Punctual and kinetic MRD analysis from the Fondazione Italiana Linfomi MCL0208 phase 3 trial in mantle cell lymphoma.

Minimal residual disease (MRD) analysis is a known predictive tool in mantle cell lymphoma (MCL). We describe MRD results from the Fondazione Italiana Linfomi phase 3 MCL0208 prospective clinical trial assessing lenalidomide (LEN) maintenance vs observation after autologous stem cell transplantation (ASCT) in the first prospective comprehensive analysis of different techniques, molecular markers, and tissues (peripheral blood [PB] and bone marrow [BM]), taken at well-defined time points. Among the 300 patients enrolled, a molecular marker was identified in 250 (83%), allowing us to analyze 234 patients and 4351 analytical findings from 10 time points. ASCT induced high rates of molecular remission (91% in PB and 83% in BM, by quantitative real-time polymerase chain reaction [RQ-PCR]). Nevertheless, the number of patients with persistent clinical and molecular remission decreased over time in both arms (up to 30% after 36 months). MRD predicted early progression and long-term outcome, particularly from 6 months after ASCT (6-month time to progression [TTP] hazard ratio [HR], 3.83; P < .001). In single-timepoint analysis, BM outperformed PB, and RQ-PCR was more reliable, while nested PCR appeared applicable to a larger number of patients (234 vs 176). To improve MRD performance, we developed a time-varying kinetic model based on regularly updated MRD results and the MIPI (Mantle Cell Lymphoma International Prognostic Index), showing an area under the ROC (Receiver Operating Characteristic) curve (AUROC) of up to 0.87 using BM. Most notably, PB reached an AUROC of up to 0.81; with kinetic analysis, it was comparable to BM in performance. MRD is a powerful predictor over the entire natural history of MCL and is suitable for models with a continuous adaptation of patient risk. The study can be found in EudraCT N. 2009-012807-25 (https://eudract.ema.europa.eu/).

Targeted locus amplification to detect molecular markers in mantle cell and follicular lymphoma.

Minimal residual disease (MRD) monitoring by PCR methods is a strong and standardized predictor of clinical outcome in mantle cell lymphoma (MCL) and follicular lymphoma (FL). However, about 20% of MCL and 40% of FL patients lack a reliable molecular marker, being thus not eligible for MRD studies. Recently, targeted locus amplification (TLA), a next-generation sequencing (NGS) method based on the physical proximity of DNA sequences for target selection, identified novel gene rearrangements in leukemia. The aim of this study was to test TLA in MCL and FL diagnostic samples lacking a classical, PCR-detectable, t(11; 14) MTC (BCL1/IGH), or t(14; 18) major breakpoint region and minor cluster region (BCL2/IGH) rearrangements. Overall, TLA was performed on 20 MCL bone marrow (BM) or peripheral blood (PB) primary samples and on 20 FL BM, identifying a novel BCL1 or BCL2/IGH breakpoint in 16 MCL and 8 FL patients (80% and 40%, respectively). These new breakpoints (named BCL1-TLA and BCL2-TLA) were validated by ASO primers design and compared as MRD markers to classical IGH rearrangements in eight MCL: overall, MRD results by BCL1-TLA were superimposable (R Pearson = 0.76) to the standardized IGH-based approach. Moreover, MRD by BCL2-TLA reached good sensitivity levels also in FL and was predictive of a primary refractory case. In conclusion, this study offers the proof of principle that TLA is a promising and reliable NGS-based technology for the identification of novel molecular markers, suitable for further MRD analysis in previously not traceable MCL and FL patients.

Immunoglobulin kappa deleting element rearrangements are candidate targets for minimal residual disease evaluation in mantle cell lymphoma.

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 of IGK-Kde rearrangements for MRD quantification in MCL patients by real-time quantitative polymerase chain reaction (RQ-PCR)/digital-droplet-PCR (ddPCR). IGK screening 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-Kde rearrangements were found in 76% (28/37) of cases, representing the sole molecular marker in 73% (8/11) of IGH-BCL1/IGH negative 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 that IGK-Kde can be a candidate target for MRD monitoring and deserves a validation of its predictive value in prospective MCL series.

Prolonged survival in the absence of disease-recurrence in advanced-stage follicular lymphoma following chemo-immunotherapy: 13-year update of the prospective, multicenter randomized GITMO-IIL trial.

