Inducible T-cell co-stimulator (ICOS) and ICOS ligand are novel players in the multiple-myeloma microenvironment.

The inducible T-cell co-stimulator (ICOS) is a T-cell receptor that, once bound to ICOS ligand (ICOSL) expressed on several cell types including the B-cell lineage, plays a decisive role in adaptive immunity by regulating the interplay between B and T cells. In addition to its immunomodulatory functions, we have shown that ICOS/ICOSL signalling can inhibit the activity of osteoclasts, unveiling a novel mechanism of lymphocyte-bone cells interactions. ICOS and ICOSL can also be found as soluble forms, namely sICOS and sICOSL. Here we show that: (i) levels of sICOS and sICOSL are increased in multiple myeloma (MM) compared to monoclonal gammopathy of undetermined significance and smouldering MM; (ii) levels of sICOS and sICOSL variably correlate with several markers of tumour burden; and (iii) sICOS levels tend to be higher in Durie-Salmon stage II/III versus stage I MM and correlate with overall survival as an independent variable. Moreover, surface ICOS and ICOSL are expressed in both myeloma cells and normal plasma cells, where they probably regulate different functional stages. Finally, ICOSL triggering inhibits the migration of myeloma cell lines in vitro and the growth of ICOSL+ MOPC-21 myeloma cells in vivo. These results suggest that ICOS and ICOSL represent novel markers and therapeutic targets for MM.

A randomized clinical study on the impact of Comprehensive Geriatric Assessment (CGA) based interventions on the quality of life of elderly, frail, onco-hematologic patients candidate to anticancer therapy: protocol of the ONCO-Aging study.

BACKGROUND: Age is considered as one of the most important risk-factor for many types of solid and hematological cancers, as their incidence increases with age in parallel to the ever-growing elderly population. Moreover, cancer incidence is constantly increasing as a consequence of the increase in life expectancy that favors the process of cellular senescence. Geriatric assessment has been increasingly recognized as predictive and prognostic instrument to detect frailty in older adults with cancer. In particular, the G8 score is a simple and reproducible instrument to identify elderly patients who should undergo full geriatric evaluation. Due to their frailty, elderly patients may be often under-treated and a therapeutic choice based also on a comprehensive geriatric assessment (CGA) is recommended. With these premises, we aim to test the impact of the CGA based interventions on the quality of life (QoL) of frail elderly onco-hematological patients, identified by the G8 screening, candidate for innovative target directed drugs or treatments including the combination of radiotherapy and chemotherapy (RT + CT). METHODS: Patients aged > 65 years, candidate to target directed agents or to RT + CT treatments are screened for frailty by the G8 test; those patients classified as frail (G8 ≤ 14) are randomized to receive a CGA at baseline or to conventional care. The primary endpoint is QoL, assessed by EORTC QLQ-C30C. As collateral biological study, the potential prognostic/predictive role of T-cell senescence and myeloid derived suppressor cells (MDSC) are evaluated on plasma samples. DISCUSSION: This trial will contribute to define the impact of CGA on the management of frail elderly onco-hematologic patients candidate to innovative biological drugs or to integrated schedules with the association of RT + CT. Furthermore, the use of plasma samples to assess the potential prognostic value of imbalance of immune-competent cells is expected to contribute to the individualized care of elderly patients, resulting into a fine tuning of the therapeutic strategies. TRIAL REGISTRATION: ClinicalTrials.gov ID: NCT04478916 . registered July 21, 2020 - retrospectively registered.

International prognostic score for asymptomatic early-stage chronic lymphocytic leukemia.

Most patients with chronic lymphocytic leukemia (CLL) are diagnosed with early-stage disease and managed with active surveillance. The individual course of patients with early-stage CLL is heterogeneous, and their probability of needing treatment is hardly anticipated at diagnosis. We aimed at developing an international prognostic score to predict time to first treatment (TTFT) in patients with CLL with early, asymptomatic disease (International Prognostic Score for Early-stage CLL [IPS-E]). Individual patient data from 11 international cohorts of patients with early-stage CLL (n = 4933) were analyzed to build and validate the prognostic score. Three covariates were consistently and independently correlated with TTFT: unmutated immunoglobulin heavy variable gene (IGHV), absolute lymphocyte count higher than 15 × 109/L, and presence of palpable lymph nodes. The IPS-E was the sum of the covariates (1 point each), and separated low-risk (score 0), intermediate-risk (score 1), and high-risk (score 2-3) patients showing a distinct TTFT. The score accuracy was validated in 9 cohorts staged by the Binet system and 1 cohort staged by the Rai system. The C-index was 0.74 in the training series and 0.70 in the aggregate of validation series. By meta-analysis of the training and validation cohorts, the 5-year cumulative risk for treatment start was 8.4%, 28.4%, and 61.2% among low-risk, intermediate-risk, and high-risk patients, respectively. The IPS-E is a simple and robust prognostic model that predicts the likelihood of treatment requirement in patients with early-stage CLL. The IPS-E can be useful in clinical management and in the design of early intervention clinical trials.

