Background: Multicenter clinical trials are producing growing amounts of clinical data. Machine Learning (ML) might facilitate the discovery of novel tools for prognostication and disease-stratification. Taking advantage of a systematic collection of multiple variables, we developed a model derived from data collected on 300 patients with mantle cell lymphoma (MCL) from the Fondazione Italiana Linfomi-MCL0208 phase III trial (NCT02354313). Methods: We developed a score with a clustering algorithm applied to clinical variables. The candidate score was correlated to overall survival (OS) and validated in two independent data series from the European MCL Network (NCT00209222, NCT00209209); Results: Three groups of patients were significantly discriminated: Low, Intermediate (Int), and High risk (High). Seven discriminants were identified by a feature reduction approach: albumin, Ki-67, lactate dehydrogenase, lymphocytes, platelets, bone marrow infiltration, and B-symptoms. Accordingly, patients in the Int and High groups had shorter OS rates than those in the Low and Int groups, respectively (Int→Low, HR: 3.1, 95% CI: 1.0–9.6; High→Int, HR: 2.3, 95% CI: 1.5–4.7). Based on the 7 markers, we defined the engineered MCL international prognostic index (eMIPI), which was validated and confirmed in two independent cohorts; Conclusions: We developed and validated a ML-based prognostic model for MCL. Even when currently limited to baseline predictors, our approach has high scalability potential.

Minimal residual disease (MRD) assessment is of high clinical relevance in patients with mantle cell lymphoma (MCL). In mature B-cell malignancies, the presence of somatic hypermutations (SHM) in Variable-Diversity-Joining Heavy chain (VDJH) rearrangements leads to frequent mismatches between primers, probes, and the target, thus impairing tumor cells quantification. Alternative targets, such as immunoglobulin kappa-deleting-element (IGK-Kde) rearrangements, might be suitable for MRD detection. We aimed at evaluating the applicability ofIGK-Kderearrangements for MRD quantification in MCL patients by real-time quantitative polymerase chain reaction (RQ-PCR)/digital-droplet-PCR (ddPCR).IGKscreening was performed on bone marrow samples from two cohorts: the first from Turin (22 patients enrolled in the FIL-MCL0208 trial, NCT02354313) and the second from Rome (15 patients).IGK-Kderearrangements were found in 76% (28/37) of cases, representing the sole molecular marker in 73% (8/11) ofIGH-BCL1/IGHnegative cases. MRD RQ-PCR monitoring was possible in 57% (16/28) of cases, showing a 100% concordance with the conventional targets. However, the frequent background amplification affected the sensitivity of the assay, that was lower in MCL compared to acute lymphoblastic leukemia and in line with multiple myeloma published results. ddPCR had a good concordance with RQ-PCR and it might help to identify false positive/negative results. From a clinical perspective, we suggest thatIGK-Kdecan be a candidate target for MRD monitoring and deserves a validation of its predictive value in prospective MCL series.

In recent years, the outcome of mantle cell lymphoma (MCL) has improved, especially in younger patients, receiving cytarabine-containing chemoimmunotherapy and autologous stem cell transplantation. Nevertheless, a proportion of MCL patients still experience early failure. To identify biomarkers anticipating failure of intensive chemotherapy in MCL, we performed target resequencing and DNA profiling of purified tumor samples collected from patients enrolled in the prospective FIL-MCL0208 phase 3 trial (high-dose chemoimmunotherapy followed by autologous transplantation and randomized lenalidomide maintenance). Mutations of KMT2D and disruption of TP53 by deletion or mutation associated with an increased risk of progression and death, both in univariate and multivariate analysis. By adding KMT2D mutations and TP53 disruption to the MIPI-c backbone, we derived a new prognostic index, the "MIPI-genetic" ("MIPI- g"). The "MIPI-g" improved the model discrimination ability compared to the MIPI-c alone, defining three risk groups: i) low-risk patients (4-year progression free survival and overall survival of 72.0% and 94.5%); ii) inter-mediate-risk patients (4-year progression free survival and overall survival of 42.2% and 65.8%) and iii) high-risk patients (4-year progression free survival and overall survival of 11.5% and 44.9%). Our results: i) confirm that TP53 disruption identifies a high-risk population characterized by poor sensitivity to conventional or intensified chemotherapy; ii) provide the pivotal evidence that patients harboring KMT2D mutations share the same poor outcome as patients harboring TP53 disruption; and iii) allow to develop a tool for the identification of high-risk MCL patients for whom novel therapeutic strategies need to be investigated. (Trial registered at clinicaltrials.gov identifier: NCT02354313).

