Ibrutinib combined with immunochemotherapy with or without autologous stem-cell transplantation versus immunochemotherapy and autologous stem-cell transplantation in previously untreated patients with mantle cell lymphoma (TRIANGLE): a three-arm, randomised, open-label, phase 3 superiority trial of the European Mantle Cell Lymphoma Network.

BACKGROUND: Adding ibrutinib to standard immunochemotherapy might improve outcomes and challenge autologous stem-cell transplantation (ASCT) in younger (aged 65 years or younger) mantle cell lymphoma patients. This trial aimed to investigate whether the addition of ibrutinib results in a superior clinical outcome compared with the pre-trial immunochemotherapy standard with ASCT or an ibrutinib-containing treatment without ASCT. We also investigated whether standard treatment with ASCT is superior to a treatment adding ibrutinib but without ASCT. METHODS: The open-label, randomised, three-arm, parallel-group, superiority TRIANGLE trial was performed in 165 secondary or tertiary clinical centres in 13 European countries and Israel. Patients with previously untreated, stage II-IV mantle cell lymphoma, aged 18-65 years and suitable for ASCT were randomly assigned 1:1:1 to control group A or experimental groups A+I or I, stratified by study group and mantle cell lymphoma international prognostic index risk groups. Treatment in group A consisted of six alternating cycles of R-CHOP (intravenous rituximab 375 mg/m2 on day 0 or 1, intravenous cyclophosphamide 750 mg/m2 on day 1, intravenous doxorubicin 50 mg/m2 on day 1, intravenous vincristine 1·4 mg/m2 on day 1, and oral prednisone 100 mg on days 1-5) and R-DHAP (or R-DHAOx, intravenous rituximab 375 mg/m2 on day 0 or 1, intravenous or oral dexamethasone 40 mg on days 1-4, intravenous cytarabine 2 × 2 g/m2 for 3 h every 12 h on day 2, and intravenous cisplatin 100 mg/m2 over 24 h on day 1 or alternatively intravenous oxaliplatin 130 mg/m2 on day 1) followed by ASCT. In group A+I, ibrutinib (560 mg orally each day) was added on days 1-19 of R-CHOP cycles and as fixed-duration maintenance (560 mg orally each day for 2 years) after ASCT. In group I, ibrutinib was given the same way as in group A+I, but ASCT was omitted. Three pairwise one-sided log-rank tests for the primary outcome of failure-free survival were statistically monitored. The primary analysis was done by intention-to-treat. Adverse events were evaluated by treatment period among patients who started the respective treatment. This ongoing trial is registered with ClinicalTrials.gov, NCT02858258. FINDINGS: Between July 29, 2016 and Dec 28, 2020, 870 patients (662 men, 208 women) were randomly assigned to group A (n=288), group A+I (n=292), and group I (n=290). After 31 months median follow-up, group A+I was superior to group A with 3-year failure-free survival of 88% (95% CI 84-92) versus 72% (67-79; hazard ratio 0·52 [one-sided 98·3% CI 0-0·86]; one-sided p=0·0008). Superiority of group A over group I was not shown with 3-year failure-free survival 72% (67-79) versus 86% (82-91; hazard ratio 1·77 [one-sided 98·3% CI 0-3·76]; one-sided p=0·9979). The comparison of group A+I versus group I is ongoing. There were no relevant differences in grade 3-5 adverse events during induction or ASCT between patients treated with R-CHOP/R-DHAP or ibrutinib combined with R-CHOP/R-DHAP. During maintenance or follow-up, substantially more grade 3-5 haematological adverse events and infections were reported after ASCT plus ibrutinib (group A+I; haematological: 114 [50%] of 231 patients; infections: 58 [25%] of 231; fatal infections: two [1%] of 231) compared with ibrutinib only (group I; haematological: 74 [28%] of 269; infections: 52 [19%] of 269; fatal infections: two [1%] of 269) or after ASCT (group A; haematological: 51 [21%] of 238; infections: 32 [13%] of 238; fatal infections: three [1%] of 238). INTERPRETATION: Adding ibrutinib to first-line treatment resulted in superior efficacy in younger mantle cell lymphoma patients with increased toxicity when given after ASCT. Adding ibrutinib during induction and as maintenance should be part of first-line treatment of younger mantle cell lymphoma patients. Whether ASCT adds to an ibrutinib-containing regimen is not yet determined. FUNDING: Janssen and Leukemia & Lymphoma Society.

