Transient Bacillary Layer Detachment During the Disease Course of Primary Vitreoretinal Lymphoma.

PURPOSE: To report the clinical course and the retinal imaging features of a case of cytology-proven primary vitreoretinal lymphoma (PVRL) presenting with a transient bacillary layer detachment (BALAD) during the disease course. METHODS: Observational case report. RESULTS: A 50 year-old woman was referred to us with a 2-month history of vitritis in both eyes, poorly responding to oral prednisolone. After discontinuation of oral prednisolone, worsening of vitritis and the appearance of multiple creamy-like subretinal infiltrates in the mid-peripheral retina of both eyes, along with the exclusion of common causes of intermediate/posterior uveitis, made us consider PVRL. Aqueous humor sampling detected MYD88 L265P mutation, and subsequent diagnostic pars plana vitrectomy in the left eye yielded a positive cytology for large B cell lymphoma consistent with PVRL. During the disease course, optical coherence tomography of the macula showed a BALAD in the right eye, which resolved during follow-up. CONCLUSION: Our case indicates that BALAD is a possible rare manifestation of PVRL, and this should be considered in the differential diagnosis process in order to avoid diagnostic delays.

Inference of genomic lesions from single-cell RNAseq in myeloma improves functional intra- and inter-clonal analysis.

Smoldering Multiple Myeloma (SMM) is an asymptomatic plasma cell (PC) neoplasm that may evolve with variable frequency into multiple myeloma (MM). SMM is initiated by chromosomal translocations involving the IgH locus or by hyperdiploidy and evolves through acquisition of additional genetic lesions. In this scenario, we aimed at establishing a reliable analysis pipeline to infer genomic lesions from transcriptomic analysis, by combining single-cell RNA sequencing (scRNA-seq) with B-cell receptor sequencing and copy- number abnormality (CNA) analysis to identify clonal PCs at the genetic level along their specific transcriptional landscape. We profiled 20,465 bone marrow (BM) PCs derived from five SMM/MM patients and unbiasedly identified clonal and polyclonal plasma cells. Hyperdiploidy, t(11;14) and t(6;14) were identified at the scRNA level by analysis of chimeric reads. Subclone functional analysis was improved by combining transcriptome with CNA analysis. As examples, we illustrate the different functional properties of a light chain escape subclone in SMM, and of different B-cell and PC subclones in a patient affected by Wäldenstrom Macroglobulinemia and SMM. Overall, our data provide a proof of principle for inference of clinically relevant genotypic data from scRNAseq, which in turn will refine functional annotation of the clonal architecture of PC dyscrasias.

Real-life monocentric experience of venetoclax-based regimens for acute myeloid leukemia.

Introduction: Combination of venetoclax and hypomethylating agents (HMAs) has become a standard of care in acute myeloid leukemia (AML) aged >75 years or who have comorbidities that preclude intensive induction chemotherapy. Methods: We conducted a monocentric retrospective analysis on adult patients affected by treatment-naïve AML not eligible for standard induction therapy or refractory/relapsed (R/R) AML treated with venetoclax combinations outside clinical trials. Venetoclax was administered at the dose of 400 mg/daily after a short ramp-up and reduced in case of concomitant CYP3A4 inhibitors. Results: Sixty consecutive AML were identified. Twenty-three patients (38%) were affected by treatment-naïve AML and 37 (62%) by R/R AML. Median age was 70 years. Among R/R AML 30% had received a prior allogeneic stem cell transplantation (allo-HSCT). In combination with venetoclax, 50 patients (83%) received azacitidine. Antifungal prophylaxis was performed in 33 patients (55%).Overall response rate was 60%, with 53% of complete remission (CR; 78% for treatment-naïve and 49% for R/R, p 0.017). Median overall survival was 130 days for R/R patients and 269 days for treatment-naïve patients; median event free survival was 145 days for R/R cohort and 199 days for treatment-naïve AML.Measurable residual disease was negative in 26% of evaluable patients in CR/CR with incomplete hematologic recovery after 2 cycles and in 50% after 4 cycles, with no significant association with survival.Eleven patients (18%) received an allo-HSCT after venetoclax combinations. Most common grade 3/4 adverse events were infectious (51% of the patients), or hematological without infections (25% of the patients). Use of CYP3A4 inhibitors was associated with a trend to shorter cytopenias and with a lower rate of infections. Invasive fungal infections were less frequent among patients receiving azole prophylaxis (6% vs 26%; p 0.0659). Discussion: Venetoclax-based regimens are a viable option for AML considered not eligible for standard induction therapy and a valid rescue therapy in the R/R setting.Azole prophylaxis did not significantly affect response and it was associated with a lower rate of invasive fungal infections. Despite a limited number of patients, the association of venetoclax and HMAs proved to be also a feasible bridging therapy to transplantation.