In 2009, the four laboratories of the Fondazione Italiana Linfomi (FIL) minimal residual disease (MRD) Network started a collaborative effort to harmonize and standardize their methodologies at the national level, performing quality control (QC) rounds for follicular lymphoma (FL) and mantle cell lymphoma (MCL) MRD assessment. In 16 QC rounds between 2010 and 2017, the four laboratories received 208 bone marrow (BM) samples (126 FL; 82 MCL); 187 were analyzed, according to the EuroMRD Consortium guidelines, by both nested (NEST) polymerase chain reaction (PCR) and real-time quantitative (RQ) PCR for BCL2/IGH MBR or IGHV rearrangements. Here, we aimed at analyzing the samples that challenged the interlaboratory reproducibility and data interpretation. Overall, 156/187 BM samples (83%) were concordantly classified as NEST+/RQ+ or NEST-/RQ- by all the four laboratories. The remaining 31 samples (17%) resulted alternatively positive and negative in the interlaboratory evaluations, independently of the method and the type of rearrangement, and were defined "borderline" (brd) samples: 12 proved NEST brd/RQ brd, 7 NEST-/RQ brd, 10 NEST brd/RQ positive not quantifiable (PNQ), and 2 NEST brd/RQ-. Results did not change even increasing the number of replicates/sample. In 6/31 brd samples, droplet digital PCR (ddPCR) was tested and showed no interlaboratory discordance. Despite the high interlaboratory reproducibility in the MRD analysis obtained and maintained by the QC round strategy, samples with the lowest MRD levels can still represent a challenge: 17% (31/187) of our samples showed discordant results in interlaboratory assessments, with 6.4% (12/187) remained brd even applying the two methods. Thus, although representing a minority, brd samples are still problematic, especially when a clinically oriented interpretation of MRD results is required. Alternative, novel methods such as ddPCR and next-generation sequencing have the potential to overcome the current limitations.

PURPOSE Data collection in clinical trials is becoming complex, with a huge number of variables that need to be recorded, verified, and analyzed to effectively measure clinical outcomes. In this study, we used data warehouse (DW) concepts to achieve this goal. A DW was developed to accommodate data from a large clinical trial, including all the characteristics collected. We present the results related to baseline variables with the following objectives: developing a data quality (DQ) control strategy and improving outcome analysis according to the clinical trial primary end points. METHODS Data were retrieved from the electronic case reporting forms (eCRFs) of the phase III, multicenter MCL0208 trial (ClinicalTrials.gov identifier: NCT02354313) of the Fondazione Italiana Linfomi for younger patients with untreated mantle cell lymphoma (MCL). The DW was created with a relational database management system. Recommended DQ dimensions were observed to monitor the activity of each site to handle DQ management during patient follow-up. The DQ management was applied to clinically relevant parameters that predicted progression-free survival to assess its impact. RESULTS The DW encompassed 16 tables, which included 226 variables for 300 patients and 199,500 items of data. The tool allowed cross-comparison analysis and detected some incongruities in eCRFs, prompting queries to clinical centers. This had an impact on clinical end points, as the DQ control strategy was able to improve the prognostic stratification according to single parameters, such as tumor infiltration by flow cytometry, and even using established prognosticators, such as the MCL International Prognostic Index. CONCLUSION The DW is a powerful tool to organize results from large phase III clinical trials and to effectively improve DQ through the application of effective engineered tools.

