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Summary: First‐line purine nucleoside analogues (PNAs) in hairy cell leukaemia (HCL) allow deep and long‐lasting responses. We retrospectively analysed 53 HCL patients treated frontline with cladribine and assessed for response at 2 and 6 months after treatment to evaluate the kinetics of response. The estimated median progression‐free survival was significantly different according to the degree of residual HCL infiltrate detected by immunohistochemistry at the bone marrow biopsy at 2 months (≤5% vs. >5%, 247 vs. 132 months, respectively, p = 0.033), but not at 6 months (p = 0.79). Our data suggest a favourable prognostic impact of early marrow HCL clearance in patients treated with cladribine. [ABSTRACT FROM AUTHOR]

Here we evaluated the epigenomic and transcriptomic profile of XPO1 mutant chronic lymphocytic leukaemia (CLL) and their clinical phenotype. By ATAC-seq, chromatin regions that were more accessible in XPO1 mutated CLL were enriched of binding sites for transcription factors regulated by pathways emanating from the B-cell receptor (BCR), including NF-κB signalling, p38-JNK and RAS-RAF-MEK-ERK. XPO1 mutant CLL, consistent with the chromatin accessibility changes, were enriched with transcriptomic features associated with BCR and cytokine signalling. By combining epigenomic and transcriptomic data, MIR155HG, the host gene of miR-155, and MYB, the transcription factor that positively regulates MIR155HG, were upregulated by RNA-seq and their promoters were more accessible by ATAC-seq. To evaluate the clinical impact of XPO1 mutations, we investigated a total of 957 early-stage CLL subdivided into 3 independent cohorts (N = 276, N = 286 and N = 395). Next-generation sequencing analysis identified XPO1 mutations as a novel predictor of shorter time to first treatment (TTFT) in all cohorts. Notably, XPO1 mutations maintained their prognostic value independent of the immunoglobulin heavy chain variable status and early-stage prognostic models. These data suggest that XPO1 mutations, conceivably through increased miR-155 levels, may enhance BCR signalling leading to higher proliferation and shorter TTFT in early-stage CLL., (© 2023 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

We explored the relevance of genomic microarrays (GM) in the refinement of prognosis in newly diagnosed low-risk chronic lymphocytic leukaemia (CLL) patients as defined by isolated del(13q) or no lesions by a standard 4 probe fluorescence in situ hybridization (FISH) analysis. Compared to FISH, additional lesions were detected by GM in 27 of the 119 patients (22.7%). The concordance rate between FISH and GM was 87.4%. Discordant results between cytogenetic banding analysis (CBA) and GM were observed in 45/119 cases (37.8%) and were mainly due to the intrinsic characteristics of each technique. The presence of additional lesions by GM was associated with age > 65 years (p = 0.047), advanced Binet stage (p = 0.001), CLL-IPI score (p < 0.001), a complex karyotype (p = 0.004) and a worse time-to-first treatment in multivariate analysis (p = 0.009). Additional lesions by GM were also significantly associated with a worse time-to-first treatment in the subset of patients with wild-type TP53 and mutated IGHV (p = 0.025). In CLL patients with low-risk features, the presence of additional lesions identified by GM helps to identify a subset of patients with a worse outcome that could be proposed for a risk-adapted follow-up and for early treatment including targeted agents within clinical trials., (© 2023 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

In chronic lymphocytic leukaemia (CLL), caution is warranted regarding the clinical implications of immunoglobulin variable heavy chain region (IGHV) rearrangements with a 'borderline' (BL) percentage of mutations (i.e. 97-97·9% IGHV identity). We analysed the IGHV mutational status in 759 untreated CLL patients (cohort 1). BL-CLL (n = 36, 5%) showed a time to first treatment (TFT) similar to that of M-CLL (n = 338) and significantly longer than that of UM-CLL (n = 385), despite the enrichment in subset #2 cases. In fact, CLLs belonging to subset #2 (n = 15/759, 2%) were significantly more frequent among BL-CLLs (n = 5/36, 14%), with a brief TFT. TFT of BL-CLL remained comparable to that of M-CLL also considering the 327 CLL patients evaluated at diagnosis. These findings were then validated in an independent cohort 2 of 759 newly diagnosed CLL patients (BL-CLL: n = 11, 1·4%) and in all newly diagnosed patients from cohorts 1 and 2 (n = 1 086, 84% stage A; BL-CLL: n = 47, 4·3%). BL-CLL at diagnosis showed a biological profile comparable to that of M-CLL with a low frequency of unfavourable prognostic markers, except for a significant enrichment in subset #2. Our data suggest that the prognosis of BL-CLL is good and similar to that of M-CLL, with the exception of subset #2 cases., (© 2020 British Society for Haematology and John Wiley & Sons Ltd.)

