Search

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/)., (© 2022 by The American Society of Hematology.)

Pancreatic ductal adenocarcinoma (PDAC) is highly refractory to systemic treatment, including radiotherapy (RT) either as alone or in combination with chemotherapy. Magnetic resonance (MR)-guided RT is a novel treatment technique which conjugates the high MR imaging contrast resolution to the possibility of re-adapting treatment plan to daily anatomical variations. Magnetic field (MF) might exert a biological effect that could be exploited to enhance radiation effect. The aim of the present study was to lay the preclinical basis of the MF effect by exploring how it modifies the response to radiation in organoid cultures established from PDAC. The short-term effect of radiation, alone or in combination with MF, was evaluated in patient-derived organoids (PDOs) and monolayer cell cultures. Cell viability, apoptotic cell death, and organoid size following exposure to the treatment were evaluated. PDOs demonstrated limited sensitivity at clinically relevant doses of radiation. The combination of radiation and MF demonstrated superior efficacy than monotherapy in almost all the PDOs tested. PDOs treated with combination of radiation and MF were significantly smaller in size and some showed increased cell death as compared to the monotherapy with radiation. Long-time exposure to 1.5T MF can increase the therapeutic efficacy of radiation in PDAC organoids.

Background: The assessment of organ motion is a crucial feature for prostate stereotactic body radiotherapy (SBRT). Rectal spacer may represent a helpful device in order to outdistance rectal wall from clinical target, but its impact on organ motion is still a matter of debate. MRI-Linac is a new frontier in radiation oncology as it allows a superior visualization of the real-time anatomy of the patient and the current highest level of adaptive radiotherapy., Methods: We present data regarding a total of 100 fractions in 20 patients who underwent MRI-guided prostate SBRT for low-to-intermediate risk prostate cancer with or without spacer. Translational and rotational shifts were computed on the pre- and post-treatment MRI acquisitions referring to the delivery position for antero-posterior, latero-lateral and cranio-caudal direction, and assessed using the Mann-Whitney U-Test., Results: All patients were treated with a five sessions schedule (35 Gy/5fx) using MRI-Linac for a median fraction treatment time of 50 min (range, 46-65). In the entire study sample, median rotational displacement was 0.1° in cranio-caudal, - 0.002° in latero-lateral and 0.01° in antero-posterior direction; median translational shift was 0.11 mm in cranio-caudal, - 0.24 mm in latero-lateral and - 0.22 mm in antero-posterior. A significant difference between spacer and no-spacer patients in terms of rotational shifts in the antero-posterior direction (p = 0.033) was observed; also for translational shifts a positive trend was detected in antero-posterior direction (p = 0.07), although with no statistical significance. We observed statistically significant differences in the pre-treatment planning phase in favor of the spacer cohort for several rectum dose constraints: rectum V32Gy < 5% (p = 0.001), V28 Gy < 10% (p = 0.001) and V18Gy < 35% (p = 0.039). Also for bladder V35 Gy < 1 cc, the use of spacer provided a dosimetric advantage compared to the no-spacer subpopulation (p = 0.04). Furthermore, PTV V33.2Gy > 95% was higher in the spacer cohort compared to the no-spacer one (p = 0.036)., Conclusion: In our experience, the application of rectal hydrogel spacer for prostate SBRT resulted in a significant impact on rotational antero-posterior shifts contributing to limit prostate intra-fraction motion. Further studies with larger sample size and longer follow-up are required to confirm this ideally favorable effect and to assess any potential impact on clinical outcomes.

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., (Copyright © 2020 the Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the European Hematology Association.)

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)., (Copyright© 2019 Ferrata Storti Foundation.)

We here describe a novel method for MYD88 L265P 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 MYD88 L265P To investigate whether MYD88 L265P 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 (r 2 =0.64). Finally, MYD88 L265P 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 MYD88 L265P 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 MYD88 L265P detection., (Copyright © 2018 Ferrata Storti Foundation.)

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., (Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Purpose: The role of the minimal residual disease (MRD) in follicular lymphoma is still debated. In this study, we assessed whether the BCL2/IGH rearrangement could have a prognostic role in patients receiving R-CHOP, R-FM, or R-CVP., Experimental Design: DNAs from 415 patients among the 504 cases enrolled in the FOLL05 trial (NCT00774826) were centralized and assessed for the BCL2/IGH at diagnosis, at the end of treatment, and after 12 and 24 months., Results: At diagnosis, the molecular marker was detected in 53% of cases. Patients without molecular marker or with a low molecular tumor burden (<1 × 10(-4) copies) showed higher complete remission (CR) rate and longer progression-free survival (PFS; 3-year PFS 80% vs. 59%; P = 0.015). PFS was significantly conditioned by the PCR status at 12 and 24 months, with 3-year PFS of 66% for MRD(-) cases versus 41% for those MRD(+) at 12 months (P = 0.015), and 84% versus 50% at 24 months (P = 0.014). The MRD negativity at 12 and 24 months resulted in an improved PFS both in CR and in partial remission (PR) patients (3-year PFS = 72% for cases CR/PCR(-) vs. 32% for those CR/PCR(+) vs. 62% for those PR/PCR(-) and 25% for patients in PR/PCR(+); P = 0.001). The prognostic value of MRD at 12 and 24 months of follow-up was confirmed also in multivariate analysis., Conclusions: In this study, standardized molecular techniques have been adopted and applied on bone marrow samples from a large cohort. Data reported show that the MRD detection is a powerful independent predictor of PFS in patients with follicular lymphoma receiving conventional chemoimmunotherapy., (©2014 American Association for Cancer Research.)

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.