Abstract Background and Aims. Minimal residual disease (MRD) detection by PCR-based methods is a relevant outcome predictor in MCL, however it is not clear which might represent the most effective methodology (nested vs real-time quantitative PCR, RQ-PCR), the most informative tissue source (bone marrow, BM, vs peripheral blood, PB), the best timing of analysis (midterm vs post-therapy) and the added value of performing multiple MRD determinations. To address these issues a systematic MRD detection program was performed in the Fondazione Italiana Linfomi (FIL) MCL0208 trial (NCT02354313), a prospective, randomized phase III trial comparing lenalidomide maintenance vs observation after an intensive citarabine containing chemo-immunotherapy (R-HDS) program followed by ASCT in 300 frontline MCL patients <66 years [Cortelazzo EHA2015]. Patients and methods. MRD was assessed with ASO primers on either IGH or BCL-1/IGH rearrangements by both nested and RQ-PCR in a Euro-MRD certified lab, both in PB and BM samples at the following time points (TP): diagnosis, after 3 R-CHOP-21 and R-high-dose cyclophosphamide (R-HD-CTX), after R-high-dose Ara-C (R-HDAC), after ASCT and every six months thereafter. Landmark analysis starting 12 months after consent using Cox models was performed based on MRD negativity at each TP. To evaluate the effect of MRD on PFS and OS, we considered also the whole follow-up (FU) period, including all available MRD evaluations as time-varying covariates, both in a dichotomous (pos vs. neg) and cumulative manner (0, 1, 2 or more consecutive MRD-negative results). Finally, the discrimination ability of MRD vs clinical evaluation after ASCT was assessed in the randomized population in terms of C index. All effects were estimated adjusting for MIPI score. Results. A total of 1476 BM and 1482 PB samples were collected, for a sampling compliance rate of 93%. 250 patients (83%) had a molecular marker and showed higher median baseline tumor infiltration by flow cytometry than no marker patients (BM 8.70% vs 0.35). 231/250 (92%) patients presented at least one FU sample and were thus studied for MRD by nested PCR, while 163/231 (71%) were studied also by RQ-PCR, according to the EuroMRD guidelines. Rates of MRD negativity in BM and PB by nested-PCR, as well as by RQ-PCR, were 29%, 46%, 36% and 49% after R-HD-CTX, 53%, 78%, 73% and 87% after R-HDAC, and 54%, 79%, 81% and 89% after ASCT, respectively. MRD positivity at every TP (either by nested or RQ-PCR, either in BM or PB) showed a two-fold higher risk of relapse or death during the six months following the sampling, independently of MIPI. Remarkably, similar two-fold HRs were recorded in terms of OS, too (Table 1A). In detail, RQ-PCR showed a higher risk increase than nested-PCR, as well as BM than PB. In the landmark analysis we found that the risk of relapse gradually increased, the more MRD negativity occurs later during therapy; actually, compared to patients with MRD response after R-HD-CTX, the HR was 1.24 for MRD responders after R-HDAC, 1.51 after ASCT and 2.04 for patients never achieving MRD response by RQ-PCR in BM (Table 1B). Therefore, 3y-PFS for patients MRD positive vs negative in BM by RQ-PCR was 53% vs 66% (HR=1.57, p=0.033) after R-HD-CTX, 47% vs 64% (HR=1.47, p=0.241) after R-HDAC and 25% vs 66% (HR=2.47, p=0.037) after ASCT. Overall, the PFS discrimination ability of MRD negativity after ASCT was better than the clinical response in terms of C-index (0.67 vs 0.62), according to Cox models including MIPI and randomization arm. Most importantly, the PFS risk seemed to follow a downward trend, according to the accumulation of MRD negative results, independently of the single TP. Actually, the presence of 2 or 3 consecutive MRD negative results conferred a significantly reduced risk of relapse, refining the risk stratification of a single MRD negativity (Table 1C). E.g., focusing on RQ-PCR in BM, the HR for relapse was 0.60 for a single negativity, 0.40 for 2 consecutive negative results and 0.27 for 3 or more. Conclusions. 1) MRD results are predictive both for PFS and OS in MCL; 2) RQ-PCR is the most reliable MRD technique and data derived from BM samples provide the best risk stratification; 3) MRD analysis performed at the TP post R-HD-CTX and post ASCT well describes patients' relapse risk, independently from MIPI, however: 4) a kinetic model, based on the combination of 2 or more MRD TP, provides a powerful risk stratification tool, suitable for MRD-guided treatment. Disclosures Vitolo: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Sandoz: Speakers Bureau; Gilead: Speakers Bureau; Takeda: Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Many biological markers at diagnosis have improved the prediction of outcome in chronic lymphocytic leukaemia (CLL) (Nabhan et al, 2015). Previous studies on telomere length (TL) have demonstrated that shorter telomeres at diagnosis are associated with poor outcome, along with unmutated IGHV status, high levels of CD38, CD49d and ZAP70 (Grabowski et al, 2005; Roos et al, 2008; Lin et al, 2014; Dos Santos et al, 2015). Moreover, our previous study, on a large CLL population (n = 401), showed that short TL (<5000 bp) at diagnosis is an independent predictor of shorter overall survival (OS), treatment-free survival (TFS) and progression to Richter syndrome (Rossi et al, 2009). Here we report TL dynamics over time and its predictive value in 90 CLL patients with a longer follow-up. Patients from the previous cohort that were willing to donate further samples were analysed. Diagnosis and treatment of CLL were managed according to institutional guidelines, based on the National Cancer Institute (NCI) Working Group (Hallek et al, 2008). Clinical and biological data were recorded (Table SI). All patients provided informed consent. TL was assessed at two time-points; telomere loss over time was calculated in terms of absolute loss, defined as the loss of telomeric DNA (in base pairs, bp) between the first and second determination of TL, and then adjusted for time, as Yearly Loss [YL = absolute loss (bp)/time (months)], in order to limit the bias of different timing in the second sample acquisition among patients. The second TL analysis was performed on peripheral blood mononuclear cells, recovered using density gradient stratification procedure (Ficoll-Hypaque, GE Healthcare, Buckinghanshire, UK), as previously described (Rossi et al, 2009). Genomic DNA was extracted using DNAzol (Invitrogen, Carlsbad, CA, USA). DNA yields and quality were measured by Nanodrop2000 (ThermoScientific, Waltham, MA, USA). TL was determined by Southern blot analysis. Primary end-point was TFS, defined as the time from the first TL evaluation to the time of first treatment. For univariate analyses, the TFS curve was estimated by the Kaplan– Meier method and compared using the log-rank test. TFS was then analysed by the Cox proportional hazards model comparing, by the Wald test, relevant risk factors (Table I). All reported P-values were two-sided, at the 5% significance level. Data were analysed as of June 2016 by R v.3.3.0 (R Foundation for Statistical Computing, Vienna, Austria). Ninety CLL patients from the original series were clinically monitored for a median time of 128 months (range, 21– 336 months). The first TL determination (baseline TL) was assessed at early stage of the disease, while the second, follow-up, determination (FU TL) was not uniform for all patients, with a median time between the two TL measures of 44 months (range: 11 5–231 months). At the time of the second determination, 26 patients had already relapsed after a first-line treatment, while 64 patients were still in Watch and Wait (WW). Among these, 33 progressed and required a treatment between FU TL and the last follow-up. Telomeres were shorter at FU TL compared to baseline TL (median YL 137 bp; range +174 to 1906 bp, P < 0 001). This was particularly evident in IGHV-mutated patients, as compared to unmutated (median YL 205 bp vs. +63 bp; P < 0 05). Patients with longer telomeres at baseline showed