Single-Cell RNA Sequencing for the Detection of Clonotypic V(D)J Rearrangements in Multiple Myeloma.

Multiple myeloma (MM) has a highly heterogeneous genetic background, which complicates its molecular tracking over time. Nevertheless, each MM patient's malignant plasma cells (PCs) share unique V(D)J rearranged sequences at immunoglobulin loci, which represent ideal disease biomarkers. Because the tumor-specific V(D)J sequence is highly expressed in bulk RNA in MM patients, we wondered whether it can be identified by single-cell RNA sequencing (scRNA-seq). To this end we analyzed CD138+ cells purified from bone marrow aspirates of 19 samples with PC dyscrasias by both a standard method based on bulk DNA and by an implementation of the standard 10x Genomics protocol to detect expressed V(D)J sequences. A dominant clonotype was easily identified in each sample, accounting on average for 83.65% of V(D)J-rearranged cells. Compared with standard methods, scRNA-seq analysis proved highly concordant and even more effective in identifying clonal productive rearrangements, by-passing limitations related to the misannealing of consensus primers in hypermutated regions. We next validated its accuracy to track 5 clonal cells with absolute sensitivity in a virtual sample containing 3180 polyclonal cells. This shows that single-cell V(D)J analysis may be used to find rare clonal cells, laying the foundations for functional single-cell dissection of minimal residual disease.

The spectrum of subclonal TP53 mutations in chronic lymphocytic leukemia: A next generation sequencing retrospective study.

Chronic lymphocytic leukemia (CLL) is a hematological disorder with complex clinical and biological behavior. TP53 mutational status and cytogenetic assessment of the deletion of the corresponding locus (17p13.1) are considered the most relevant biomarkers associated with pharmaco-predictive response, chemo-refractoriness, and worse prognosis in CLL patients. The implementation of Next Generation Sequencing (NGS) methodologies in the clinical laboratory allows for comprehensively analyzing the TP53 gene and detecting mutations with allele frequencies ≤10%, that is, "subclonal mutations". We retrospectively studied TP53 gene mutational status by NGS in 220 samples from 171 CLL patients. TP53 mutations were found in 60/220 (27.3%) samples and 47/171 (27.5%) patients. Interestingly, subclonal mutations could be detected in 31/60 samples (51.7%) corresponding to 25 patients (25/47, 53.2%). We identified 44 distinct subclonal TP53 mutations clustered in the central DNA-binding domain of p53 protein (exons 5-8, codons 133-286). Missense mutations were predominant (>80%), whereas indels, nonsense, and splice site variants were less represented. All subclonal TP53 variants but one [p.(Pro191fs)] were already described in NCI and/or Seshat databases as "damaging" and/or "probably damaging" mutations (38/44, 86% and 6/44, 14%, respectively). Longitudinal samples were available for 37 patients. Almost half of them displayed at least one TP53 mutant subclone, which could be alone (4/16, 25%) or concomitant with other TP53 mutant clonal ones (12/16, 75%); different patterns of mutational dynamics overtimes were documented. In conclusion, utilization of NGS in our "real-life" cohort of CLL patients demonstrated an elevated frequency of subclonal TP53 mutations. This finding indicates the need for precisely identifying these mutations during disease since the clones carrying them may become predominant and be responsible for therapy failures.

MiR-146b-5p regulates IL-23 receptor complex expression in chronic lymphocytic leukemia cells.

