High rate of durable responses with undetectable minimal residual disease with front-line venetoclax and rituximab in young, fit patients with chronic lymphocytic leukemia and an adverse biological profile: results of the GIMEMA phase II LLC1518 - VERITAS study.

The GIMEMA phase II LLC1518 VERITAS trial investigated the efficacy and safety of front-line, fixed-duration venetoclax and rituximab (VenR) in combination in young (≤65 years), fit patients with chronic lymphocytic leukemia and unmutated IGHV and/or TP53 disruption. Treatment consisted of the venetoclax ramp-up, six monthly courses of the VenR combination, followed by six monthly courses of venetoclax as a single agent. A centralized assessment of minimal residual disease (MRD) was performed by allele-specific oligonucleotide polymerase chain reaction assay on the peripheral blood and bone marrow at the end of treatment (EOT) and during the follow-up. The primary endpoint was the complete remission rate at the EOT. Seventy-five patients were enrolled; the median age was 54 years (range, 38-65), 96% had unmutated IGHV, 12% had TP53 disruption, and 4% had mutated IGHV with TP53 disruption. The overall response rate at the EOT was 94.7%, with a complete remission rate of 76%. MRD was undetectable in the peripheral blood of 69.3% of patients and in the bone marrow of 58.7% of patients. The 12-month MRD-free survival in the 52 patients with undetectable MRD in the peripheral blood at the EOT was 73.1%. After a median follow-up of 20.8 months, no cases of disease progression were observed. Three patients had died, two due to COVID-19 and one due to tumor lysis syndrome. The first report of the VERITAS study shows that front-line VenR was associated with a high rate of complete remissions and durable response with undetectable MRD in young patients with chronic lymphocytic leukemia and unfavorable genetic characteristics. ClinicalTrials.gov identifier: NCT03455517.

Minimal Residual Disease Analysis by Monitoring Immunoglobulin and T-Cell Receptor Gene Rearrangements by Quantitative PCR and Droplet Digital PCR.

Analysis of immunoglobulin and T-cell receptor gene rearrangements by real-time quantitative polymerase chain reaction (RQ-PCR) is the gold standard for sensitive and accurate minimal residual disease (MRD) monitoring; it has been extensively standardized and guidelines have been developed within the EuroMRD consortium ( www.euromrd.org ). However, new generations of PCR-based methods are standing out as potential alternatives to RQ-PCR, such as digital PCR technology (dPCR), the third-generation implementation of conventional PCR, which has the potential to overcome some of the limitations of RQ-PCR such as allowing the absolute quantification of nucleic acid targets without the need for a calibration curve. During the last years, droplet digital PCR (ddPCR) technology has been compared to RQ-PCR in several hematologic malignancies showing its proficiency for MRD analysis. So far, no established guidelines for ddPCR MRD analysis and data interpretation have been defined and its potential is still under investigation. However, a major standardization effort is underway within the EuroMRD consortium ( www.euromrd.org ) for future application of ddPCR in standard clinical practice.

Does MRD have a role in the management of iNHL?

Among indolent non-Hodgkin lymphomas (iNHLs), the analysis of measurable/minimal residual disease (MRD) has been extensively applied to follicular lymphoma (FL). Treatment combinations have deeply changed over the years, as well as the techniques to measure MRD, which is currently evaluated only in the setting of clinical trials. Here, we discuss the evidence on the role of molecular monitoring in the management of FL. Mature data support the quantification of molecular tumor burden at diagnosis as a tool to stratify patients in risk categories and of MRD evaluation at the end of treatment to predict progression-free survival and overall survival. Moreover, MRD deserves further studies as a tool to refine the clinical/metabolic response and to modulate treatment intensity/duration. Patients with a higher relapse probability can be identified, but the relevance of continuous molecular follow-up should be clarified by kinetic models of MRD analysis. Being the BCL2/heavy chain immunoglobulin gene hybrid rearrangement detectable in about 50% to 60% of advanced FL and in 30% of positron emission tomography/computed tomography-staged localized FL, technical advancements such as next-generation sequencing/target locus amplification may allow broadening the FL population carrying a molecular marker. Droplet digital polymerase chain reaction can better quantify MRD at low levels, and novel sources of DNA, such as cell-free DNA, may represent a noninvasive tool to monitor MRD. Finally, MRD in other iNHLs, such as lymphoplasmacytic lymphoma/Waldenström macroglobulinemia and marginal zone lymphoma, is beginning to be explored.

Adolescent and young adult acute lymphoblastic leukemia. Final results of the phase II pediatric-like GIMEMA LAL-1308 trial.

Adolescents and young adults (AYA) with acute lymphoblastic leukemia (ALL) represent a unique patient population with specific characteristics and needs. Growing evidences suggest that pediatric-inspired approaches improve the outcome in AYA. These results prompted the design of a pediatric AIEOP-BFM ALL 2000-based regimen - the GIMEMA LAL-1308 protocol - for newly diagnosed AYA (range 18-35 years) with Philadelphia negative (Ph-) ALL. The protocol included minimal residual disease (MRD) analysis at two different time-points (TP), that is, at the end of induction IA and consolidation IB, and a modulation in post-consolidation intensity according to MRD. Seventy-six patients were eligible between September 2010 and October 2014. The regimen was well tolerated, with 2.7% induction deaths and no deaths in the post-consolidation phase. The complete response (CR) rate was 92%; the 48-month overall survival (OS) and disease-free survival (DFS) were 60.3% and 60.4%. Both OS and DFS were significantly better in T-ALL than B-ALL. A molecular MRD <10-3 at TP1 was associated with a significantly better OS and DFS (77% vs 39% and 71.9% vs 34.4%, respectively);similar results were documented at TP2 (OS and DFS 74.5% vs 30.6% and 71.5% vs 25.7%, respectively). The LAL-1308 results were compared to those from similar historic AYA populations undergoing the two previous GIMEMA LAL-2000 and LAL-0904 protocols. Both OS and DFS improved significantly compared to the two previous protocols. These results indicate that this pediatric-inspired and MRD-oriented protocol is feasible and effective for Ph- AYA ALL patients, and underline the prognostic value of MRD determinations at specific TPs.