Background: Minimal residual disease (MRD) monitoring by PCR methods is a strong and standardized predictor of clinical outcome in mantle cell (MCL) and follicular (FL) lymphoma [Pott C., Blood 2010; Ladetto M, Blood 2013]. However, some technical limitations hamper its feasibility in daily clinical routine. First of all, current techniques allow the identification of a molecular marker only in 75-80% of MCL and 55-60% of FL patients. All the other “no marker” cases cannot be studied for MRD purposes. Next generation sequencing (NGS) tools might overcome these limitations. Among them, the recently developed targeted locus amplification (TLA) technology [de Vree P.J., Nat Biotechnol 2014] that selectively amplifies and sequences entire genes on the basis of the crosslinking of physically proximal DNA loci, was able to identify novel candidate oncogenes and gene fusions in acute lymphoblastic leukemia. [Kuiper R.P.; Vroegindeweij EM, 57th ASH Communication, Blood 2015].

BACKGROUND:Mantle Cell Lymphoma (MCL) is a B cell aggressive neoplasia accounting for about the 6% of all lymphomas. The most common molecular marker of clonality in MCL, as in other B lymphoproliferative disorders, is the ImmunoGlobulin Heavy chain (IGH) rearrangement, occurring in B-lymphocytes. The patient-specific IGH rearrangement is extensively used to monitor the Minimal Residual Disease (MRD) after treatment through the standardized Allele-Specific Oligonucleotides Quantitative Polymerase Chain Reaction based technique. Recently, several studies have suggested that the IGH monitoring through deep sequencing techniques can produce not only comparable results to Polymerase Chain Reaction-based methods, but also might overcome the classical technique in terms of feasibility and sensitivity. However, no standard bioinformatics tool is available at the moment for data analysis in this context.RESULTS:In this paper we present HashClone, an easy-to-use and reliable bioinformatics tool that provides B-cells clonality assessment and MRD monitoring over time analyzing data from Next-Generation Sequencing (NGS) technique. The HashClone strategy-based is composed of three steps: the first and second steps implement an alignment-free prediction method that identifies a set of putative clones belonging to the repertoire of the patient under study. In the third step the IGH variable region, diversity region, and joining region identification is obtained by the alignment of rearrangements with respect to the international ImMunoGenetics information system database. Moreover, a provided graphical user interface for HashClone execution and clonality visualization over time facilitate the tool use and the results interpretation. The HashClone performance was tested on the NGS data derived from MCL patients to assess the major B-cell clone in the diagnostic samples and to monitor the MRD in the real and artificial follow up samples.CONCLUSIONS:Our experiments show that in all the experimental settings, HashClone was able to correctly detect the major B-cell clones and to precisely follow them in several samples showing better accuracy than the state-of-art tool.