Biological and clinical implications of BIRC3 mutations in chronic lymphocytic leukemia.

BIRC3 is a recurrently mutated gene in chronic lymphocytic leukemia (CLL) but the functional implications of BIRC3 mutations are largely unexplored. Furthermore, little is known about the prognostic impact of BIRC3 mutations in CLL cohorts homogeneously treated with first-line fludarabine, cyclophosphamide, and rituximab (FCR). By immunoblotting analysis, we showed that the non-canonical nuclear factor-κB pathway is active in BIRC3-mutated cell lines and in primary CLL samples, as documented by the stabilization of MAP3K14 and by the nuclear localization of p52. In addition, BIRC3-mutated primary CLL cells are less sensitive to flu-darabine. In order to confirm in patients that BIRC3 mutations confer resistance to fludarabine-based chemoimmunotherapy, a retrospective multicenter cohort of 287 untreated patients receiving first-line FCR was analyzed by targeted next-generation sequencing of 24 recurrently mutated genes in CLL. By univariate analysis adjusted for multiple comparisons BIRC3 mutations identify a poor prognostic subgroup of patients in whom FCR treatment fails (median progression-free survival: 2.2 years, P<0.001) similar to cases harboring TP53 mutations (median progression-free survival: 2.6 years, P<0.0001). BIRC3 mutations maintained an independent association with an increased risk of progression with a hazard ratio of 2.8 (95% confidence interval 1.4-5.6, P=0.004) in multivariate analysis adjusted for TP53 mutation, 17p deletion and IGHV mutation status. If validated, BIRC3 mutations may be used as a new molecular predictor to select high-risk patients for novel frontline therapeutic approaches.

An update on: molecular genetics of high-risk chronic lymphocytic leukemia.

Introduction: During the past few years, new genomic approaches have elucidated the molecular genetics of chronic lymphocytic leukemia (CLL) to a large extent. As a consequence, specific high-risk genetic features of the disease, e.g. TP53 disruption, have become the backbone of the treatment algorithm for CLL and serve as robust biomarkers for a precision medicine approach to this leukemia.Areas covered: This review covers the genetics of CLL and highlights the translational implications of molecular biomarkers that characterize patients with a high risk of disease progression. Knowledge of the genetic landscape of CLL has allowed the identification of the main molecular features associated with chemo-refractoriness, as well as resistance to BCR inhibitors and BCL2 inhibitors. The molecular basis of Richter transformation has also been characterized.Expert opinion: The term 'high risk CLL' has been changing over time, and might be subject to further changes in the future. With the advent of new therapeutic strategies targeting pathogenetic pathways of the disease, the definition is shifting from the historical view of refractoriness to chemo-immunotherapy, to refractoriness to BCR inhibitors and/or to BCL2 inhibitors. Patients failing these novel medicines are those for whom new therapeutic approaches are still highly needed.

Digital PCR improves the quantitation of DMR and the selection of CML candidates to TKIs discontinuation.

Treatment-free remission (TFR) by tyrosine kinase inhibitors (TKI) discontinuation in patients with deep molecular response (DMR) is a paramount goal in the current chronic myeloid leukemia (CML) therapeutic strategy. The best DMR level by real-time quantitative PCR (RT-qPCR) for TKI discontinuation is still a matter of debate. To compare the accuracy of digital PCR (dPCR) and RT-qPCR for BCR-ABL1 transcript levels detection, 142 CML patients were monitored for a median time of 24 months. Digital PCR detected BCR-ABL1 transcripts in the RT-qPCR undetectable cases. The dPCR analysis of the samples, grouped by the MR classes, revealed a significant difference between MR4.0 and MR4.5 (P = 0.0104) or MR5.0 (P = 0.0032). The clinical and hematological characteristics of the patients grouped according to DMR classes (MR4.0 vs MR4.5-5.0 ) were superimposable. Conversely, patients with dPCR values <0.468 BCR-ABL1 copies/µL (as we previously described) showed a longer DMR duration (P = 0.0220) and mainly belonged to MR4.5-5.0 (P = 0.0442) classes compared to patients with higher dPCR values. Among the 142 patients, 111 (78%) discontinued the TKI treatment; among the 111 patients, 24 (22%) lost the MR3.0 or MR4.0 . RT-qPCR was not able to discriminate patients with higher risk of MR loss after discontinuation (P = 0.8100). On the contrary, according to dPCR, 12/25 (48%) patients with BCR-ABL1 values ≥0.468 and 12/86 (14%) patients with BCR-ABL1 values <0.468 lost DMR in this cohort, respectively (P = 0.0003). Treatment-free remission of patients who discontinued TKI with a dPCR <0.468 was significantly higher compared to patients with dPCR ≥ 0.468 (TFR at 2 years 83% vs 52% P = 0.0017, respectively). In conclusion, dPCR resulted in an improved recognition of stable DMR and of candidates to TKI discontinuation.