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/).

Genetic and phenotypic attributes of splenic marginal zone lymphoma.

Splenic marginal zone B-cell lymphoma (SMZL) is a heterogeneous clinico-biological entity. The clinical course is variable, multiple genes are mutated with no unifying mechanism, and essential regulatory pathways and surrounding microenvironments are diverse. We sought to clarify the heterogeneity of SMZL by resolving different subgroups and their underlying genomic abnormalities, pathway signatures, and microenvironment compositions to uncover biomarkers and therapeutic vulnerabilities. We studied 303 SMZL spleen samples collected through the IELSG46 multicenter international study (NCT02945319) by using a multiplatform approach. We carried out genetic and phenotypic analyses, defined self-organized signatures, validated the findings in independent primary tumor metadata and in genetically modified mouse models, and determined correlations with outcome data. We identified 2 prominent genetic clusters in SMZL, termed NNK (58% of cases, harboring NF-κB, NOTCH, and KLF2 modules) and DMT (32% of cases, with DNA-damage response, MAPK, and TLR modules). Genetic aberrations in multiple genes as well as cytogenetic and immunogenetic features distinguished NNK- from DMT-SMZLs. These genetic clusters not only have distinct underpinning biology, as judged by differences in gene-expression signatures, but also different outcomes, with inferior survival in NNK-SMZLs. Digital cytometry and in situ profiling segregated 2 basic types of SMZL immune microenvironments termed immune-suppressive SMZL (50% of cases, associated with inflammatory cells and immune checkpoint activation) and immune-silent SMZL (50% of cases, associated with an immune-excluded phenotype) with distinct mutational and clinical connotations. In summary, we propose a nosology of SMZL that can implement its classification and also aid in the development of rationally targeted treatments.

End of induction [18F]FDG PET is prognostic for progression-free survival and overall survival in follicular lymphoma patients enrolled in the FOLL12 trial.

PURPOSE: To evaluate the reliability of the Deauville score (DS) in therapy response assessment and to define the prognostic value of the metabolic response of end of induction (EOI) [18F]FDG PET (PET) in follicular lymphoma patients. METHODS: Adult patients with untreated grade 1-3a FL/ stage II-IV enrolled in the multicentre, prospective, phase III FOLL12 trial (NCT02063685) were randomized to receive standard immunochemotherapy followed by rituximab maintenance (standard arm) versus standard immunochemotherapy followed by response-adapted post-induction management (experimental arm). Baseline and EOI PET were mandatory for the study. All PET scans were centralized on the WIDEN® platform and classified according to DS in a blind independent central review. DS1-3 was considered negative (CMR), whereas DS4-5 was considered positive (not CMR). The primary endpoint was PFS. The main secondary endpoint was overall survival (OS). RESULTS: Overall, 807 follicular lymphoma patients-52% women, 89% stage III-IV disease, 40% with a high-risk FLIPI-2 score (3-5)-were enrolled in the study; 729 (90.4%) baseline and EOI PET were available for the analysis. EOI PET was positive (DS4-5) in 88/729 (12.1%) cases. Overall inter-reviewer agreement on PET pos/neg result was 0.92, while agreement on positive and negative cases was 0.77 and 0.94, respectively. The median follow-up was 69 months; 247 events were registered in the 5-yr follow-up, with a 5-yr PFS of 67% (95%CI: 63%-70%). The 5-yr PFS rate for PET neg (DS1-3) and PET pos (DS4-5) patients was 71% (95%CI: 67%-75%) and 36% (95%CI: 25%-46%), respectively, with HR 3.49 (95%CI: 2.57-4.72). Five-year PFS was worse as DS increased, with 74% (70%-78%), 58% (48%-67%; HR 1.71; p = 0.001)] and 36% (25%-46%; HR 3.88; p < 0.001) in DS1-2, DS3 and DS4-5, respectively. EOI PET maintained its prognostic value in both the standard and experimental arms. In the whole population, 5-yr OS was 94% (95%CI: 92%-96%), with 96% (95%CI: 94-97) and 82% (95%CI: 72%-89%) in EOI PET negative (DS1-3) and positive (DS4-5), respectively (HR 4.48; p < 0.001). When DS was associated with FLIPI-2, patients with DS3 or DS1-2 with high FLIPI-2 (3-5) experienced worse OS than patients with DS1-2 and low FLIPI-2 (1-2) (p = 0.003). CONCLUSION: This study shows that DS is a reliable prognostic tool to evaluate EOI PET in follicular lymphoma patients, with prognostic value maintained both in the standard and experimental arms, making metabolic imaging a robust tool to assess response in FL. Moreover, although preliminary, this study provides further information on DS3 patients, who are considered as CMR but show a less favourable PFS than DS1-2 patients.