Background and aims. Data collection in clinical trials is becoming complex, with huge amount of clinical and biological variables that need to be recorded, verified and analyzed to confirm obvious and identify unexpected associations. Outside the medical field, data warehouses (DWs) are widely employed to achieve these objectives. To verify whether DWs might be useful for data quality and association analysis, a team of clinicians, biologists, statisticians and biomedical engineers developed DELPHI (“Data ELaboration to Predict Hypothetical assocIations”). The tool has been suited to a large clinical trial from the Fondazione Italiana Linfomi (FIL), including all the clinical, biological and mutational features collected both in the trial and in the ancillary studies. Herein we present the first results, related to baseline variables: 1) creation of a broad DW (including 350 variables for 300 patients, thus > 105data); 2) data harmonization and quality control; 3) discovery of unexpected associations and (in a later phase) outcome correlations; 4) results visualization.

Background The combination of rituximab, bendamustine, and cytarabine (R-BAC) was highly active in a pilot trial of mantle cell lymphoma, but its use was restricted by high haematological toxicity. We aimed to assess the efficacy and safety of an R-BAC regimen with low-dose cytarabine (RBAC500).Methods In this multicentre, phase 2 trial, we recruited previously untreated patients with an established histological diagnosis of mantle cell lymphoma from 29 Fondazione Italiana Linfomi centres in Italy. Patients had to be older than 65 years and fit according to the comprehensive geriatric assessment, or aged 60-65 years if they were ineligible for high-dose chemotherapy plus autologous stem-cell transplantation and were fit or unfit. All patients received RBAC500 (rituximab 375 mg/m(2) on day 1, bendamustine 70 mg/m(2) on days 2 and 3, and cytarabine 500 mg/m(2) on days 2-4; all administered intravenously) every 4 weeks for up to six cycles. Primary endpoints were the proportion of patients achieving complete response at the end of treatment and toxicity, defined as the occurrence of any of the stop treatment criteria or of any episode of relevant toxicity. All patients who started at least one cycle of RBAC500 were included in the primary and safety analyses. Using efficacy and toxicity as a composite primary endpoint, we considered the final conclusion positive if more than 28 of 57 patients achieve complete response and fewer than 18 of 57 patients report toxicities. This study is registered with EudraCT, number 2011-005739-23, and ClinicalTrials.gov, number NCT01662050, and is completed.Findings Between May 2, 2012, and Feb 25, 2014, we enrolled 57 patients (median age 71 years, IQR 67-75). 54 (95%) patients received at least four RBAC500 cycles (three discontinued because of toxicity), and 38 (67%) completed six cycles. Two (4%) had disease progression (one after the fourth cycle and one after the sixth cycle). All 52 (91%, lower limit of one-sided 95% CI 85%) remaining patients achieved complete response at the end of treatment. 23 (40%, upper limit of one-sided 95% CI 53%) of 57 patients had at least one episode of relevant toxicity. The most frequent grade 3-4 haematological toxicities were neutropenia (149 [49%] of 304 cycles) and thrombocytopenia (158 [52%]). Most treatment-related non-haematological adverse events were of grade 1-2, with the most frequent ones being fatigue (14 [25%] patients), nausea or vomiting (12 [21%]), and infusion-related reactions or tumour lysis syndrome (12 [21%]). 41 (72%) patients required a dose reduction. 12 patients died during the study, but no deaths were related to treatment.Interpretation RBAC500 is an effective treatment for elderly patients with mantle cell lymphoma and, despite not meeting our prespecified safety boundary, haematological toxicity was manageable with appropriate supportive care and dose reduction. Since maintenance therapy is not required, RBAC500 could be considered an option and should be studied in phase 3 trials.