Since 2000, we have investigated 67 consecutive patients with stage I/II follicular lymphoma (FL) for the presence of BCL2/IGH rearrangements by polymerase chain reaction (PCR), real time quantitative PCR (RQ-PCR) and digital droplet PCR (ddPCR). All patients were treated with involved-field radiotherapy (IF-RT) (24-30 Gy). From 2005, patients with minimal residual disease (MRD) after IF-RT received rituximab (R) (375 mg/m 2 , 4 weekly administrations). The median follow-up is 82 months (17-196). At diagnosis, 72% of patients were BCL2/IGH+. Progression-free survival (PFS) was significantly better in patients with undetectable/low levels (<10 -5 ) of circulating BCL2/IGH+ cells at diagnosis and in those who were persistently MRD- during follow-up (P = 0·0038). IF-RT induced an MRD- status in 50% of cases; 16/19 (84%) MRD+ patients after IF-RT became MRD- after R treatment. A significantly longer PFS was observed in MRD+ patients treated with R compared to untreated MRD+ patients (P = 0·049). In early stage FL, both circulating levels of BCL2/IGH+ cells at diagnosis and MRD status during follow-up bear prognostic implications. Standard IF-RT fails to induce an MRD-negative status in half of patients. Most patients become MRD- following treatment with R and this is associated with a significantly better PFS., (© 2019 British Society for Haematology and John Wiley & Sons Ltd.)

TP53-disrupted chronic lymphocytic leukaemia (CLL) patients show a suboptimal long-term response to ibrutinib. We hereby report that ibrutinib-induced in vitro apoptosis and proliferation inhibition were significantly lower in TP53-mutated (TP53-M) CLL cells compared to TP53 wild-type cells. Contrariwise, venetoclax effectively killed TP53-M cells. Gene expression profile analysis of TP53-M cells revealed a downmodulation of B-cell receptor (BCR)-related genes and an upmodulation of genes with anti-apoptotic/pro-survival activity, suggesting that the survival and proliferation of TP53-M cells are less dependent on the BCR pathway. These observations further support the use of drug combinations for the optimal management of TP53-M CLL patients., (© 2018 British Society for Haematology and John Wiley & Sons Ltd.)

BCL2/IGH rearrangements were analysed by polymerase chain reaction (PCR) at diagnosis in paired peripheral blood (PB) and bone marrow (BM) samples from 67 patients with stage I/II follicular lymphoma (FL). Real time quantitative PCR (RQ-PCR) and digital droplet PCR (ddPCR) were performed in cases with a major breakpoint region (MBR+) at diagnosis and after localized radiotherapy and rituximab administration in order to investigate the applicability of ddPCR. The overall ddPCR/RQ-PCR concordance was 81·9% (113/138 samples) and 97·5% in the 40/138 with quantifiable disease (RQ-PCR≥10 -5 ). At baseline, ddPCR allowed the recovery of a MBR+ marker in 8/18 (44·4%) samples that resulted MBR-negative/minor cluster region-negative/minor BCL2-negative by qualitative PCR. Moreover, the tumour burden at diagnosis significantly predicted progression-free survival (PSF) only when quantified by ddPCR. Paired PB and BM samples analysis demonstrated a high concordance in the detection of BCL2/IGH+ cells by qualitative and quantitative methods; in particular, 40/62 samples were positive by ddPCR (25 PB+/BM+; 9 PB+/BM-; 6 PB-/BM+), with 34/40 (85%) identified by the study of PB only. In conclusion, in localized FL, ddPCR is a promising tool for monitoring minimal residual disease (MRD) that is at least comparable to RQ-PCR and potentially more accurate. PB is a suitable source for serial BCL2/IGH MRD assessments, regardless of the methodology utilized., (© 2017 John Wiley & Sons Ltd.)

Real-time quantitative polymerase chain reaction (RQ-PCR) is a standardized tool for minimal residual disease (MRD) monitoring in acute lymphoblastic leukaemia (ALL). The applicability of this technology is limited by the need of a standard curve based on diagnostic DNA. The digital droplet PCR (ddPCR) technology has been recently applied to various medical fields, but its use in MRD monitoring is under investigation. In this study, we analysed 50 ALL cases by both methods in two phases: in the first, we established analytical parameters to investigate the applicability of this new technique; in the second, we analysed MRD levels in 141 follow-up (FU) samples to investigate the possible use of ddPCR for MRD monitoring in ALL patients. We documented that ddPCR has sensitivity and accuracy at least comparable to those of RQ-PCR. Overall, the two methods gave concordant results in 124 of the 141 analysed MRD samples (88%, P = 0·94). Discordant results were found in 12% borderline cases. The results obtained prove that ddPCR is a reliable method for MRD monitoring in ALL, with the advantage of quantifying without the need of the calibration curves. Its application in a cohort of patients with a longer FU will conclusively define its clinical predictive value., (© 2016 John Wiley & Sons Ltd.)