Circulating tumor DNA reveals genetics, clonal evolution, and residual disease in classical Hodgkin lymphoma.

The rarity of neoplastic cells in the biopsy imposes major technical hurdles that have so far limited genomic studies in classical Hodgkin lymphoma (cHL). By using a highly sensitive and robust deep next-generation sequencing approach for circulating tumor DNA (ctDNA), we aimed to identify the genetics of cHL in different clinical phases, as well as its modifications on treatment. The analysis was based on specimens collected from 80 newly diagnosed and 32 refractory patients with cHL, including longitudinal samples collected under ABVD (adriamycin, bleomycin, vinblastine, dacarbazine) chemotherapy and longitudinal samples from relapsing patients treated with chemotherapy and immunotherapy. ctDNA mirrored Hodgkin and Reed-Sternberg cell genetics, thus establishing ctDNA as an easily accessible source of tumor DNA for cHL genotyping. By identifying STAT6 as the most frequently mutated gene in ∼40% of cases, we refined the current knowledge of cHL genetics. Longitudinal ctDNA profiling identified treatment-dependent patterns of clonal evolution in patients relapsing after chemotherapy and patients maintained in partial remission under immunotherapy. By measuring ctDNA changes during therapy, we propose ctDNA as a radiation-free tool to track residual disease that may integrate positron emission tomography imaging for the early identification of chemorefractory patients with cHL. Collectively, our results provide the proof of concept that ctDNA may serve as a novel precision medicine biomarker in cHL.

Diffuse large B-cell lymphoma genotyping on the liquid biopsy.

Accessible and real-time genotyping for diagnostic, prognostic, or treatment purposes is increasingly impelling in diffuse large B-cell lymphoma (DLBCL). Cell-free DNA (cfDNA) is shed into the blood by tumor cells undergoing apoptosis and can be used as source of tumor DNA for the identification of DLBCL mutations, clonal evolution, and genetic mechanisms of resistance. In this study, we aimed at tracking the basal DLBCL genetic profile and its modification upon treatment using plasma cfDNA. Ultra-deep targeted next generation sequencing of pretreatment plasma cfDNA from DLBCL patients correctly discovered DLBCL-associated mutations that were represented in >20% of the alleles of the tumor biopsy with >90% sensitivity and ∼100% specificity. Plasma cfDNA genotyping also allowed for the recovery of mutations that were undetectable in the tissue biopsy, conceivably because, due to spatial tumor heterogeneity, they were restricted to clones that were anatomically distant from the biopsy site. Longitudinal analysis of plasma samples collected under rituximab-cyclophosphamide-doxorubicin-vincristine-prednisone (R-CHOP) chemotherapy showed a rapid clearance of DLBCL mutations from cfDNA among responding patients. Conversely, among patients who were resistant to R-CHOP, basal DLBCL mutations did not disappear from cfDNA. In addition, among treatment-resistant patients, new mutations were acquired in cfDNA that marked resistant clones selected during the clonal evolution. These results demonstrate that cfDNA genotyping of DLBCL is as accurate as genotyping of the diagnostic biopsy to detect clonally represented somatic tumor mutations and is a real-time and noninvasive approach to tracking clonal evolution and the emergence of treatment-resistant clones.

Clinical impact of small TP53 mutated subclones in chronic lymphocytic leukemia.

