When Waldenström macroglobulinemia hits the kidney: Description of a case series and management of a "rare in rare" scenario.

BACKGROUND: Renal injury related to Waldenström macroglobulinemia (WM) occurs in approximately 3% of patients. Kidney biopsy is crucial to discriminate between distinct histopathological entities such as glomerular (amyloidotic and non-amyloidotic), tubulo-interstitial and non-paraprotein mediated renal damage. In this context, disease characterization, management, relationship between renal, and hematological response have been poorly explored. We collected clinical, genetic and laboratory data of seven cases of biopsy-proven renal involvement by WM managed at our academic center and focused on three cases we judged paradigmatic discussing their histopathological patterns, clinical features, and therapeutic options. CASE: In this illustrative case series, we confirm that serum creatinine levels and 24 h proteinuria are parameters that when altered should prompt the clinical suspicion of WM-related renal involvement, even if at present there are not precise cut-off levels recommending the execution of a renal biopsy. In our series AL Amyloidosis (n = 3/7) and tubulo-interstitial infiltration by lymphoma cells (n = 3/7) were the two more represented entities. BTKi did not seem to improve renal function (Case 1), while bortezomib-based regimens demonstrated a beneficial activity on the hematological and organ response, even when used as second-line therapy after chemoimmunotherapy (Case 3) and also with coexistence of anti-MAG neuropathy (Case 2). In case of poor response to bortezomib, standard chemoimmunotherapy (CIT), such as rituximab-bendamustine, represents an effective option (Case 1, 6, and 7). In our series, CIT generates durable responses more frequently in cases with amyloidogenic renal damage (Case 1, 5, and 7). CONCLUSION: In this illustrative case series, we confirm that serum creatinine levels and 24 h proteinuria are parameters that when altered should prompt the clinical suspicion of WM-related renal involvement, even if at present there are not precise cut-off levels recommending the execution of a renal biopsy. Studies with higher numerosity are needed to better clarify the pathological and clinical features of renal involvement during WM and to determine the potential benefit of different therapeutic regimens according to the histopathological subtypes.

Innovative Combinations, Cellular Therapies and Bispecific Antibodies for Chronic Lymphocytic Leukemia: A Narrative Review.

In the last few years, several agents targeting molecules that sustain the survival and the proliferation of chronic lymphocytic leukemia (CLL) cells have become clinically available. Most of these drugs target surface proteins, such as CD19 or CD20, via monoclonal or bispecific monoclonal antibodies (BsAbs), CAR T cells, intracellular proteins like BTK by using covalent or non-covalent inhibitors or BCL2 with first or second generation BH3-mimetics. Since the management of CLL is evolving quickly, in this review we highlighted the most important innovative treatments including novel double and triple combination therapies, CAR T cells and BsAbs for CLL. Recently, a large number of studies on novel combinations and newer strategic options for CLL therapy have been published or presented at international conferences, which were summarized and linked together. Although the management of treatment with a single continuous agent is easier, the emergence of protein mutations, long-term toxicities and costs are important concerns that favor the use of a fixed duration therapy. In the future, a measurable residual disease (MRD)-guided treatment cessation and MRD-based re-initiation of targeted therapy seems to be a more feasible approach, allowing identification of the patients who might benefit from continuous therapy or who might need a consolidation with BsAbs or CAR T cells to clear the neoplastic clone.

Chronic Lymphocytic Leukemia (CLL) with Borderline Immunoglobulin Heavy Chain Mutational Status, a Rare Subgroup of CLL with Variable Disease Course.