The identification of patients at high risk of relapse is a critical goal of modern translational research in oncohematology. Minimal residual disease (MRD) detection by polymerase chain reaction-based methods is routinely employed in the management of patients with acute lymphoblastic leukemia. Current knowledge indicates that it is also a useful prognostic tool in several mature lymphoproliferative disorders and particularly in follicular lymphoma (FL). Based on this evidence clinical trials employing MRD-based risk stratification are currently ongoing in FL. In this review the 'state of the art' of MRD evaluation in FL is discussed. A short description of technical issues and recent methodological advances is provided. Then, the bulk of the review focuses on critical take-home messages for clinicians working in the field. Finally, we discuss future perspectives of MRD detection and more generally outcome prediction in FL.

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.

Molecular remission (MR) is associated with improved outcome in mantle cell lymphoma (MCL). If MR is not achieved, patients are at high risk of relapse. We retrospectively describe the molecular and clinical follow-ups of 4 patients with molecular relapses (M-rels) who were treated with rituximab. The 4 patients received rituximab-supplemented, high-dose sequential chemotherapy and autologous stem cell transplantation as induction treatment and achieved clinical remission and MR. M-rel was defined as polymerase chain reaction (PCR) positivity in 2 consecutive samples in the absence of clinical relapse. M-rels occurred at 3, 6, 39, and 52 months and were always confirmed by direct sequencing of the clonal rearrangement. Minimal residual disease was monitored by qualitative and real-time quantitative PCR. All patients received 4 courses of rituximab, with 2 additional infusions if PCR positivity remained. After 4-6 courses of rituximab, all patients re-entered MR. No clinical relapses were recorded at 3, 6, 18, and 62 months from treatment, although 1 patient had a second M-rel that was sensitive to rituximab. Our results indicate that rituximab is active against residual MCL cells and suggest that molecularly tailored maintenance therapy needs to be investigated in clinical trials.

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.

Cyclooxygenase 2 (COX-2) is an inflammation-associated enzyme involved in the pathogenesis of many solid tumors, but little is known about its presence and role in hematologic neoplasms. Multiple myeloma (MM) is known to involve a deregulated cytokine network with secretion of inflammatory mediators. We thus decided to investigate the involvement of COX-2 in this neoplasm. Western blotting (WB) was used to evaluate 142 bone marrow (BM) specimens, including MM and monoclonal gammopathy of undetermined significance (MGUS). Selected cases under-went further evaluation by WB on purified CD138(+) cells, immunohistochemistry (IC), and real-time polymerase chain reaction (PCR) for mRNA expression. COX-2 was expressed in 11% (2 of 18) of MGUS specimens, 31% (29 of 94) of MM at diagnosis, and 47% (14 of 30) of MM with relapsed/refractory disease. COX-2 positivity was associated with a poor outcome in terms of progression-free (18 vs 36 months; P < .001) and overall survival (28 vs 52 months; P < .05). Real-time PCR showed COX-2 mRNA overexpression. IC and cell separation studies demonstrated COX-2 expression to be restricted to malignant plasma cells. This is the first report of the presence and prognostic role of COX-2 expression in MM. Future studies will assess COX-2 involvement in other hematologic tumors and its potential use as a therapeutic or chemo-preventive target in onco-hematology.

In this study we investigated telomere restriction fragment (TRF) length in a panel of mature B-cell lymphoproliferative disorders (MBCLDs) and correlated this parameter with histology and histopathogenesis in relation to the germinal center (GC). We assessed 123 MBCLD samples containing 80% or more tumor cells. TRF length was evaluated by Southern blot analysis using a chemiluminescence-based assay. GC status was assessed through screening for stable and ongoing somatic mutations within the immunoglobulin heavy-chain genes. Median TRF length was 6170 bp (range, 1896-11 200 bp) and did not correlate with patient age or sex. TRF length was greater in diffuse large cell lymphoma, Burkitt lymphoma, and follicular lymphoma (medians: 7789 bp, 9471 bp, and 7383 bp, respectively) than in mantle cell lymphoma and chronic lymphocytic leukemia (medians: 3582 bp and 4346 bp, respectively). GC-derived MBCLDs had the longest telomeres, whereas those arising from GC-inexperienced cells had the shortest (P < 10(-9)). We conclude that (1) TRF length in MBCLD is highly heterogeneous; (2) GC-derived tumors have long telomeres, suggesting that minimal telomere erosion occurs during GC-derived lymphomagenesis; and (3) the short TRF lengths of GC-inexperienced MBCLDs indicates that these neoplasms are good candidates for treatment with telomerase inhibitors, a class of molecules currently the subject of extensive preclinical evaluation.

Twenty-six Candida dubliniensis and 27 Candida albicans oral strains isolated from patients infected by the human immunodeficiency virus (HIV) were tested for germ tube production and 21 extracellular enzymatic activities. Assessment of the enzymatic profile was performed by using the API-ZYM commercial kit system (bioMerieux, France), which tests 19 different enzymes. Protease activity was expressed during the first days of incubation by 100% of the strains studied and resulted higher than phospholipase activity in the C. dubliniensis and C. albicans strains tested. The API-ZYM profile of the C. dubliniensis and C. albicans strains differs with respect to the number and percentage of the enzymes considered, as well as with the intensity of the substrate metabolized by the strains, in particular for the enzymes n 8 (cystine-arylamidase), n 12 (naphtol-AS-BI-phosphohydrolase) and n 16 (alpha-glucosidase). These enzymes may be useful to differentiate C. dubliniensis and C. albicans together with other phenotypic characteristics proposed in the literature. No relationship among protease, phospholipase and other extracellular enzymatic activities was observed in C. dubliniensis. The average percentage of strains filamentation after 4 h was between 32 and 42%.

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.

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.