Chronic lymphocytic leukemia (CLL) cells express the interleukin-23 receptor (IL-23R) chain, but the expression of the complementary IL-12Rß1 chain requires cell stimulation via surface CD40 molecules (and not via the B-cell receptor [BCR]). This stimulation induces the expression of a heterodimeric functional IL-23R complex and the secretion of IL-23, initiating an autocrine loop that drives leukemic cell expansion. Based on the observation in 224 untreated Binet stage A patients that the cases with the lowest miR-146b-5p concentrations had the shortest time to first treatment (TTFT), we hypothesized that miR-146b-5p could negatively regulate IL-12Rß1 side chain expression and clonal expansion. Indeed, miR-146b-5p significantly bound to the 3'-UTR region of the IL-12Rß1 mRNA in an in vitro luciferase assay. Downregulation of miR-146b-5p with specific miRNA inhibitors in vitro led to the upregulation of the IL-12Rß1 side chain and expression of a functional IL-23R complex similar to that observed after stimulation of the CLL cell through the surface CD40 molecules. Expression of miR-146b-5p with miRNA mimics in vitro inhibited the expression of the IL-23R complex after stimulation with CD40L. Administration of a miR-146b-5p mimic to NSG mice, successfully engrafted with CLL cells, caused tumor shrinkage, with a reduction of leukemic nodules and of IL-12Rß1-positive CLL cells in the spleen. Our findings indicate that IL-12Rß1 expression, a crucial checkpoint for the functioning of the IL-23 and IL-23R complex loop, is under the control of miR-146b-5p, which may represent a potential target for therapy since it contributes to the CLL pathogenesis. This trial is registered at www.clinicaltrials.gov as NCT00917540.

Target Therapies for Systemic Mastocytosis: An Update.

Systemic mastocytosis (SM) results from a clonal proliferation of abnormal mast cells (MCs) in extra-cutaneous organs. It could be divided into indolent SM, smoldering SM, SM with an associated hematologic (non-MC lineage) neoplasm, aggressive SM, and mast cell leukemia. SM is generally associated with the presence of a gain-of-function somatic mutation in KIT at codon 816. Clinical features could be related to MC mediator release or to uncontrolled infiltration of MCs in different organs. Whereas indolent forms have a near-normal life expectancy, advanced diseases have a poor prognosis with short survival times. Indolent forms should be considered for symptom-directed therapy, while cytoreductive therapy represents the first-line treatment for advanced diseases. Since the emergence of tyrosine kinase inhibitors (TKIs), KIT inhibition has been an attractive approach. Initial reports showed that only the rare KITD816V negative cases were responsive to first-line TKI imatinib. The development of new TKIs with activity against the KITD816V mutation, such as midostaurin or avapritinib, has changed the management of this disease. This review aims to focus on the available clinical data of therapies for SM and provide insights into possible future therapeutic targets.

Vaccination Therapy for Acute Myeloid Leukemia: Where Do We Stand?

Immunotherapy is changing the therapeutic landscape of many hematologic diseases, with immune checkpoint inhibitors, bispecific antibodies, and CAR-T therapies being its greatest expression. Unfortunately, immunotherapy in acute myeloid leukemia (AML) has given less brilliant results up to now, and the only approved drug is the antiCD33 antibody-drug conjugate gemtuzumab ozogamicin. A promising field of research in AML therapy relies on anti-leukemic vaccination to induce remission or prevent disease relapse. In this review, we analyze recent evidence on AML vaccines and their biological mechanisms. The principal proteins that have been exploited for vaccination strategies and have reached clinical experimental phases are Wilm's tumor 1, proteinase 3, and RHAMM. the majority of data deals with WT1-base vaccines, given also the high expression and mutation rates of WT1 in AML cells. Stimulators of immune responses such as TLR7 agonist and interleukin-2 have also proven anti-leukemic activity both in vivo and in vitro. Lastly, cellular vaccines mainly based on autologous or allogeneic off-the-shelf dendritic cell-based vaccines showed positive results in terms of T-cell response and safety, also in elderly patients. Compared to other immunotherapeutic strategies, anti-AML vaccines have the advantage of being a less toxic and a more manageable approach, applicable also to elderly patients with poorer performance status, and may be used in combination with currently available therapies. As for the best scenario in which to use vaccination, whether in a therapeutic, prophylactic, or preemptive setting, further studies are needed, but available evidence points to poorer results in the presence of active or high-burden disease. Given the poor prognosis of relapsed/refractory or high-risk AML, further research is urgently needed to better understand the biological pathways that sustain its pathogenesis. In this setting, research on novel frontiers of immunotherapy-based agents, among which vaccines represent important actors, is warranted to develop new and efficacious strategies to obtain long-term disease control by immune patrolling.

