monocytic lineage (CD33, CD14) markers (median 0.98%, range 0.032-38%). In non swALLs median of this population was 0.059% (range 0.0025-1.1%) and the cells did not form a clear cluster. According to RNAseq data, majority of swALL patients (n=42/68) belong to DUX4 subgroup (chi square p< 0.00001). The distribution into other molecular genetic subtypes is summarized in table 1.Correlation coefficient (Spearman) of all included samples with both available values (n=552) was 0.82 (p<0.0001), Concordance in categorization of positivity and negativity with cut-off 1e-4 was 85%. We observed worse correlation between FC and PCR MRD in patients with swALL (d8 R=0.58, d15 R=0.6, d33 R=0.36, w12 n.s.) compared to non swALL (d8 R=0.83, d15 R=0.91, d33 R=0.69, w12 R=0.37). However, concordance in swALL in categorization of positivity and negativity with cut-off 1e-4 was still ≥80% in each analyzed timepoint apart from d33 with concordance only 44% showing significant discrepancy of both methods (Figure 1a). On the contrary, concordance in non swALL was ≥87% for each analyzed timepoint (d33 with concordance 87% shown in Figure 1b). Poor correlation between B-cell oriented FC MRD and PCR MRD at d33 was also obvious when analyzed DUX4 subgroup separately (R=0.31 (p=0.04), concordance 45%).We observed significantly higher MRD in swALLs compared to non swALLs at all analyzed timepoints: d8 (p=0.0021), d15 (p=0.0088, d33 (p<0.0001) and w12 (p=0.008). Higher MRD levels were also found in DUX4 patients when compared to non DUX4 (all timepoints p<0.05). Interestingly, when compared swALL and non swALLs pts in DUX4 subgroup only, the DUX4 swALLs are those with poorer treatment response (all timepoints p<0.05). With respect to protocolar cut-off values, FC MRD at d15 was above 10% in 18/67 swALL patients (in 13/52 DUX4 patients), d33 PCR MRD was above 0.1% in 33/57 swALLs (24/44 DUX4 pts) and at w12 PCR MRD was above 0.01% in 12/55 swALLs (10/44 DUX4 pts).Conclusions:DUX4 subgroup is the most prevalent genetic subtype among swALLs. SwALLs and/or DUX4 subgroup have poorer treatment response at d15, d33 and w12. However, it remains to be elucidated whether poor initial treatment response is eventually reflected in treatment outcome. In majority of swALL patients B cell phenotype of blasts is preserved at day 15 enabling correct classification. Prominent discrepancy between FC and PCR MRD is present especially at d33 and development of different FC MRD strategies focused on monocytic compartment is needed.Supported by Ministry of Health of the Czech Republic, grant nr. 15-28525A and NV18-03-00343; Czech Science Foundation nr.P302/12/G101, UNCE204012 [Display omitted] DisclosuresBrüggemann:Affimed: Research Funding; Regeneron: Research Funding; Amgen: Consultancy, Research Funding, Speakers Bureau; Roche: Speakers Bureau; Pfizer: Speakers Bureau; Incyte: Consultancy; PRMA: Consultancy. Ritgen:abbvie: Research Funding; Roche: Honoraria, Research Funding.

Monitoring of childhood ALL using BCR-ABL1 genomic breakpoints identifies a subgroup with CML-like biology

AbstractWe used the genomic breakpoint between BCR and ABL1 genes for the DNA-based monitoring of minimal residual disease (MRD) in 48 patients with childhood acute lymphoblastic leukemia (ALL). Comparing the results with standard MRD monitoring based on immunoglobulin/T-cell receptor (Ig/TCR) gene rearrangements and with quantification of IKZF1 deletion, we observed very good correlation for the methods in a majority of patients; however, >20% of children (25% [8/32] with minor and 12.5% [1/8] with major-BCR-ABL1 variants in the consecutive cohorts) had significantly (>1 log) higher levels of BCR-ABL1 fusion than Ig/TCR rearrangements and/or IKZF1 deletion. We performed cell sorting of the diagnostic material and assessed the frequency of BCR-ABL1-positive cells in various hematopoietic subpopulations; 12% to 83% of non–ALL B lymphocytes, T cells, and/or myeloid cells harbored the BCR-ABL1 fusion in patients with discrepant MRD results. The multilineage involvement of the BCR-ABL1-positive clone demonstrates that in some patients diagnosed with BCR-ABL1-positive ALL, a multipotent hematopoietic progenitor is affected by the BCR-ABL1 fusion. These patients have BCR-ABL1-positive clonal hematopoiesis resembling a chronic myeloid leukemia (CML)–like disease manifesting in “lymphoid blast crisis.” The biological heterogeneity of BCR-ABL1-positive ALL may impact the patient outcomes and optimal treatment (early stem cell transplantation vs long-term administration of tyrosine-kinase inhibitors) as well as on MRD testing. Therefore, we recommend further investigations on CML-like BCR-ABL1-positive ALL.