Prognostic impact of TP53 mutation in newly diagnosed diffuse large B-cell lymphoma patients treated in the FIL-DLCL04 trial.

The prognostic role of TP53 disruption has been established in diffuse large B-cell lymphoma (DLBCL). Aim of this analysis was to correlate TP53 mutations by Sanger sequencing, cell of origin (COO) profile by Lymph2Cx panel on the NanoString platform and MYC, BCL2 and BCL6 overexpression or re-arrangements by immunohistochemistry (IHC) and fluorescent in-situ hybridization (FISH), with outcome in DLBCL patients enrolled into the FIL-DLCL04 trial (NCT00499018). One hundred and twenty-five DLBCL patients with tumour block available were analyzed. TP53 was mutated in 11/125 (9%) cases; 60/125 patients received high-dose chemoimmunotherapy up-front, as for the randomization arm; COO was reported in 88 patients: 48 germinal centre B-cell like, 25 activated B-cell like and 17 unclassified; 26 patients were double expressors in IHC and 11 double hit in FISH. After a median follow-up of 72 months, five-year failure-free survival (FFS) for TP53 mutated versus wild-type was 24% and 72%, and five-year overall survival (OS) was 34% and 83%, respectively. Adjusted hazard ratio (HR) was 2·28 [95% confidence interval (CI) 0·89-5·86, p = 0·086] and 4·05 (95% CI 1·37-11·97, p = 0·011) for FFS and OS, respectively. In this series of young DLBCL patients, TP53 gene mutation identified a poor prognosis subgroup, regardless of treatment and other biological markers.

Application of the Euro Clonality next-generation sequencing-based marker screening approach to detect immunoglobulin heavy chain rearrangements in mantle cell lymphoma patients: first data from the Fondazione Italiana Linfomi MCL0208 trial.

Minimal residual disease (MRD) determined by classic polymerase chain reaction (PCR) methods is a powerful outcome predictor in mantle cell lymphoma (MCL). Nevertheless, some technical pitfalls can reduce the rate of of molecular markers. Therefore, we applied the EuroClonality-NGS IGH (next-generation sequencing immunoglobulin heavy chain) method (previously published in acute lymphoblastic leukaemia) to 20 MCL patients enrolled in an Italian phase III trial sponsored by Fondazione Italiana Linfomi. Results from this preliminary investigation show that EuroClonality-NGS IGH method is feasible in the MCL context, detecting a molecular IGH target in 19/20 investigated cases, allowing MRD monitoring also in those patients lacking a molecular marker for classical screening approaches.

A brief rituximab, bendamustine, mitoxantrone (R-BM) induction followed by rituximab consolidation in elderly patients with advanced follicular lymphoma: a phase II study by the Fondazione Italiana Linfomi (FIL).

Treatment for follicular lymphoma (FL) in the elderly is not well standardized. A phase II, multicentre, single arm trial was conducted in this setting with a brief chemoimmunotherapy regimen. Treatment consisted in four monthly courses of rituximab, bendamustine and mitoxantrone (R-BM) followed by 4 weekly rituximab as consolidation; rituximab maintenance was not applied because the drug was not licensed at the time of enrolment. The primary endpoint was the complete remission rate (CR). Seventy-six treatment-naive FL patients (aged 65-80 and a "FIT" score, according to the Comprehensive Geriatric Assessment) were enrolled. CR was documented in 59/76 patients (78%), partial remission in 12 (16%) and stable/progressive disease in five (6%) with an overall response rate in 71/76 (94%). Median follow-up was 44 months with 3-year progression-free-survival (PFS) and overall-survival of 67% and 92% respectively. Nine deaths occurred, three of progressive disease. The regimen was well tolerated and the most frequent severe toxicity was neutropenia (18% of the cycles). Bcl-2/IGH rearrangement was found in 40/75 (53%) of evaluated patients. R-BM was highly effective in clearing polymerase chain reaction-detectable disease: 29/31 (96%) evaluated patients converted to bcl-2/IGH negativity at the end of treatment. A brief R-BM regimen plus rituximab consolidation is effective and safe in "FIT" elderly, treatment-naïve, FL patients, inducing high CR and molecular remission rates with prolonged PFS.