LINC00152 expression in normal and Chronic Lymphocytic Leukemia B cells.

Long non-coding RNAs are emerging as essential regulators of gene expression, but their role in normal and neoplastic B cells is still largely uncharacterized. Here, we report on the expression pattern of the LINC00152 in normal B cells and Chronic Lymphocytic Leukemia B cell clones. Higher LINC00152 levels were consistently observed in memory B cell populations when compared to naïve B cells in the normal tissues analyzed [peripheral blood (PB), tonsils, and spleen]. In addition, independent stimulation via Immunoglobulins (IG), CD40, or Toll-like Receptor 9 (TLR9) upregulated LINC00152 in PB B cells. The expression of LINC00152 in a cohort of 107 early stage Binet A CLL patients was highly variable and did not correlate with known prognostic markers or clinical evolution. TLR9 stimulation, but not CD40 or IG challenge, was able to upregulate LINC00152 expression in CLL cells. In addition, LINC00152 silencing in CLL cell lines expressing LINC00152 failed to induce significant cell survival or apoptosis changes. These data suggest that, in normal B cells, the expression of LINC00152 is regulated by immunomodulatory signals, which are only partially effective in CLL cells. However, LINC00152 does not appear to contribute to CLL cell expansion and/or survival in a cohort of newly diagnosed CLL patients.

Case Report: Evolution of KIT D816V-Positive Systemic Mastocytosis to Myeloid Neoplasm With PDGFRA Rearrangement Responsive to Imatinib.

Systemic mastocytosis (SM) is a rare neoplasm resulting from extracutaneous infiltration of clonal mast cells (MC). The clinical features of SM are very heterogenous and treatment should be highly individualized. Up to 40% of all SM cases can be associated with another hematological neoplasm, most frequently myeloproliferative neoplasms. Here, we present a patient with indolent SM who subsequently developed a myeloid neoplasm with PDGFRA rearrangement with complete response to low-dose imatinib. The 63-year-old patient presented with eosinophilia and elevated serum tryptase level. Bone marrow analysis revealed aberrant MCs in aggregates co-expressing CD2/CD25 and KIT D816V mutation (0.01%), and the FIP1L1-PDGFRA fusion gene was not identified. In the absence of 'B' and 'C' findings, we diagnosed an indolent form of SM. For 2 years after the diagnosis, the absolute eosinophil count progressively increased. Bone marrow evaluation showed myeloid hyperplasia and the FIP1L1-PDGFRA fusion gene was detected. Thus, the diagnosis of myeloid neoplasm with PDGFRA rearrangement was established. The patient was treated with imatinib 100 mg daily and rapidly obtained a complete molecular remission. The clinical, biological, and therapeutic aspects of SM might be challenging, especially when another associated hematological disease is diagnosed. Little is known about the underlying molecular and immunological mechanisms that can promote one entity prevailing over the other one. Currently, the preferred concept of SM pathogenesis is a multimutated neoplasm in which KIT mutations represent a "phenotype modifier" toward SM. Our patient showed an evolution from KIT mutated indolent SM to a myeloid neoplasm with PDGFRA rearrangement; when the eosinophilic component expanded, a regression of the MC counterpart was observed. In conclusion, extensive clinical monitoring associated with molecular testing is essential to better define these rare diseases and consequently their prognosis and treatment.

Lymphocyte Doubling Time As A Key Prognostic Factor To Predict Time To First Treatment In Early-Stage Chronic Lymphocytic Leukemia.