Chronic lymphocytic leukemia (CLL) exhibits substantial variability in disease course. The mutational status of the B-cell receptor immunoglobulin heavy variable (IGHV) chain is a critical prognostic factor, categorizing patients into mutated (M-IGHV) and unmutated (U-IGHV) groups. Recently, a third subgroup with borderline mutational status (BL-IGHV) has been identified, comprising approximately 5% of CLL cases. This study retrospectively analyzes the outcomes of 30 BL-IGHV mutated patients among a cohort of 653 CLL patients, focusing on time to first treatment (TTFT) and overall survival (OS). BL-IGHV patients had a short TTFT similar to U-IGHV patients (median 30.2 vs. 34 months; p = 0.9). Conversely, the OS of BL-IGHV patients resembled M-IGHV patients (median NR vs. 258 months; p = 1). Despite a similar incidence in unfavorable prognostic factors, the TTFT was shorter compared to other published cohorts. However, striking similarities with other experiences suggest that BL-IGHV mutated patients share common biological characteristics, biased IGHV gene usage and BCR subset frequency. These findings also underscore the need for multicentric efforts aggregating data on BL-IGHV CLL in order to elucidate its disease course and optimize therapeutic approaches for this rare subgroup. Until then, predicting outcomes and optimal management of BL-IGHV CLL will remain challenging.

Real-World Outcome of Treatment with Single-Agent Ibrutinib in Italian Patients with Chronic Lymphocytic Leukemia: Final Results of the EVIdeNCE Study.

Real-world data in clinical practice are needed to confirm the efficacy and safety that ibrutinib has demonstrated in clinical trials of patients with chronic lymphocytic leukemia (CLL). We described the real-world persistence rate, patterns of use, and clinical outcomes in 309 patients with CLL receiving single-agent ibrutinib in first line (1L, n = 118), 2L (n = 127) and ≥3L (n = 64) in the prospective, real-world, Italian EVIdeNCE study. After a median follow-up of 23.9 months, 29.8% of patients discontinued ibrutinib (1L: 24.6%, 2L: 29.9%, ≥3L: 39.1%), mainly owing to adverse events (AEs)/toxicity (14.2%). The most common AEs leading to discontinuation were infections (1L, ≥3L) and cardiac events (2L). The 2-year retention rate was 70.2% in the whole cohort (1L: 75.4%, 2L: 70.1%, ≥3L: 60.9%). The 2-year PFS and OS were, respectively, 85.4% and 91.7% in 1L, 80.0% and 86.2% in 2L, and 70.1% and 80.0% in ≥3L. Cardiovascular conditions did not impact patients' clinical outcomes. The most common AEs were infections (30.7%), bleeding (12.9%), fatigue (10.0%), and neutropenia (9.7%), while grade 3-4 atrial fibrillation occurred in 3.9% of patients. No new safety signals were detected. These results strongly support ibrutinib as a valuable treatment option for CLL.

p66Shc deficiency in CLL cells enhances PD-L1 expression and suppresses immune synapse formation.

Introduction: Escape from immunosurveillance is a hallmark of chronic lymphocytic leukemia (CLL) cells. In the protective niche of lymphoid organs, leukemic cells suppress the ability of T lymphocytes to form the immune synapse (IS), thereby hampering T-cell mediated anti-tumoral activities. By binding its cognate receptor PD-1 at the surface of T lymphocytes, the inhibitory ligand PD-L1, which is overexpressed in CLL cells, mediates the T-cell suppressive activities of CLL cells. However, the molecular mechanism underlying PD-L1 overexpression in CLL cells remains unknown. We have previously reported a defective expression of the pro-apoptotic and pro-oxidant adaptor p66Shc in CLL cells, which is causally related to an impairment in intracellular reactive oxygen species (ROS) production and to the activation of the ROS-sensitive transcription factor NF-κB. The fact that PD-L1 expression is regulated by NF-κB suggests a mechanistic relationship between p66Shc deficiency and PD-L1 overexpression in CLL cells. Methods: 62 treatment-naive CLL patients and 43 healthy donors were included in this study. PD-L1 and p66Shc expression was quantified in B cells by flow cytometry and qRT-PCR. IS architecture and local signaling was assessed by flow cytometry and confocal microscopy. CD8+ cell killing activity was assessed by flow cytometry. Results: Here we show that residual p66Shc expression in leukemic cells isolated both from CLL patients and from the CLL mouse model Eµ-TCL1 inversely correlated with PD-L1 expression. We also show that the PD-L1 increase prevented leukemic cells from forming ISs with T lymphocytes. Reconstitution of p66Shc, but not of a ROS-defective mutant, in both CLL cells and the CLL-derived cell line MEC-1, enhanced intracellular ROS and decreased PD-L1 expression. Similar results were obtained following treatment of CLL cells with H2O2 as exogenous source of ROS, that normalized PD-L1 expression and recovered IS formation. Discussion: Our data provide direct evidence that the p66Shc-deficiency-related ROS depletion in CLL cells concurs to enhance PD-L1 expression and provides a mechanistic basis for the suppression of T cell-mediated anti-tumoral functions in the immunosuppressive lymphoid niche.