Abstract Background: The combination of rituximab (R, 375 mg/m2 intravenously [IV], day 1), bendamustine (B, 70 mg/m2IV, days 2 and 3), and cytarabine (800 mg/m2, IV on days 2 to 4) was highly active in patients with mantle-cell lymphoma (MCL) in a phase 2 study [R-BAC; Visco et al, JCO 2013]. This regimen was well tolerated, but hematologic toxicity was quite relevant, especially in terms of transient grade 3 to 4 thrombocytopenia (76% of cycles). Aiming at reducing hematologic toxicity, the Fondazione Italiana Linfomi (FIL) designed a phase 2 trial adopting the R-BAC schedule, but lowering cytarabine dose to 500 mg/m2 (RBAC500). Materials and Methods: Patients with newly diagnosed MCL, aged 61 to 80 years, not eligible for autologous transplant and fit according to the comprehensive geriatric assessment, were enrolled. Patients presenting with non-nodal leukemic disease were excluded. The primary endpoints were complete remission rate (CR) measured by FDG-PET according to Cheson criteria 2007, and safety. Secondary endpoints included rate of molecular response (MR) by nested-PCR using patient specific IGH or BCL1 based targets, progression-free (PFS) and overall survival (OS). The study was conducted according to the Bryant and Day two-stage design. Results: From May 2012 to February 2014, 57 patients with MCL from 29 centers were recruited and treated. Central pathology revision was performed in 87% of cases. Median age was 71 years (range 61-79), 75% were males, and 91% had Ann Arbor stage III/IV disease. Mantle Cell International Prognostic Index (MIPI) was low in 15%, intermediate in 40%, high in 45%, Ki-67 was ≥30% in 31%, and 9% had the blastoid cytological variant. Overall, 53 patients (91%) received at least 4 cycles, while 36 (63%) had 6 cycles (median 5.3 cycles per patient). Fifteen patients (26%) discontinued treatment before reaching cycle 6 because of toxicity/adverse events, that mainly consisted of prolonged hemato-toxicity between cycles. Only one patient discontinued due to progressive disease. Grade 3 or 4 neutropenia and thrombocytopenia were observed in 49% and 52% of administered cycles, respectively. Febrile neutropenia occurred in 6% of cycles. Extra-hematologic toxicity was mainly cardiac (5%). Overall response rate was 96%, and CR was 93%. The MR rate at the end of treatment was 76% on peripheral blood and 55% on bone marrow (BM) samples. With a median follow-up of 34 months (28-52), the 2-years PFS (± confidence interval) was 81%±5% and the OS 85%±4%. Elevated Ki-67 (≥30%), and the blastoid variant were the strongest independent predictors of adverse PFS. Patients with either of these two features (33%), had a significantly inferior PFS (41% vs 97% after 34 months) compared to patients with classical/pleomorphic variants and low proliferative index (p<0.0001, Figure 1). Conclusions: The R-BAC500 regimen can be safely administered as first line therapy to elderly patients with MCL. Hematologic toxicity is substantially reduced compared to our previous experience. With 93% of FDG-PET negative CR, and a 2-years PFS of 81% without maintenance therapy, the R-BAC500 regimen is a highly effective treatment for patients with MCL, and compares favourably with previously reported regimens in this patient population, including R-bendamustine. Figure 1 Figure 1. Disclosures Visco: Gilead: Speakers Bureau; Lundbeck: Consultancy; Mundipharma: Research Funding; Celgene: Speakers Bureau. Spina:Mundipharma: Membership on an entity's Board of Directors or advisory committees, Other: Speaker Fee; Teva Pharmaceuticals Industries: Membership on an entity's Board of Directors or advisory committees, Other: Speaker Fee. Di Rocco:Celgene: Honoraria. Carella:Millenium: Speakers Bureau; Genentech: Speakers Bureau. Vitolo:Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria; Gilead: Honoraria; Celgene: Honoraria.

Real-time quantitative PCR (qPCR) is a well-established tool for minimal residual disease (MRD) detection in mature lymphoid malignancies. Despite remarkable sensitivity and specificity, qPCR has some limitations, particularly in the need for a reference standard curve, based on target serial dilutions. In this study, we established droplet digital PCR (ddPCR) for MRD monitoring in multiple myeloma, mantle cell lymphoma, and follicular lymphoma and compared it head-to-head with qPCR. We observed that ddPCR has sensitivity, accuracy, and reproducibility comparable with qPCR. We then compared the two approaches in 69 patients with a documented molecular marker at diagnosis (18 multiple myelomas, 21 mantle cell lymphomas assessed with the immunoglobutin gene rearrangement, and 30 follicular lymphomas with the use of the BCL2/immunoglobulin gene major breakpoint region rearrangement). ddPCR was successful in 100% of cases, whereas qPCR failed to provide a reliable standard curve in three patients. Overall, 222 of 225 samples were evaluable by both methods. The comparison highlighted a good concordance (r = 0.94, P < 0.0001) with 189 of 222 samples (85.1%; 95% CI, 80.4 %-89.8%) being fully concordant. We found that ddPCR is a reliable tool for MRD detection with greater applicability and reduced labor intensiveness than qPCR. It wilt be necessary to authorize ddPCR as an outcome predictor tool in controlled clinical settings and multilaboratory standardization programs.