The prognostic role of lymphocyte doubling time (LDT) in chronic lymphocytic leukemia (CLL) was recognized more than three decades ago when the neoplastic clone's biology was almost unknown. LDT was defined as the time needed for the peripheral blood lymphocyte count to double the of the initial observed value. Herein, the LDT prognostic value for time to first treatment (TTFT) was explored in our prospective O-CLL cohort and validated in in two additional CLL cohorts. Specifically, newly diagnosed Binet stage A CLL patients from 40 Italian Institutions, representative of the whole country, were prospectively enrolled into the O-CLL1-GISL protocol (clinicaltrial.gov identifier: NCT00917540). Two independent cohorts of newly diagnosed CLL patients recruited respectively at the Division of Hematology in Novara, Italy, and at the Hospital Clinic in Barcelona, Spain, were utilized as validation cohorts. In the training cohort, TTFT of patients with LDT >12 months was significantly longer related to those with a shorter LDT. At Cox multivariate regression model, LDT ≤ 12 months maintained a significant independent relationship with shorter TTFT along with IGHV unmutated (IGHVunmut) status, 11q and 17p deletions, elevated ß2M, Rai stage I-II, and NOTCH1 mutations. Based on these statistics, two regression models were constructed including the same prognostic factors with or without the LDT. The model with the LTD provided a significantly better data fitting (χ2 = 8.25, P=0.0041). The risk prediction developed including LDT had better prognostic accuracy than those without LDT. Moreover, the Harrell'C index for the scores including LDT were higher than those without LDT, although the accepted 0.70 threshold exceeded in both cases. These findings were also confirmed when the same analysis was carried out according to TTFT's explained variation. When data were further analyzed based on the combination between LDT and IGHV mutational status in the training and validation cohorts, IGHVunmut and LDT>12months group showed a predominant prognostic role over IGHVmut LTD ≤ 12 months (P=0.006) in the O-CLL validation cohort. However, this predominance was of borden-line significance (P=0.06) in the Barcelona group, while the significant prognostic impact was definitely lost in the Novara group. Overall, in this study, we demonstrated that LDT could be re-utilized together with the more sophisticated prognostic factors to manage the follow-up plans for Binet stage A CLL patients.

Triple-Negative Essential Thrombocythemia: Clinical-Pathological and Molecular Features. A Single-Center Cohort Study.

Lack of demonstrable mutations affecting JAK2, CALR, or MPL driver genes within the spectrum of BCR-ABL1-negative myeloproliferative neoplasms (MPNs) is currently referred to as a triple-negative genotype, which is found in about 10% of patients with essential thrombocythemia (ET) and 5-10% of those with primary myelofibrosis (PMF). Very few papers are presently available on triple-negative ET, which is basically described as an indolent disease, differently from triple-negative PMF, which is an aggressive myeloid neoplasm, with a significantly higher risk of leukemic evolution. The aim of the present study was to evaluate the bone marrow morphology and the clinical-laboratory parameters of triple-negative ET patients, as well as to determine their molecular profile using next-generation sequencing (NGS) to identify any potential clonal biomarkers. We evaluated a single-center series of 40 triple-negative ET patients, diagnosed according to the 2017 WHO classification criteria and regularly followed up at the Hematology Unit of our Institution, between January 1983 and January 2019. In all patients, NGS was performed using the Illumina Ampliseq Myeloid Panel; morphological and immunohistochemical features of the bone marrow trephine biopsies were also thoroughly reviewed. Nucleotide variants were detected in 35 out of 40 patients. In detail, 29 subjects harbored one or two variants and six cases showed three or more concomitant nucleotide changes. The most frequent sequence variants involved the TET2 gene (55.0%), followed by KIT (27.5%). Histologically, most of the cases displayed a classical ET morphology. Interestingly, prevalent megakaryocytes morphology was more frequently polymorphic with a mixture of giant megakaryocytes with hyperlobulated nuclei, normal and small sized maturing elements, and naked nuclei. Finally, in five cases a mild degree of reticulin fibrosis (MF-1) was evident together with an increase in the micro-vessel density. By means of NGS we were able to identify nucleotide variants in most cases, thus we suggest that a sizeable proportion of triple-negative ET patients do have a clonal disease. In analogy with driver genes-mutated MPNs, these observations may prevent issues arising concerning triple-negative ET treatment, especially when a cytoreductive therapy may be warranted.

Time to first treatment and P53 dysfunction in chronic lymphocytic leukaemia: results of the O-CLL1 study in early stage patients.