Whole exome sequencing and copy number aberration (CNA) analysis were performed on cells taken from peripheral blood (PB) and lymph nodes (LN) of patients with chronic lymphocytic leukaemia (CLL). Of 64 non-silent somatic mutations, 54 (84·4%) were clonal in both compartments, 3 (4·7%) were PB-specific and 7 (10·9%) were LN-specific. Most of the LN- or PB-specific mutations were subclonal in the other corresponding compartment (variant frequency 0·5-5·3%). Of 41 CNAs, 27 (65·8%) were shared by both compartments and 7 (17·1%) were LN- or PB-specific. Overall, 6 of 9 cases (66·7%) showed genomic differences between the compartments. At subsequent relapse, Case 10, with 6 LN-specific lesions, and Case 100, with 6 LN-specific and 8 PB-specific lesions, showed, in the PB, the clonal expansion of LN-derived lesions with an adverse impact: SF3B1 mutation, BIRC3 deletion, del8(p23·3-p11·1), del9(p24·3-p13·1) and gain 2(p25·3-p14). CLL shows an intra-patient clonal heterogeneity according to the disease compartment, with both LN and PB-specific mutations/CNAs. The LN microenvironment might contribute to the clonal selection of unfavourable lesions, as LN-derived mutations/CNAs can appear in the PB at relapse., (© 2015 John Wiley & Sons Ltd.)

Minimal residual disease (MRD) is becoming increasingly important in chronic lymphocytic leukaemia (CLL) as treatment strategies are progressively improving. The primary objective of this study was to compare the applicability of three different flow cytometric approaches: basic 4-colour analysis, European Research Initiative in CLL (ERIC) consensus method and 8-colour analysis. Secondly, we investigated the sensitivity and specificity of flow cytometry (FC) compared to molecular analyses for MRD detection. A total of 462 CLL samples were evaluated by basic FC; in 143, ERIC consensus method was also performed and all three FC methodologies were applied in a subgroup of 10 cases. No discordance in defining MRD-positive/negative samples was observed between the FC methods; within positive samples, the ERIC consensus method and 8-colour analysis showed the most accurate results. MRD was analysed by FC and polymerase chain reaction (PCR) in 243 cases: concordant results were obtained in 199/243 samples (81·9%); 42/243 were FC-/PCR+. Overall, the sensitivity and specificity of FC compared to PCR was 96·5% and 77·2%, respectively. Both FC and PCR proved suitable for the detection of MRD and prediction of progression-free survival, which was significantly reduced in MRD-positive patients, regardless of the methodology. These results offer the rationale for a strategy to monitor MRD in CLL patients., (© 2014 John Wiley & Sons Ltd.)

Accurate genomic characterization requires sufficient amounts of optimal quality DNA. An approach for increasing the DNA amount is the whole-genome amplification (WGA) method. We applied WGA to the molecular quantification and minimal residual disease (MRD) evaluation of acute lymphoblastic leukaemia (ALL), aiming to compare the results obtained from genomic DNA and amplified DNA with WGA, and to evaluate the applicability and the reliability of WGA-DNA. Twenty paired samples from adult ALL patients were sequenced to identify the functional germline V-D-J segment at diagnosis; real-time quantitative polymerase chain reaction (RQ-PCR) quantitative analysis was performed both at diagnosis and follow-up. Genomic DNA and WGA-DNA screening identified equivalent 87 rearrangements. At diagnosis, the quantitative evaluation of genomic DNA samples showed 1 logarithm difference to WGA-DNA samples; these levels are comparable, being within the degree of acceptability and confidence. In the follow-up samples, RQ-PCR analysis on genomic DNA and WGA showed concordant MRD results in 16/18 samples, while 2/18 were MRD-positive outside the quantitative range by RQ-PCR (i.e. <5 × 10(-5)) on genomic DNA and MRD-negative on WGA-DNA. WGA-DNA enables: (i) the design of accurate targets for MRD evaluation in ALL patients, (ii) accurate disease quantification at diagnosis, (iii) MRD quantification comparable to genomic DNA., (© 2014 John Wiley & Sons Ltd.)