A prospective trial conducted in the period 2000-2005 showed no survival advantage for high-dose chemotherapy with rituximab and autograft (R-HDS) versus conventional chemotherapy with rituximab (CHOP-R) as first-line therapy in 134 high-risk follicular lymphoma patients aged <60 years. The study has been updated at the 13-year median follow up. As of February 2017, 88 (66%) patients were alive, with overall survival of 66.4% at 13 years, without a significant difference between R-HDS (64.5%) and CHOP-R (68.5%). To date, 46 patients have died, mainly because of disease progression (47.8% of all deaths), secondary malignancies (3 solid tumor, 9 myelodysplasia/acute leukemia; 26.1% of all deaths), and other toxicities (21.7% of all deaths). Complete remission was documented in 98 (73.1%) patients and associated with overall survival, with 13-year estimates of 77.0% and 36.8% for complete remission versus no-complete remission, respectively. Molecular remission was documented in 39 (65%) out of 60 evaluable patients and associated with improved survival. In multivariate analysis, complete remission achievement had the strongest effect on survival (P<0.001), along with younger age (P=0.002) and female sex (P=0.013). Overall, 50 patients (37.3%) survived with no disease recurrence (18 CHOP-R, 32 R-HDS). This follow up is the longest reported on follicular lymphoma treated upfront with rituximab-chemotherapy and demonstrates an unprecedented improvement in survival compared to the pre-rituximab era, regardless of the use of intensified or conventional treatment. Complete remission was the most important factor for prolonged survival and a high proportion of patients had prolonged survival in their first remission, raising the issue of curability in follicular lymphoma. (Registered at clinicaltrials.gov identifier: 00435955).

Minimal residual disease (MRD) in non-Hodgkin lymphomas: Interlaboratory reproducibility on marrow samples with very low levels of disease within the FIL (Fondazione Italiana Linfomi) MRD Network.

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.

Highly sensitive MYD88L265P mutation detection by droplet digital polymerase chain reaction in Waldenström macroglobulinemia.

We here describe a novel method for MYD88L265P mutation detection and minimal residual disease monitoring in Waldenström macroglobulinemia, by droplet digital polymerase chain reaction, in bone marrow and peripheral blood cells, as well as in circulating cell-free DNA. Our method shows a sensitivity of 5.00×10-5, which is far superior to the widely used allele-specific polymerase chain reaction (1.00×10-3). Overall, 291 unsorted samples from 148 patients (133 with Waldenström macroglobulinemia, 11 with IgG lymphoplasmacytic lymphoma and 4 with IgM monoclonal gammopathy of undetermined significance) were analyzed: 194 were baseline samples and 97 were followup samples. One hundred and twenty-two of 128 (95.3%) bone marrow and 47/66 (71.2%) baseline peripheral blood samples scored positive for MYD88L265P To investigate whether MYD88L265P detection by droplet digital polymerase chain reaction could be used for minimal residual disease monitoring, mutation levels were compared with IGH-based minimal residual disease analysis in 10 patients, and was found to be as informative as the classical, standardized, but not yet validated in Waldenström macroglobulinemia, IGH-based minimal residual disease assay (r2=0.64). Finally, MYD88L265P detection by droplet digital polymerase chain reaction on plasma circulating tumor DNA from 60 patients showed a good correlation with bone marrow findings (bone marrow median mutational value 1.92×10-2, plasma circulating tumor DNA value: 1.4×10-2, peripheral blood value: 1.03×10-3). This study indicates that droplet digital polymerase chain reaction assay of MYD88L265P is a feasible and sensitive tool for mutation screening and minimal residual disease monitoring in Waldenström macroglobulinemia. Both unsorted bone marrow and peripheral blood samples can be reliably tested, as can circulating tumor DNA, which represents an attractive, less invasive alternative to bone marrow for MYD88L265P detection.

Persistence of minimal residual disease in bone marrow predicts outcome in follicular lymphomas treated with a rituximab-intensive program.

We assessed the prognostic value of minimal residual disease (MRD) within the ML17638 phase 3 trial from the Fondazione Italiana Linfomi, investigating the role of rituximab maintenance in elderly follicular lymphoma (FL) patients after a brief first-line chemoimmunotherapy. MRD for the bcl-2/IgH translocation was determined on bone marrow cells in a centralized laboratory belonging to the Euro-MRD consortium, using qualitative and quantitative polymerase chain reactions (PCRs). Of 234 enrolled patients, 227 (97%) were screened at diagnosis. A molecular marker (MM) was found in 51%. Patients with an MM were monitored at 8 subsequent times. Of the 675 expected follow-up samples, 83% were analyzed. Conversion to PCR negativity predicted better progression-free survival (PFS) at all post-treatment times (eg, end of therapy: 3-year PFS, 72% vs 39%; P < .007). MRD was predictive in both maintenance (83% vs 60%; P < .007) and observation (71% vs 50%; P < .001) groups. PCR positivity at the end of induction was an independent adverse predictor (hazard ratio, 3.1; 95% confidence interval, 1.36-7.07). MRD is a powerful independent outcome predictor in FL patients who receive rituximab-intensive programs, suggesting a need to investigate its value for decision-making. This trial was registered at www.clinicaltrial.gov as #NCT01144364.