Chronic lymphocytic leukaemia (CLL) is characterised by a heterogeneous clinical course. Such heterogeneity is associated with a number of markers, including TP53 gene inactivation. While TP53 gene alterations determine resistance to chemotherapy, it is not clear whether they can influence early disease progression. To clarify this issue, TP53 mutations and deletions of the corresponding locus [del(17p)] were evaluated in 469 cases from the O-CLL1 observational study that recruited a cohort of clinically and molecularly characterised Binet stage A patients. Twenty-four cases harboured somatic TP53 mutations [accompanied by del(17p) in 9 cases], 2 patients had del(17p) only, and 5 patients had TP53 germ-line variants. While del(17p) with or without TP53 mutations was capable of significantly predicting the time to first treatment, a reliable measure of disease progression, TP53 mutations were not. This was true for cases with high or low variant allele frequency. The lack of predictive ability was independent of the functional features of the mutant P53 protein in terms of transactivation and dominant negative potential. TP53 mutations alone were more frequent in patients with mutated IGHV genes, whereas del(17p) was associated with the presence of adverse prognostic factors, including CD38 positivity, unmutated-IGHV gene status, and NOTCH1 mutations.

Deregulation of miRNAs-cMYC circuits is a key event in refractory celiac disease type-2 lymphomagenesis.

A percentage of celiac disease (CD) patients develop refractory type-2 disease (RCD2), a condition associated with increased risk of enteropathy-associated T-cell-lymphoma (EATL) and without therapeutic option. Therefore, we profiled the miRNome in series of peripheral T-cell lymphomas (PTCLs), CD, RCD1 or 2 and in the murine interleukin-15 (IL15)-transgenic (TG) model of RCD. The transcriptome was analyzed in 18 intestinal T-cell lymphomas (ITLs). Bioinformatics pipelines provided significant microRNA (miRNA) lists and predicted targets that were confirmed in a second set of patients. Our data show that ITLs have a unique miRNA profile with respect to other PTCLs. The c-MYC regulated miR-17/92 cluster distinguishes monomorphic epitheliotropic ITL (MEITL) from EATL and prognosticates EATL outcome. These miRNAs are decreased in IL15-TG mice upon Janus kinase (JAK) inhibition. The random forest algorithm identified a signature of 38 classifier miRNAs, among which, the miR-200 and miR-192/215 families were progressively lost in RCD2 and ITL-CD, whereas miR-17/92 and C19MC miRNAs were up-regulated. Accordingly, SMAD3, MDM2, c-Myc and activated-STAT3 were increased in RCD2 and EATL tissues while JAK inhibition in IL15-TG mice restored their levels to baseline. Our data suggest that miRNAs circuit supports activation of STAT3 and c-Myc oncogenic signaling in RCD2, thus contributing to lymphomagenesis. This novel understanding might pave the way to personalized medicine approaches for RCD and EATL.

NEAT1 Long Isoform Is Highly Expressed in Chronic Lymphocytic Leukemia Irrespectively of Cytogenetic Groups or Clinical Outcome.

The biological role and therapeutic potential of long non-coding RNAs (lncRNAs) in chronic lymphocytic leukemia (CLL) are still open questions. Herein, we investigated the significance of the lncRNA NEAT1 in CLL. We examined NEAT1 expression in 310 newly diagnosed Binet A patients, in normal CD19+ B-cells, and other types of B-cell malignancies. Although global NEAT1 expression level was not statistically different in CLL cells compared to normal B cells, the median ratio of NEAT1_2 long isoform and global NEAT1 expression in CLL samples was significantly higher than in other groups. NEAT1_2 was more expressed in patients carrying mutated IGHV genes. Concerning cytogenetic aberrations, NEAT1_2 expression in CLL with trisomy 12 was lower with respect to patients without alterations. Although global NEAT1 expression appeared not to be associated with clinical outcome, patients with the lowest NEAT1_2 expression displayed the shortest time to first treatment; however, a multivariate regression analysis showed that the NEAT1_2 risk model was not independent from other known prognostic factors, particularly the IGHV mutational status. Overall, our data prompt future studies to investigate whether the increased amount of the long NEAT1_2 isoform detected in CLL cells may have a specific role in the pathology of the disease.