Immunoglobulin/T-Cell Receptor (Ig/TCR) Allele Usage in Normal and on Treatment Bone Marrow Samples in Childhood Acute Lymphoblastic Leukaemia - Implications for NGS Based MRD Analysis

Non-specific amplification of background DNA may cause false positive MRD results using conventional Ig/TCR gene rearrangement RQ-PCR; non-amplification control (NAC) samples taken from pooled buffy coat lymphocytes are thus routinely analysed in parallel to test samples. In contrast NGS-based MRD relies solely on DNA sequence specificity to avoid false positives. However at certain alleles gene rearrangement diversity is limited and false positive results remain a risk (the same DNA sequence from normal not leukaemic lymphocytes). We therefore assessed the relevant parameters of Ig/TCR gene rearrangement (5'/3' gene selection, deletion/insertion size) across all loci screened by the Biomed-2 PCR panel, in pooled buffy coat (24 donors), single blood donor cones and patient samples at end of induction (EOI). This subsequently allows us to determine an 'e-score', the probability that a given sequence is generated by chance. This allows investigators to avoid selecting gene rearrangements with poor diversity for MRD analysis in a systematic manner.In a 1st round PCR DNA was amplified with primers covering 6 Ig/TCR loci (IgH, inc. IgH, IgK, TRB, TRD and TRG) containing partial MiSeq adaptor sequences. The resulting amplicons were purified using Agencourt AMPure XP beads (Beckman Coulter, Jersey City, NJ). The amplicons acted as templates in a 2nd round PCR, using primers containing index and full MiSeq adaptor sequences. Samples were purified, then quantitated by Qubit (InvitroGen, Carlsbad, CA) and TapeStation analysis (Agilent, Santa Clara, CA). This data was used to normalise DNA and construct the final sequencing library which was quantified using the Kapa Library Quantification Kit (Kapa Biosystems, Wilmington, MA) to ensure ideal cluster density. PhiX Sequencing Control v3 (Illumina, San Diego, CA) was added to the sequencing library at 8% to ensure early cycle high sequence diversity. Bi-directional sequencing was completed using Illumina MiSeq 500 cycle cartridge and Nano Reagent Kit (Illumina, San Diego, CA). The Vidjil bioinformatic pipeline (Bonsai team, CRIStAL, Lille, France) was used for data analysis.As illustrated in Fig 1 for TRG, 5' and 3' allele selection was found to be biased at all loci. However, the biases were stable across NAC, cone and importantly EOI samples. EOI samples were somewhat more oligoclonal than those of normal individuals (146 vs 212 vs 255 clones/1000 reads for EOI, NAC and cone respectively) leading to slightly greater variance around mean allele usage. Mean 5' and 3' deletion lengths and N insertion size varied both across loci and between alleles of a given gene. The patterns were stable across NAC, cone and EOI samples (fig 2).As the data from NAC samples is representative of background DNA in typical end of induction test samples, an e-score for defining rearrangement probability is therefore calculable (fig 3).Example e-scores for typical rearrangements are: -VH2.5-DH3.22-JH4.02 (-4/7/-7)(-5/9/-6) = 2.03x10-11DD2-DD3 (-3/5/-3) = 1.06x10-4TRBV6-2*01-TRBD2*01-TRBJ2-3*01 (-3/7/-4)(-2/10/-5) = 2.05x10-11VKINT-KDE (-5/6/-1) = 9.94x10-5IGKV3-20*01 (-3/5/-6) KDE = 4.32x10-6The worst e-score would be found for rearrangements with poor diversity including IGK-KDE rearrangements (VKInt-KDE 0/0/0 e = 1.7x10-01) and certain TRG rearrangements (V8*01/J1*02 0/0/0 e=1.3x10-03). In contrast the worst e-score for IgH (V3-23*01-D6-13-J4*02(0/0/0)(0/0/-4) is 3.5x10-07.Incorporating a 1 log margin of safety discrimination of specific amplification over background amplification as per Ig/TCR RQ-PCR MRD criteria would mean that an e-score of <1x10-07 is required to confirm a true negative result at 1 in 1,000,000 cells. Thus in practice the vast majority of IgH rearrangements meet those criteria, whilst only a minority of IGK and TRG rearrangements (<10%) would achieve an e-score of 10-05, low enough to be used as a single target for conventional MRD stratification at the commonly used threshold for low risk disease of 10-04). Nevertheless, a multi-loci approach for MRD target identification incorporating loci likely to yield poorer e-scores remains a worthwhile strategy that increases the probability of finding an MRD target in a given patient.In conclusion the e-score provides a standardised methodology for robust target selection that is useful for quality control purposes and one that should be incorporated into clinical NGS-MRD pipelines. [Display omitted] [Display omitted] [Display omitted] DisclosuresMoppett:Jazz Pharmaceuticals: Honoraria.