TP53 mutations are strong predictors of poor survival and refractoriness in chronic lymphocytic leukemia (CLL) and have direct implications for disease management. Clinical information on TP53 mutations is limited to lesions represented in >20% leukemic cells. Here, we tested the clinical impact and prediction of chemorefractoriness of very small TP53 mutated subclones. The TP53 gene underwent ultra-deep-next generation sequencing (NGS) in 309 newly diagnosed CLL. A robust bioinformatic algorithm was established for the highly sensitive detection of few TP53 mutated cells (down to 3 out of ∼1000 wild-type cells). Minor subclones were validated by independent approaches. Ultra-deep-NGS identified small TP53 mutated subclones in 28/309 (9%) untreated CLL that, due to their very low abundance (median allele frequency: 2.1%), were missed by Sanger sequencing. Patients harboring small TP53 mutated subclones showed the same clinical phenotype and poor survival (hazard ratio = 2.01; P = .0250) as those of patients carrying clonal TP53 lesions. By longitudinal analysis, small TP53 mutated subclones identified before treatment became the predominant population at the time of CLL relapse and anticipated the development of chemorefractoriness. This study provides a proof-of-principle that very minor leukemia subclones detected at diagnosis are an important driver of the subsequent disease course.

Alterations of negative regulators of cytokine signalling in immunodeficiency-related non-Hodgkin lymphoma.

We investigated immunodeficiency-related non-Hodgkin lymphoma for the presence of molecular alterations affecting negative regulators of the Janus family protein tyrosine kinase/signal transducer and activator of transcription pathway. Protein tyrosine phosphatase, non-receptor type 6/Src homology 2-containing tyrosine phosphatase-1 epigenetic silencing was recurrent in primary effusion lymphoma (100%), and diffuse large B-cell lymphoma (63%), with a higher prevalence in the non-germinal centre subtype, and was associated with the activation of the Janus family protein tyrosine kinase/signal transducer and activator of transcription 3 pathway. Suppressor of cytokine signalling (SOCS)1 and SOCS3 epigenetic silencing were occasionally detected, whereas SOCS1 was frequently mutated in diffuse large B-cell lymphoma and polymorphic post-transplant lymphoproliferative disorders, possibly as a cause of aberrant somatic hypermutation. However, the mutation profile of the coding region of the gene was different from that expected from the aberrant somatic hypermutation process, suggesting that, at least in some cases, SOCS1 mutations may have been selected for their functional activity.

Integrated mutational and cytogenetic analysis identifies new prognostic subgroups in chronic lymphocytic leukemia.

The identification of new genetic lesions in chronic lymphocytic leukemia (CLL) prompts a comprehensive and dynamic prognostic algorithm including gene mutations and chromosomal abnormalities and their changes during clonal evolution. By integrating mutational and cytogenetic analysis in 1274 CLL samples and using both a training-validation and a time-dependent design, 4 CLL subgroups were hierarchically classified: (1) high-risk, harboring TP53 and/or BIRC3 abnormalities (10-year survival: 29%); (2) intermediate-risk, harboring NOTCH1 and/or SF3B1 mutations and/or del11q22-q23 (10-year survival: 37%); (3) low-risk, harboring +12 or a normal genetics (10-year survival: 57%); and (4) very low-risk, harboring del13q14 only, whose 10-year survival (69.3%) did not significantly differ from a matched general population. This integrated mutational and cytogenetic model independently predicted survival, improved CLL prognostication accuracy compared with FISH karyotype (P < .0001), and was externally validated in an independent CLL cohort. Clonal evolution from lower to higher risk implicated the emergence of NOTCH1, SF3B1, and BIRC3 abnormalities in addition to TP53 and 11q22-q23 lesions. By taking into account clonal evolution through time-dependent analysis, the genetic model maintained its prognostic relevance at any time from diagnosis. These findings may have relevant implications for the design of clinical trials aimed at assessing the use of mutational profiling to inform therapeutic decisions.

The coding genome of splenic marginal zone lymphoma: activation of NOTCH2 and other pathways regulating marginal zone development.

Splenic marginal zone lymphoma (SMZL) is a B cell malignancy of unknown pathogenesis, and thus an orphan of targeted therapies. By integrating whole-exome sequencing and copy-number analysis, we show that the SMZL exome carries at least 30 nonsilent gene alterations. Mutations in NOTCH2, a gene required for marginal-zone (MZ) B cell development, represent the most frequent lesion in SMZL, accounting for ∼20% of cases. All NOTCH2 mutations are predicted to cause impaired degradation of the NOTCH2 protein by eliminating the C-terminal PEST domain, which is required for proteasomal recruitment. Among indolent B cell lymphoproliferative disorders, NOTCH2 mutations are restricted to SMZL, thus representing a potential diagnostic marker for this lymphoma type. In addition to NOTCH2, other modulators or members of the NOTCH pathway are recurrently targeted by genetic lesions in SMZL; these include NOTCH1, SPEN, and DTX1. We also noted mutations in other signaling pathways normally involved in MZ B cell development, suggesting that deregulation of MZ B cell development pathways plays a role in the pathogenesis of ∼60% SMZL. These findings have direct implications for the treatment of SMZL patients, given the availability of drugs that can target NOTCH, NF-κB, and other pathways deregulated in this disease.