Comparison of two real-time quantitative polymerase chain reaction strategies for minimal residual disease evaluation in lymphoproliferative disorders: correlation between immunoglobulin gene mutation load and real-time quantitative polymerase chain reaction performance.

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.

Rituximab-based pre-emptive treatment of molecular relapse in follicular and mantle cell lymphoma.

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.

Multiple myeloma shows no intra-disease clustering of immunoglobulin heavy chain genes.

BACKGROUND: Characterization of the immunoglobulin gene repertoire has improved our understanding of the immunopathogenesis of lymphoid tumors. Early B-lymphocyte precursors of multiple myeloma are known to exist and might be susceptible to antigenic drive. DESIGN AND METHODS: To verify this hypothesis, we collected a database of 345 fully readable multiple myeloma immunoglobulin sequences. We characterized the immunoglobulin repertoire, analyzed the somatic hypermutation load, and investigated for stereotyped receptor clusters. RESULTS: Compared to the normal immunoglobulin repertoire, multiple myeloma displayed only modest differences involving only a few genes, showing that the myeloma immunoglobulin repertoire is the least skewed among mature B-cell tumors. Median somatic hypermutation load was 7.8%; median length of complementarity determining-region 3 was 15.5 amino acids. Clustering analysis showed the absence of myeloma specific clusters and no similarity with published chronic lymphocytic leukemia or lymphoma subsets. CONCLUSIONS: Analysis of multiple myeloma immunoglobulin repertoire does not support a pathogenetic role for antigen selection in this tumor.

Minimal residual disease detection in lymphoma and multiple myeloma: impact on therapeutic paradigms.

Early identification of patients at high risk of relapse is a major goal of current translational research in oncohematology. Minimal residual disease (MRD) detection by polymerase chain reaction-based methods is currently part of the routine clinical management of patients with acute lymphoblastic leukemia. However, the current knowledge indicates that it is also a useful prognostic tool in several mature lymphoproliferative disorders. Its utility is currently well established in follicular lymphoma, mantle cell lymphoma, and multiple myeloma. In some of these entities, clinical trials employing MRD as a decision-making tool are currently ongoing. In the present review, we will discuss the 'state of the art' of MRD evaluation in these three neoplasms with the ultimate aim of providing critical take-home messages for clinicians working in the field. Moreover, we will outline the role of MRD detection in the design of future clinical trials.

Droplet Digital PCR Quantification of Mantle Cell Lymphoma Follow-up Samples From Four Prospective Trials of the European MCL Network.

Minimal residual disease (MRD) has been increasingly investigated in mantle cell lymphoma (MCL), including for individual therapeutic stratification and pre-emptive treatment in clinical trials. Although patient/allele specific real-time quantitative polymerase chain reaction (qPCR) of IGH or BCL1-IGH clonal markers is the gold-standard method, its reliance on a standard curve for relative quantification limits quantification of low-level positivity within the 1E-4 to 1E-5 range; over half of positive MRD samples after treatment fall below the quantitative range (BQR) of the standard curve. Droplet digital PCR (ddPCR), in contrast, allows absolute quantification, including for samples with no baseline determination of tumor infiltration by multicolor flow cytometry (MFC), avoiding the need for a reference standard curve. Using updated, optimized, ddPCR criteria we compared it with qPCR in 416 MRD samples (and with MFC in 63), with over-representation (61%) of BQR results by qPCR, from a total of 166 patients from four prospective MCL clinical trials. ddPCR, qPCR and MFC gave comparable results in MRD samples with at least 0.01% (1E-4) positivity. ddPCR was preferable to qPCR since it provided more robust quantification at positivity between 1E-4 and 1E-5. Amongst 240 BQR samples with duplicate or triplicate analysis, 39% were positive by ddPCR, 49% negative and only 12% remained positive below quantifiable ddPCR limits. The prognostic relevance of ddPCR is currently under assessment in the context of prospective trials within the European MCL Network.

Cells carrying nonlymphoma-associated bcl-2/IgH rearrangements (NLABR) are phenotypically related to follicular lymphoma and can establish as long-term persisting clonal populations.