Proposal for the standardization of flow cytometry protocols to detect minimal residual disease in acute lymphoblastic leukemia.

Minimal residual disease is the most powerful predictor of outcome in acute leukemia and is useful in therapeutic stratification for acute lymphoblastic leukemia protocols. Nowadays, the most reliable methods for studying minimal residual disease in acute lymphoblastic leukemia are multiparametric flow cytometry and polymerase chain reaction. Both provide similar results at a minimal residual disease level of 0.01% of normal cells, that is, detection of one leukemic cell in up to 10,000 normal nucleated cells. Currently, therapeutic protocols establish the minimal residual disease threshold value at the most informative time points according to the appropriate methodology employed. The expertise of the laboratory in a cancer center or a cooperative group could be the most important factor in determining which method should be used. In Brazil, multiparametric flow cytometry laboratories are available in most leukemia treatment centers, but multiparametric flow cytometry processes must be standardized for minimal residual disease investigations in order to offer reliable and reproducible results that ensure quality in the clinical application of the method. The Minimal Residual Disease Working Group of the Brazilian Society of Bone Marrow Transplantation (SBTMO) was created with that aim. This paper presents recommendations for the detection of minimal residual disease in acute lymphoblastic leukemia based on the literature and expertise of the laboratories who participated in this consensus, including pre-analytical and analytical methods. This paper also recommends that both multiparametric flow cytometry and polymerase chain reaction are complementary methods, and so more laboratories with expertise in immunoglobulin/T cell receptor (Ig/TCR) gene assays are necessary in Brazil.

Clinical Implication of BCR/ABL Fusion Transcript Monitoring in Addition to Ig/TCR Gene Rearrangement-Based Minimal Residual Disease in Philadelphia Chromosome-Positive Childhood Acute Lymphoblastic Leukemia

▪Background: Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) accounts for about 3% of pediatric ALL and has a poor prognosis. Advances of treatment due to the tyrosine kinase inhibitor imatinib have improved the cure rates. According to recent guidelines in the amended European intergroup trial on Ph+ ALL (EsPhALL), patients with rapid minimal residual disease (MRD) response and negativity during further treatment are no longer eligible for allogeneic stem cell transplantation (alloSCT). This down-grading of therapy in a circumscribed patient cohort with favorable prognosis is a desirable development as stem cell transplantation still implies a considerable risk of toxicity. These guidelines refer to MRD by immunoglobulin/T-cell receptor (Ig/TCR) gene rearrangements and do not consider monitoring of the BCR/ABL fusion transcript as long as informative results for Ig/TCR MRD are available. However, discrepancies between the results of the two methods occur. This complicates the decision on alloSCT indication if Ig/TCR MRD becomes negative while the BCR/ABL fusion transcript remains detectable.Objectives/Methods: We therefore evaluated the prognostic relevance of this specific combination of findings, i.e. the continuous negativity for Ig/TCR MRD and persistently positive results for BCR/ABL after the second intensive consolidation block or later, in 16 pediatric patients with Ph+ ALL. They were identified among 139 German and Austrian Ph+ patients treated in the ALL-BFM 2000 or EsPhALL trial from August 1, 1999 to July 31, 2013. Twelve out of the 16 patients received imatinib in first-line treatment intermittently as previously described in the EsPhALL protocol (Biondi A et al Lancet Oncol. 2012) or continuously as recommended by the amended EsPhALL protocol.Results: Eight of the 16 identified patients received an alloSCT in first complete remission (1st CR), whereas the remaining eight patients were treated with chemotherapy only. Of the eight patients with alloSCT, seven are in first continuous complete remission (1st CCR) with median EFS of 7.6 years, one patient died after second relapse. In the group of eight patients without alloSCT three are in 1st CCR with a median EFS of 2.6 years, four patients are in 2nd CR after relapse (3/4 had alloSCT in 2nd CR, median EFS 4.7 years), and one patient with Down syndrome died of an infectious complication. Remarkably, two patients of the latter group (both with M-BCR) showed a protracted increase of BCR/ABL copy numbers over several years with neither morphological signs of relapse nor Ig/TCR MRD based reappearance. One of them eventually suffered a relapse 5 years after diagnosis, one is still in 1st CCR with EFS of 5.2 years.Conclusion: The data suggest that patients with Ig/TCR MRD negativity and persistently detectable BCR/ABL fusion transcript have a high risk of relapse when treated with chemotherapy only and may benefit from alloSCT. However, patient numbers are currently too small to deduce recommendations from this observation. Further investigation of a larger cohort with longer follow-up is needed to confirm the prognostic importance of BCR/ABL fusion transcript monitoring in addition to Ig/TCR MRD, especially considering a potential additional impact of the recently implemented continuous imatinib treatment. One additional patient would have met the diagnostic inclusion criteria of this analysis. He had an extensive increase of BCR/ABL fusion transcript at the end of maintenance treatment while being in morphological remission and negative for Ig/TCR MRD. This patient proved to be BCR/ABL positive in granulocytes revealing a chronic myeloid leukemia (CML) misdiagnosed as ALL during initial blast crisis. This indicates that an underlying CML might be taken into consideration also in other patients of the analyzed cohort. In consequence, BCR/ABL in granulocytes is now tested in all newly diagnosed Ph+ ALL patients in Germany to ensure the differentiation of BCR/ABL positive ALL vs. CML in blast crisis. DisclosuresNo relevant conflicts of interest to declare.