COVID-19 elicits an impaired antibody response against SARS-CoV-2 in patients with haematological malignancies.

COVID-19 is associated with high mortality in patients with haematological malignancies (HM) and rate of seroconversion is unknown. The ITA-HEMA-COV project (NCT04352556) investigated patterns of seroconversion for SARS-CoV-2 IgG in patients with HMs. A total of 237 patients, SARS-CoV-2 PCR-positive with at least one SARS-CoV-2 IgG test performed during their care, entered the analysis. Among these, 62 (26·2%) had myeloid, 121 (51·1%) lymphoid and 54 (22·8%) plasma cell neoplasms. Overall, 69% of patients (164 of 237) had detectable IgG SARS-CoV-2 serum antibodies. Serologically negative patients (31%, 73 of 237) were evenly distributed across patients with myeloid, lymphoid and plasma cell neoplasms. In the multivariable logistic regression, chemoimmunotherapy [odds ratio (OR), 3·42; 95% confidence interval (CI), 1·04-11·21; P = 0·04] was associated with a lower rate of seroconversion. This effect did not decline after 180 days from treatment withdrawal (OR, 0·35; 95% CI: 0·11-1·13; P = 0·08). This study demonstrates a low rate of seroconversion in HM patients and indicates that treatment-mediated immune dysfunction is the main driver. As a consequence, we expect a low rate of seroconversion after vaccination and thus we suggest testing the efficacy of seroconversion in HM patients.

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.

A Phase III study of zanubrutinib plus rituximab versus bendamustine plus rituximab in transplant-ineligible, untreated mantle cell lymphoma.

Mantle cell lymphoma is an aggressive B-cell malignancy. Current frontline chemoimmunotherapies produce high response rates but relapse is inevitable. Furthermore, the elderly and those with comorbidities are precluded from standard regimens and stem cell transplant, leaving them with limited options. Targeted therapies, including Bruton tyrosine kinase inhibitors, are an effective treatment strategy in mantle cell lymphoma. Zanubrutinib is a potent next-generation Bruton tyrosine kinase inhibitor that has demonstrated complete and sustained Bruton tyrosine kinase occupancy, minimal off-target effects and favorable pharmacokinetic/pharmacodynamic properties. Described herein is an ongoing Phase III study comparing the efficacy and safety of zanubrutinib plus rituximab followed by zanubrutinib monotherapy versus bendamustine plus rituximab followed by observation in transplant-ineligible patients with previously untreated mantle cell lymphoma. Clinical Trial Registration: NCT04002297 (ClinicalTrials.gov).

KMT2D mutations and TP53 disruptions are poor prognostic biomarkers in mantle cell lymphoma receiving high-dose therapy: a FIL study.

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).

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.

STAble: a novel approach to de novo assembly of RNA-seq data and its application in a metabolic model network based metatranscriptomic workflow.