Background. Recently, the somatic MYD88L265P mutation has been found as the hallmark of Waldenström Macroglobulinemia (WM), being detectable in nearly 90% of cases, as well as in up to 50% of IgM MGUS, rarely in other non-Hodgkin lymphomas and never in multiple myeloma (MM). Beyond its potential diagnostic role, this mutation has been associated with tumor growth and therapy resistance. Moreover, MYD88L265P might represent an ideal marker for minimal residual disease (MRD) monitoring in a disease whose therapeutic scenario has been rapidly changing, with many new available and highly effective drugs (nucleoside analogues, proteasome and BTK-inhibitors). However, the current MYD88L265P allele-specific quantitative PCR (ASqPCR) diagnostic tool lacks sensitivity (1.00E-03) and thus is not suitable for MRD. Moreover, is not useful to test peripheral blood (PB), that harbors low concentrations of circulating tumor cells (especially after immunochemotherapy), neither to assess cell-free DNA (cfDNA), usually present at very low amount in plasma. Therefore, our study aims: 1) to assess whether a highly sensitive tool as droplet digital PCR (ddPCR) might be helpful in MYD88L265P screening; 2) to evaluate whether MYD88L265P might be a suitable marker for MRD monitoring in WM.

Background. Recent studies have described the landscape of recurrently mutated genes in mantle cell lymphoma (MCL), including genes involved in DNA damage response/cell cycle (ATM, TP53, CCND1), epigenetic regulation (KMT2D also known as MLL2, WHSC1), and cell signaling (BIRC3, TRAF2, NOTCH1). However, with the exception of TP53 abnormalities, little is known about the clinical relevance of recurrent mutations in MCL. Thus, we performed deep sequencing analysis of a MCL gene panel in the prospective series of patients enrolled in the ongoing FIL-MCL0208 phase III trial (EudraCTNumber: 2009-012807-25).

Pre-emptive rituximab (pRTX) might represent an effective approach for patients with follicular (FL) and mantle cell lymphoma (MCL) experiencing molecular relapse (M-rel). However, available experience is still limited. We retrospectively collected FL and MCL cases that underwent pRTX with four weekly rituximab infusions (375 mg/m2) due to molecular persistence or M-rel. M-rel was assessed using nested polymerase chain reaction (PCR) and real-time quantitative PCR using the Bcl-1/IGH, Bcl-2/IGH or the immunoglobulin heavy chain rearrangement. Twenty-three occurrences of M-rel or persistence were treated in 18 patients (nine MCL and nine FL). The pRTX reinduced molecular remission (MR) in 17/23 cases (7/9 FL and 10/14 MCL). The median time to MR reinduction was 4.5 months (range 3–12), and the median duration of the first MR reinduction was 34 months (range 12–72). In five MCL cases, pRTX was used to treat subsequent M-rels, with success in four cases. No clinical relapses were seen within 2 years of successful reinduction of MR. Progression-free survival after pRTX was 64 % at a median follow-up of 6 years. pRTX was feasible and safe and effectively reinduced MR in FL and MCL patients (74 %). Prospective trials are needed to verify the clinical benefit of similar approaches.