Significance of BIOMED-2 standardized IG/TCR gene rearrangement detection in paraffin-embedded section in lymphoma diagnosis

To explore the feasibility of detecting lymphoma with the application of BIOMED-2 standardized immunoglobulin/T cell receptor (IG/TCR) gene rearrangement system in formalin fixed paraffin-embedded (FFPE) tissue samples, and to discuss the relationship between the longest amplification fragment of extracted DNA and positive detection rate of different IGH V-J primers. DNA was extracted from 50 cases of FFPE tissue samples. Multiplex-PCR amplifications were performed and then the IG/TCR gene rearrangements were analyzed using BIOMED-2 standardized clonality analysis system. (1)When the DNA concentration was diluted to 50-100 ng/μl from 100-500 ng/μl, the proportion of the longest amplification fragment (300-400 bp) of DNA was improved from 10.0% to 90.0% in 30 cases of diffuse large B cell lymphoma (DLBCL) wax roll samples (P<0.01). The positive rate of IGH+IGK was increased from 46.7% to 83.3%, the difference was statistically significant (P=0.006). The lengths of the longest amplification fragments of DNA were all longer than 300 bp in the paraffin section samples of DLBCL. The positive rate of IGH+IGK of these samples was 96.7%. The difference of the positive rate of IGH+IGK between the wax roll samples and the paraffin section samples had no statistical significance (P=0.195). (2)When the concentration of DNA was high, most of the longest amplification fragments of extracted DNA were 100 bp or 200 bp, and the detection rate of short fragment IGH FR3 was more stable than that of long fragment IGH FR1. (3)The clonality analysis of TCRG+TCRB in all 13 cases of peripheral T cell lymphoma samples showed positive results, while no positive IG/TCR clones were found in 7 cases of reactive lymphoid tissue hyperplasia in control group. Dilution of DNA is the only method to improve not only the proportion of longest fragment amplification but also the detection rate of clonality. The detection rate of IGH FR3 would not be affected by the concentration of DNA. The application of BIOMED-2 standardized IG/TCR gene rearrangement system in FFPE tissue samples plays an important role in the lymphoma diagnosis.

Advanced generation anti‐prostate specific membrane antigen designer T Cells for prostate cancer immunotherapy