The efficacy of bendamustine versus chlorambucil in a phase III trial of previously untreated patients with Binet stage B/C chronic lymphocytic leukaemia (CLL) was re-evaluated after a median observation time of 54 months in May 2010. Overall survival (OS) was analysed for the first time. At follow-up, investigator-assessed complete response (CR) rate (21·0% vs 10·8%), median progression-free survival (21·2 vs 8·8 months; P < 0·0001; hazard ratio 2·83) and time to next treatment (31·7 vs 10·1 months; P < 0·0001) were improved for bendamustine over chlorambucil. OS was not different between groups for all patients or those ≤65 years, >65 years, responders and non-responders. However, patients with objective response or a CR experienced a significantly longer OS than non-responders or those without a CR. Significantly more patients on chlorambucil progressed to second/further lines of treatment compared with those on bendamustine (78·3% vs 63·6%; P = 0·004). The benefits of bendamustine over chlorambucil were achieved without reducing quality of life. In conclusion, bendamustine is significantly more effective than chlorambucil in previously untreated CLL patients, with the achievement of a CR or objective response appearing to prolong OS. Bendamustine should be considered as a preferred first-line option over chlorambucil for CLL patients ineligible for fludarabine, cyclophosphamide and rituximab., (© 2012 Blackwell Publishing Ltd.)

Mantle cell lymphoma (MCL) is an aggressive neoplasm with a short survival. Cases with leukaemic MCL and splenomegaly without adenopathies (non-nodal MCL) may have a more indolent course. To gain insights into the biological features underlying this presentation, we investigated the gene expression profile (GEP) and the IGHV mutational status in a cohort of leukaemic MCL cases. Comparison of MCL with other lymphoproliferative disorders (i.e. splenic marginal zone lymphoma, follicular lymphoma, chronic lymphocytic leukaemia) revealed a MCL signature enriched for the following gene categories: mitochondrion, oxidoreductase activity, response to stress, to DNA damage and TP53-pathway. Furthermore, GEP analysis revealed that non-nodal MCL cases were characterized by the down-modulation of the following gene categories: cell projection, actin cytoskeleton organization, cell adhesion (ITGAE, CELSR1, PCDH9) and tumour invasion/progression (PGF, ST14, ETS1, OCIAD1, EZR). Many down-modulated genes were related to the TP53-pathway and to DNA damage response. IGHV status proved unmutated in all nodal and mutated in all non-nodal MCL. Non-nodal leukaemic MCLs display a peculiar clinical presentation, with distinctive biological features, such as mutated IGHV and a transcriptional profile lacking tumour invasion properties, that might contribute to the absence of nodal involvement and to the less aggressive clinical course., (© 2011 Blackwell Publishing Ltd.)

The immunoglobulin heavy chain variable gene (IGHV) mutational status has been recognized as an important predictor of prognosis in chronic lymphocytic leukaemia (CLL) since 1999. More recently, other features of the B-cell receptor, such as stereotypy, have been identified as capable of refining the prognostic potential of IGHV status in the clinical assessment of CLL patients. In this context, different genes belonging to the IGHV3 subgroup, the most frequently used subgroup in CLL, have been shown to denote disease subsets that either display a bad prognosis (i.e. IGHV3-21, IGHV3-23) or are associated with particularly good clinical outcomes, including a highly stable/indolent clinical course, even prone to spontaneous regression (i.e. IGHV3-72, IGHV3-30). The present review focuses on the molecular and biological features of CLL-expressing specific genes belonging to the IGHV3 subgroup that are known to mark disease subsets with completely different clinical courses, and may be possibly related to CLL pathogenesis via antigen and/or superantigen involvement., (© 2011 Blackwell Publishing Ltd.)

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.

The outcome of children with acute lymphoblastic leukaemia (ALL) and early relapse remains unsatisfactory. In January 1995, the AIEOP (Associazione Italiana di Oncologia ed Ematologia Pediatrica) group opened a trial for children with ALL in first isolated or combined bone marrow relapse defined at high risk according to the length of first remission and the immunophenotype. The treatment plan included the combination of a single high-dose idarubicin and high-dose cytarabine as induction therapy followed by an intensive consolidation and stem cell transplant (SCT). In total, 100 children from 16 Italian centres were enrolled; 80 out of the 99 evaluable patients (81%) achieved second complete remission; eight (8%) died during induction and 11 (11%) failed to respond. A total of 42 out of the 80 responders (52.5%) received a SCT: 19 from an identical sibling, 11 from a matched unrelated donor and 12 from umbilical cord blood cells. The estimated 4-year overall survival and event-free survival were 25% and 21% respectively. Disease-free survival at 4 years was 25.8% for the 80 responders. At 4 years, 39 out of 100 children remain alive, with 27 of them free of leukaemia. This induction therapy has shown antileukaemic efficacy with acceptable toxicity; moreover, all responders proved eligible for intensive consolidation.