Abstract Background In mature lymphoid disorders, minimal residual disease (MRD) detection based on real time quantitative PCR (RQ-PCR) of immunoglobulin heavy chain gene rearrangement (IgH) has a well-established role in prognostic assessment, particularly in Mantle cell Lymphoma (MCL) and Multiple Myeloma (MM). RQ-PCR has excellent sensitivity and specificity but has a major limitation in its relative quantification nature, as it requires a reference standard curve usually built with dilutions of diagnostic tumor DNA or on plasmids containing the target rearrangement. Droplet Digital PCR (DD-PCR), applying the principle of limiting dilution of DNA and single molecule detection allows a reliable absolute quantification of target. In this study we compared IgH-based MRD detection by RQ-PCR and DD-PCR, to assess whether DD-PCR could achieve the same performances of RQ-PCR in the absence of the limitation mentioned above. Methods Bone marrow (BM) and peripheral blood (PB) samples were collected from patients affected by MCL and MM in which RQ-PCR based MRD analysis was already performed in the context of prospective clinical trials. In all trials patients gave the informed consent for MRD determination. IgH-based MRD detection by RQ-PCR was carried out as previously described [Ladetto et al. BBMT 2000] and results were interpreted according to the Euro-MRD guidelines [van der Velden et al. Leukemia 2007]. DD-PCR was performed by the QX100 Droplet Digital PCR system (Bio-RAD Inc.) on 500 ng of genomic DNA combined with the same Allele Specific Oligonucleotides (ASO)-primers and TaqMan-probes used in the RQ-PCR. Droplets were generated by QX100 droplet generator. End-point PCR (40 cycles) was performed on a T100 Thermal cycler (Bio-RAD Inc). The PCR product was loaded in the QX100 droplet reader and analyzed by QuantaSoft 1.2 (Bio-Rad Inc). For data interpretation RQ-PCR and DD-PCR results were expressed as amount of target copies per 1E+05 cells. Comparability of MRD results by DD-PCR and RQ-PCR was assessed by means of bivariate correlations between methods analysis (R2.15.1 package irr). Discordances were classified as follows: a positive/negative discordance was defined as major when the positive result was >1E-04 and minor when ≤1E-04; a quantitative discordance was defined as the presence of two positive results with a quantitative discrepancy >1 log. Results Overall, 161 samples belonging to 35 patients (18 MCL and 17 MM), 66 MCL and 95 MM were analyzed. 35 samples were taken at diagnosis and 126 at follow-up. 118 were BM while 43 were PB. A significant correlation was found between DD-PCR and RQ-PCR (R2=0.89, p<0.0001) (fig). DD-PCR and RQ-PCR showed superimposable sensitivity (10-5). Specificity in terms of appearance of non-specific amplifications signals in no-template samples (tested for all patients) and reproducibility on 30 replicates (4 samples) were superimposable. 128 out of 161 samples were fully concordant (Choen's K=0.80). MRD detection was concordantly positive in 106/161 (65.8%) samples and concordantly negative in 22/161 samples (13.7%). Only 5/161 (3.1%) samples showed major qualitative discordance. 28/161 (17.4%) samples showed minor qualitative discordance (which might be related to Poisson's statistics). Quantitative discordances were observed in 5/161 (3.1%) of cases (positive non quantifiable (PNQ) cases were conventionally placed to a value intermediate between sensitivity and quantitative range). Interestingly, 17 samples negative by RQ-PCR were scored positive by DD-PCR (median 6 copies, range 2-74) while 16 samples positive by RQ-PCR (median 5 copies, range 2-44) were negative by DD-PCR. Conclusions Here we report for the first time the use of DD-PCR in the context of IgH-based MRD evaluation in lymphoproliferative disorders. DD-PCR is a feasible tool for IGH-based MRD monitoring in MCL and MM, reaching similar sensitivities compared to standardized RQ-PCR. Moreover DD-PCR allows bypassing the need of building a standard curve thus considerably reducing the complexity of IgH-based RQ-PCR (need of purified diagnostic tissue or Flow Cytometry-based quantification of tumor load or diagnosis, or building of a plasmid-derived standard curve). Finally DD-PCR might potentially overcome the problem of positive non-quantifiable samples. These features make DD-PCR a feasible and attractive alternative method for IgH-based MRD assessment. Disclosures: Kubiczkovà: GAP304/10/1395 : Research Funding; MUNI/11/InGA17/2012: Research Funding.

We compared two strategies for minimal residual disease evaluation of B-cell lymphoproliferative disorders characterized by a variable immunoglobulin heavy chain (IGH) genes mutation load. Twenty-five samples from chronic lymphocytic leukaemia (n=18) or mantle cell lymphoma (n=7) patients were analyzed. Based on IGH variable region genes, 22/25 samples carried >2% mutations, 20/25>5%. In the IGH joining region genes, 23/25 samples carried >2% mutations, 18/25>5%. Real-time quantitative polymerase chain reaction was performed on IGH genes using two strategies: method A utilizes two patient-specific primers, whereas method B employs one patient-specific and one germline primer, with different positions on the variable, diversity and joining regions. Twenty-three samples (92%) resulted evaluable using method A, only six (24%) by method B. Method B poor performance was specifically evident among mutated IGH variable/joining region cases, although no specific mutation load above, which the real-time quantitative polymerase chain reaction failed was found. The molecular strategies for minimal residual disease evaluation should be adapted to the B-cell receptor features of the disease investigated. Copyright (c) 2013 John Wiley & Sons, Ltd.