Adoptive immunotherapy by infusion of designer T cells (dTc) engineered with chimeric antigen receptors (CARs) for tumoricidal activity represents a potentially highly specific modality for the treatment of cancer. In this study, 2nd generation (gen) anti-prostate specific membrane antigen (PSMA) dTc were developed for improving the efficacy of previously developed 1st gen dTc for prostate cancer immunotherapy. The 1st gen dTc are modified with chimeric immunoglobulin-T cell receptor (IgTCR) while the 2nd gen dTc are engineered with an immunoglobulin-CD28-T cell receptor (IgCD28TCR), which incorporates a CD28 costimulatory signal for optimal T cell activation. A 2nd gen anti-PSMA IgCD28TCR CAR was constructed by inserting the CD28 signal domain into the 1st gen CAR. 1st and 2nd gen anti-PSMA dTc were created by transducing human T cells with anti-PSMA CARs and their antitumor efficacy was compared for specific activation on PSMA-expressing tumor contact, cytotoxicity against PSMA-expressing tumor cells in vitro, and suppression of tumor growth in an animal model. The 2nd gen dTc can be optimally activated to secrete larger amounts of cytokines such as IL2 and IFNγ than 1st gen and to proliferate more vigorously on PSMA-expressing tumor contact. More importantly, the 2nd gen dTc preserve the PSMA-specific cytotoxicity in vitro and suppress tumor growth in animal models with significant higher potency. Our results demonstrate that 2nd gen anti-PSMA designer T cells exhibit superior antitumor functions versus 1st gen, providing a rationale for advancing this improved agent toward clinical application in prostate cancer immunotherapy.

T‐cell acute lymphoblastic leukaemia: recent molecular biology findings

For many years, T-cell acute lymphoblastic leukaemia (T-ALL) has been considered and treated as a single malignancy, but divergent outcomes in T-ALL patients receiving uniform treatment protocols encouraged intensive research on the molecular biology of this disease. Recent findings in the field demonstrate that T-ALL is much more heterogeneous than originally believed and extremely diverse outcomes of patients require refinement of T-ALL classification, leading to subtype-specific adjustment of treatment. Many different biological features of T-ALL blast cells have recently been found to contribute to disease development and patient outcome and their analysis could potentially be introduced into improved diagnostics and classification of the disease. This review focuses on five key issues of T-ALL biology: chromosome aberrations, gene expression profiles, gene mutations, DNA methylation patterns, and immunoglobulin/T cell receptor (Ig/TCR) gene rearrangements. Additionally, molecular monitoring of minimal residual disease, by far the most reliable independent prognostic factor in T-ALL, has been highlighted in the context of Ig/TCR gene rearrangements. Translation of this biological information into better prognostic classification and more effective treatment should lead to improvement of outcome in T-ALL patients.

The EuroClonality website: information, education and support on clonality testing

Clonality assessment is an established tool in the diagnosis of malignant lymphoma; however, the evaluation of immunoglobulin/T-cell receptor (Ig/TCR) gene rearrangement profiles is not always straightforward. Therefore, the EuroClonality group supports and advises diagnostic laboratories in their clonality assessments on samples from patients suspicious for lymphoma, who are seen in the routine diagnostic setting. The support for clonality assessment is provided via the EuroClonality website: http://www.euroclonality.org. The features and procedures of the website are presented in this paper.

Anti-GD3 Chimeric sFv-CD28/T-Cell Receptor ζ Designer T Cells for Treatment of Metastatic Melanoma and Other Neuroectodermal Tumors

The aims of this study are to compare antitumor activities of two generations of GD3-specific chimeric antigen receptors (CAR) in human primary T lymphocytes in vitro and to evaluate the antitumor efficacy of using a combination of systemic infusion of interleukin-2 (IL2) and designer T cells to eradicate subcutaneous established GD3+ melanoma in nude mice. Antitumor activities were compared for two generations of designer T cells, the progenitor first-generation with immunoglobulin T-cell receptor (TCR) with Signal 1 and the second-generation designer T cells with Signal 1+2. Osmotic IL2 pumps were used to deliver the maximum tolerated dose of IL2 to enhance the antitumor effects of designer T cells on subcutaneous established melanoma in nude mice. Melanoma is associated with high expression of ganglioside GD3, which has been targeted with modest effect in antibody therapies. We previously showed that an anti-GD3 CAR (sFv-TCRzeta) will recruit T cells to target this non-T-dependent antigen, with potent killing of melanoma cells. Here, we report the addition of a CD28 costimulation domain to create a second-generation CAR, called Tandem for two signals. We show that this Tandem sFv-CD28/TCRzeta receptor on T cells confers advantages of improved cytokine secretion, cytotoxicity, proliferation, and clonal expansion on tumor contact versus the same CAR without costimulation. In an adoptive transfer model using established melanoma tumors, designer T cells with CD28 showed a 50% rate of complete remissions but only where IL2 was supplemented. As a reagent for clinical development, the second-generation product is shown to have superior properties to warrant its preference for clinical designer T-cell immunotherapy for melanoma and other tumors. Systemic IL2 was required for optimal activity in an established tumor model.