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.

Chlorambucil plus rituximab with or without maintenance rituximab as first-line treatment for elderly chronic lymphocytic leukemia patients.

In a phase II trial, we evaluated chlorambucil and rituximab (CLB-R) as first-line induction treatment with or without R as maintenance for elderly chronic lymphocytic leukemia (CLL) patients. Treatment consisted of eight 28-day cycles of CLB (8 mg/m(2) /day, days 1-7) and R (day 1 of cycle 3, 375 mg/m(2) ; cycles 4-8, 500 mg/m(2) ). Responders were randomized to 12 8-week doses of R (375 mg/m(2) ) or observation. As per intention-to-treat analysis, 82.4% (95% CI, 74.25-90.46%) of 85 patients achieved an overall response (OR), 16.5% a complete response (CR), 2.4% a CR with incomplete bone marrow recovery. The OR was similar across Binet stages (A 86.4%, B 81.6%, and C 78.6%) and age categories (60-64 years, 92.3%; 65-69, 85.2%; 70-74, 75.0%; ≥75, 81.0%). CLB-R was well tolerated. After a median follow-up of 34.2 months, the median progression-free survival (PFS) was 34.7 months (95% CI, 33.1-39.5). TP53 abnormalities, complex karyotype, and low CD20 gene expression predicted lack of response; SF3B1 mutation and BIRC3 disruption low CR rates. IGHV mutations significantly predicted PFS. R maintenance tended towards a better PFS than observation and was safe and most beneficial for patients in partial response and for unmutated IGHV cases. CLB-R represents a promising option for elderly CLL patients.

Histopathological and molecular features of persistent polyclonal B-cell lymphocytosis (PPBL) with progressive splenomegaly.

Five cases of persistent polyclonal B-cell lymphocytosis (PPBL) with progressive splenomegaly are reported; three were splenectomized. BCL2/IGH rearrangements were found in three cases; HLA-DRB1*07 in all. Bone marrow (BM) trephines showed a moderate lymphoid infiltrate with intrasinusoidal distribution resembling a splenic marginal-zone lymphoma. Splenic white pulp revealed an enlargement of the marginal-zone area; red pulp was infiltrated by the same lymphocytes engulfing the sinuses. Splenic and BM B-lymphocytes were CD79a(+)/CD20(+)/IgM(+)/IgD(+)/bcl-2(+)/CD27(+)/DBA.44(-)/CD31(-) and polyclonal by immunophenotype/polymerase chain reaction. PPBL features an expansion of splenic marginal-zone B-lymphocytes, which infiltrate BM sinusoids and circulate in the blood with no evidence of clonality, even in cases with progressive splenomegaly.

Stage I/II follicular lymphoma: spread of bcl-2/IgH+ cells in blood and bone marrow from primary site of disease and possibility of clearance after involved field radiotherapy.

Stage I/IIA follicular lymphoma (FL) is considered a localised disease that can be adequately treated with radiotherapy alone. Bone marrow (BM) and peripheral blood (PB) involvement in FL was investigated by polymerase chain reaction (PCR) in a series of 24 consecutive patients with histologically revised diagnosis and treated with involved field radiotherapy. Despite the limited stage, Bcl-2/IgH+ cells were found at diagnosis in PB and/or BM of 16 patients (66.6%). After treatment, in 9/15 Bcl-2/IgH positive evaluable patients, a disappearance of Bcl-2/IgH+ cells was observed, which persisted after a median follow-up of 43.5 months (range 11-70) in all but one patient. Quantitative PCR demonstrated the feasibility of clearing PB and BM Bcl-2+ cells after local irradiation of the primary site of the disease only when the basal number of lymphoma cells was <1:100 000. Patients with Bcl-2/IgH+ cells at diagnosis or after treatment had a higher likelihood of relapse. Thus, despite a negative BM biopsy, the majority of localised FL Bcl-2/IgH+ cells were found in the PB and BM. Lymphoma cells can reversibly spread from the affected lymph node to PB and BM and, in a proportion of cases, durably disappear after irradiation. The possibility of a persistent lymphoma cell clearance is proportional to the amount of cells detected at presentation by quantitative PCR.

Gamma-delta hepatosplenic T-cell lymphoma. Description of a case with immunophenotypic and molecular follow-up successfully treated with chemotherapy alone.

This study hereby reports the case of a 19-year old boy with a gamma-delta hepatosplenic T-cell lymphoma (HSTCL). Initial therapy consisted of four cycles of the IEV (Ifosphamide, Epirubicin and Etoposide) scheme. Further treatment strategy was then adapted according to minimal residual disease monitoring by immunophenotypic and T-cell receptor gamma chain gene evaluation. The patient remains in complete clinical, immunological and molecular remission and in good clinical conditions 48 months after diagnosis and 40 months after stopping therapy.