Disruption of BIRC3 associates with fludarabine chemorefractoriness in TP53 wild-type chronic lymphocytic leukemia.

The genetic lesions identified to date do not fully recapitulate the molecular pathogenesis of chronic lymphocytic leukemia (CLL) and do not entirely explain the development of severe complications such as chemorefractoriness. In the present study, BIRC3, a negative regulator of noncanonical NF-κB signaling, was investigated in different CLL clinical phases. BIRC3 lesions were absent in monoclonal B-cell lymphocytosis (0 of 63) and were rare in CLL at diagnosis (13 of 306, 4%). Conversely, BIRC3 disruption selectively affected 12 of 49 (24%) fludarabine-refractory CLL cases by inactivating mutations and/or gene deletions that distributed in a mutually exclusive fashion with TP53 abnormalities. In contrast to fludarabine-refractory CLL, progressive but fludarabine-sensitive patients were consistently devoid of BIRC3 abnormalities, suggesting that BIRC3 genetic lesions associate specifically with a chemorefractory phenotype. By actuarial analysis in newly diagnosed CLL (n = 306), BIRC3 disruption identified patients with a poor outcome similar to that associated with TP53 abnormalities and exerted a prognostic role that was independent of widely accepted clinical and genetic risk factors. Consistent with the role of BIRC3 as a negative regulator of NF-κB, biochemical studies revealed the presence of constitutive noncanonical NF-κB activation in fludarabine-refractory CLL patients harboring molecular lesions of BIRC3. These data identify BIRC3 disruption as a recurrent genetic lesion of high-risk CLL devoid of TP53 abnormalities.

Mutations of the SF3B1 splicing factor in chronic lymphocytic leukemia: association with progression and fludarabine-refractoriness.

The genetic lesions identified in chronic lymphocytic leukemia (CLL) do not entirely recapitulate the disease pathogenesis and the development of serious complications, such as chemorefractoriness. While investigating the coding genome of fludarabine-refractory CLL, we observed that mutations of SF3B1, encoding a splicing factor and representing a critical component of the cell spliceosome, were recurrent in 10 of 59 (17%) fludarabine-refractory cases, with a frequency significantly greater than that observed in a consecutive CLL cohort sampled at diagnosis (17/301, 5%; P = .002). Mutations were somatically acquired, were generally represented by missense nucleotide changes, clustered in selected HEAT repeats of the SF3B1 protein, recurrently targeted 3 hotspots (codons 662, 666, and 700), and were predictive of a poor prognosis. In fludarabine-refractory CLL, SF3B1 mutations and TP53 disruption distributed in a mutually exclusive fashion (P = .046). The identification of SF3B1 mutations points to splicing regulation as a novel pathogenetic mechanism of potential clinical relevance in CLL.

Genome-wide DNA profiling better defines the prognosis of chronic lymphocytic leukaemia.

The integration of molecular and clinical information to tailor treatments remains an important research challenge in chronic lymphocytic leukaemia (CLL). This study aimed to identify genomic lesions associated with a poor outcome and a higher risk of histological transformation. A mono-institutional cohort of 147 cases was used as the test series, and a multi-institutional cohort of 176 cases as a validation series. Genomic profiles were obtained using Affymetrix SNP 6.0. The impact of the recurrent minimal common regions (MCRs) on overall survival was evaluated by univariate analysis followed by multiple-test correction. The independent prognostic significance was assessed by multivariate analysis. Eight MCRs showed a prognostic impact: gains at 2p25.3-p22.3 (MYCN), 2p22.3, 2p16.2-p14 (REL), 8q23.3-q24.3 (MYC), losses at 8p23.1-p21.2, 8p21.2, and of the TP53 locus. Gains at 2p and 8q and TP53 inactivation maintained prognostic significance in multivariate analysis and a hierarchical model confirmed their relevance. Gains at 2p also determined a higher risk of Richter syndrome transformation. The prediction of outcome for CLL patients might be improved by evaluating the presence of gains at 2p and 8q as novel genomic regions besides those included in the 'standard' fluorescence in situ hybridization panel.