OBJECTIVE: Nonlymphoma-associated bcl-2/IgH rearrangements (NLABRs) are frequently amplified by PCR in blood of lymphoma-free subjects (LFS), but the temporal kinetics and phenotypic nature of NLABR-positive cells are unknown. To address these issues we prospectively monitored a panel of NLABR-positive LFS. METHODS: LFS have been studied by nested PCR, real-time PCR, and DNA sequencing. Cell selection studies were also performed to define the nature of NLABR-bearing clones. RESULTS: Of 125 donors, 16 (12.8%) were found to be bcl-2/IgH positive and were monitored at least every 6 months for a median time of 22 months (range 6-50). In half of the subjects the same NLABR detected initially was again reamplified at follow-up thrice or more. In 5, the same NLABR was constantly amplified in every follow-up sample. With a median follow-up of 22 months (range 9-50), no stable disappearance of a recurrent clone has been so far recorded. Real-time PCR indicated that persistent NLABR-positive clones are stable over time in the same subject. Cell separation studies indicate that NLABRs belong to CD19+, CD5-, CD23-, CD10+/- cells. CONCLUSIONS: Our results indicate that NLABR-positive clones are persistent populations phenotypically related to follicular lymphoma (FL). This suggests the existence of a FL-related clonal expansion of undetermined significance, which might be either a premalignant or a nonmalignant counterpart of FL.

Comparison of different DNA extraction methods from peripheral blood cells: advice from the Fondazione Italiana Linfomi Minimal Residual Disease Network.

Genomic DNA extraction is a primary component of genomic research and diagnostic routine analysis. Recently, the importance of this process has been highlighted by the necessity to standardize the diagnostic procedure. In this regard, the Minimal Residual Disease (MRD) Network of the Fondazione Italiana Linfomi (FIL MRD Network) has performed a comparative study of four different commercially available kits for DNA extraction, applying them on a panel of cellular pellets, with the aim of defining possible technical recommendations in order to harmonize and standardize diagnostic procedures in the clinical setting. Overall, all four kits usually allowed the recovery of a significant quantity of high-quality DNA (in most conditions), although specific indications could be addressed for cellular pellets of different sizes.

Real-time polymerase chain reaction of immunoglobulin rearrangements for quantitative evaluation of minimal residual disease in myeloma.

The evaluation of minimal residual disease (MRD) is critical in the evaluation of treatments aimed at maximal cytoreduction in multiple myeloma (MM). Qualitative evaluation of MRD now has a 10-yr-long history, but it remains a relatively sophisticated procedure. More recently, real-time quantitative approaches have also been developed. These approaches allow a very effective monitoring of disease but introduce additional complexity and costs to the procedure. This chapter describes how we currently perform real-time polymerase chain reaction (PCR) in MM. Compared to the first description of the assay in June 2000, significant improvements have been made. Although real-time PCR is the main focus of the chapter, most of the information suitable for a proper setup of a qualitative approach is also provided.

Recurrence of Bcl-2/IgH polymerase chain reaction positivity following a prolonged molecular remission can be unrelated to the original follicular lymphoma clone.

OBJECTIVE: The aim of this study was to evaluate whether reappearance of polymerase chain reaction (PCR) positivity for the Bcl-2/IgH translocation following a phase of molecular remission in autografted follicular lymphoma (FL) patients is always associated with reappearance of the original neoplastic clone. PATIENTS AND METHODS: The molecular follow-up of 119 autografted Bcl-2/IgH positive patients was evaluated by nested PCR. In case of molecular recurrence, direct sequencing of involved rearrangements has been performed both at diagnosis and at the time of recurrence. The two sequences then were compared in terms of breakpoints, N insertions, and JH usage. RESULTS: Seventy-five patients achieving molecular remission were identified in our patient sample (63%). Of these patients, eight (10.6%) experienced molecular recurrence. Direct sequencing of the Bcl-2/IgH translocation performed at diagnosis and recurrence showed identical rearrangements in six subjects and unrelated rearrangements in two. As opposed to most true molecular relapses, unrelated rearrangements always occurred several years after transplantation. To date, the two subjects carrying unrelated rearrangements show no signs of active lymphoproliferative disease. CONCLUSIONS: This report is the first evidence that Bcl-2/IgH rearrangements unrelated to the original tumor clone can lead to false-positive results during the molecular follow-up of autografted FL patients. Based on these results, we recommend confirmation by direct sequencing, at least for patients experiencing molecular relapse 2 or more years after the end of treatment. This will be particularly important for patients enrolled in clinical trials that schedule additional treatment in case of molecular evidence of persistent disease activity.

Minimal Residual Disease Detection by Droplet Digital PCR in Multiple Myeloma, Mantle Cell Lymphoma, and Follicular Lymphoma: A Comparison with Real-Time PCR.

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 immunoglobulin 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 will be necessary to authorize ddPCR as an outcome predictor tool in controlled clinical settings and multilaboratory standardization programs.

Adherence of Candida albicans and Candida dubliniensis to buccal and vaginal cells.

Twenty-seven Candida albicans strains and 26 Candida dubliniensis strains, isolated from HIV patients, were tested for their adherence to buccal and vaginal epithelial cells. Both species showed important levels of adhesion to buccal and vaginal epithelial cells, although C. albicans showed the highest levels of adhesion. These results suggest that both Candida species are well adapted, in terms of adhesion capability, to the oral and vaginal environment.