BACKGROUND: De novo assembly of RNA-seq data allows the study of transcriptome in absence of a reference genome either if data is obtained from a single organism or from a mixed sample as in metatranscriptomics studies. Given the high number of sequences obtained from NGS approaches, a critical step in any analysis workflow is the assembly of reads to reconstruct transcripts thus reducing the complexity of the analysis. Despite many available tools show a good sensitivity, there is a high percentage of false positives due to the high number of assemblies considered and it is likely that the high frequency of false positive is underestimated by currently used benchmarks. The reconstruction of not existing transcripts may false the biological interpretation of results as - for example - may overestimate the identification of "novel" transcripts. Moreover, benchmarks performed are usually based on RNA-seq data from annotated genomes and assembled transcripts are compared to annotations and genomes to identify putative good and wrong reconstructions, but these tests alone may lead to accept a particular type of false positive as true, as better described below. RESULTS: Here we present a novel methodology of de novo assembly, implemented in a software named STAble (Short-reads Transcriptome Assembler). The novel concept of this assembler is that the whole reads are used to determine possible alignments instead of using smaller k-mers, with the aim of reducing the number of chimeras produced. Furthermore, we applied a new set of benchmarks based on simulated data to better define the performance of assembly method and carefully identifying true reconstructions. STAble was also used to build a prototype workflow to analyse metatranscriptomics data in connection to a steady state metabolic modelling algorithm. This algorithm was used to produce high quality metabolic interpretations of small gene expression sets obtained from already published RNA-seq data that we assembled with STAble. CONCLUSIONS: The presented results, albeit preliminary, clearly suggest that with this approach is possible to identify informative reactions not directly revealed by raw transcriptomic data.

Outcome of a Salvage Third Autologous Stem Cell Transplantation in Multiple Myeloma.

To evaluate the outcomes of salvage third autologous stem cell transplantation (ASCT) in patients with relapsed multiple myeloma. We analyzed 570 patients who had undergone a third ASCT between 1997 and 2010 (European Society for Blood and Marrow Transplantation data), of whom 482 patients underwent tandem ASCT and a third ASCT at first relapse (AARA group) and 88 patients underwent an upfront ASCT with second and third transplantations after subsequent relapses (ARARA group). With a median follow-up after salvage third ASCT of 61 months in the AARA group and 48 months in the ARARA group, the day +100 nonrelapse mortality in the 2 groups was 4% and 7%, the incidence of second primary malignancy was 6% and 7%, the median progression-free survival was 13 and 8 months, and median overall survival (OS) was 33 and 15 months. In the AARA group, according to the relapse-free interval (RFI) from the second ASCT, the median OS after the third ASCT was 17 months if the RFI was <18 months, 37 months if the RFI was between 18 and 36 months, and 64 months if the RFI was ≥36 months (P < .001). In the ARARA group, the median OS after the third ASCT was 7 months if the RFI was <6 months, 13 months if the RFI was between 6 and 18 months, and 27 months if the RFI was ≥18 months (P < .001). In a multivariate analysis of the AARA group, the favorable prognostic factor was an RFI after second ASCT of ≥18 months. Progressive disease and a Karnofsky Performance Status score of <70 at third ASCT were unfavorable factors. A salvage third ASCT is of value for patients with relapsed myeloma, particularly for those with a long duration of response and chemosensitive disease at the time of transplantation.

Combinatorial targeting of nuclear export and translation of RNA inhibits aggressive B-cell lymphomas.

Aggressive double- and triple-hit (DH/TH) diffuse large B-cell lymphomas (DLBCLs) feature activation of Hsp90 stress pathways. Herein, we show that Hsp90 controls posttranscriptional dynamics of key messenger RNA (mRNA) species including those encoding BCL6, MYC, and BCL2. Using a proteomics approach, we found that Hsp90 binds to and maintains activity of eIF4E. eIF4E drives nuclear export and translation of BCL6, MYC, and BCL2 mRNA. eIF4E RNA-immunoprecipitation sequencing in DLBCL suggests that nuclear eIF4E controls an extended program that includes B-cell receptor signaling, cellular metabolism, and epigenetic regulation. Accordingly, eIF4E was required for survival of DLBCL including the most aggressive subtypes, DH/TH lymphomas. Indeed, eIF4E inhibition induces tumor regression in cell line and patient-derived tumorgrafts of TH-DLBCL, even in the presence of elevated Hsp90 activity. Targeting Hsp90 is typically limited by counterregulatory elevation of Hsp70B, which induces resistance to Hsp90 inhibitors. Surprisingly, we identify Hsp70 mRNA as an eIF4E target. In this way, eIF4E inhibition can overcome drug resistance to Hsp90 inhibitors. Accordingly, rational combinatorial inhibition of eIF4E and Hsp90 inhibitors resulted in cooperative antilymphoma activity in DH/TH DLBCL in vitro and in vivo.

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.