Leukemic cell-secreted interleukin-9 suppresses cytotoxic T cell-mediated killing in chronic lymphocytic leukemia.

The tumor microenvironment (TME) plays a central role in the pathogenesis of chronic lymphocytic leukemia (CLL), contributing to disease progression and chemoresistance. Leukemic cells shape the TME into a pro-survival and immunosuppressive niche through contact-dependent and contact-independent interactions with the cellular components of the TME. Immune synapse (IS) formation is defective in CLL. Here we asked whether soluble factors released by CLL cells contribute to their protection from cytotoxic T cell (CTL)-mediated killing by interfering with this process. We found that healthy CTLs cultured in media conditioned by leukemic cells from CLL patients or Eµ-TCL1 mice upregulate the exhaustion marker PD-1 and become unable to form functional ISs and kill target cells. These defects were more pronounced when media were conditioned by leukemic cells lacking p66Shc, a proapoptotic adapter whose deficiency has been implicated in disease aggressiveness both in CLL and in the Eµ-TCL1 mouse model. Multiplex ELISA assays showed that leukemic cells from Eµ-TCL1 mice secrete abnormally elevated amounts of CCL22, CCL24, IL-9 and IL-10, which are further upregulated in the absence of p66Shc. Among these, IL-9 and IL-10 were also overexpressed in leukemic cells from CLL patients, where they inversely correlated with residual p66Shc. Using neutralizing antibodies or the recombinant cytokines we show that IL-9, but not IL-10, mediates both the enhancement in PD-1 expression and the suppression of effector functions in healthy CTLs. Our results demonstrate that IL-9 secreted by leukemic cells negatively modulates the anti-tumor immune abilities of CTLs, highlighting a new suppressive mechanism and a novel potential therapeutical target in CLL.

Ibrutinib as first line therapy in chronic lymphocytic leukemia patients over 80 years old: A retrospective real-life multicenter Italian cohort.

Although chronic lymphocytic leukemia (CLL) predominantly affects the elderly, limited data exists about the outcomes of over 80-year-old patients, usually underrepresented in clinical trials. We conducted a multicenter study enrolling 79 consecutive CLL patients ≥80 years at the time of frontline therapy, all treated with ibrutinib. Nearly 48% of cases exhibited unmutated IGHV genes, 32% 17p deletion, and 39.2% TP53 mutations; 63.3% displayed a cumulative illness rating scale (CIRS) > 6. The overall response rate on ibrutinib, computed in 74/79 patients (5 patients excluded for early withdrawal), was 89.9%. After a median follow-up of 28.9 months, the median progression-free survival (PFS) and overall survival (OS) were 42.5 and 51.8 months, respectively. CIRS>6 and temporary discontinuation of ibrutinib lasting for 7-30 days were the only parameters associated with a significantly shorter PFS and were both relevant in predicting a shorter PFS compared to patients with CIRS≤6 and therapy discontinuation ≤7 days. The most common grade≥3 adverse events were infections (25.5%), neutropenia (10.1%), and anemia (2.5%). Eighteen patients (22.8%) experienced a cardiovascular event, including grade-2 atrial fibrillation (n = 9; 11%), grade-2 hypertension (n = 5; 6%), heart failure (n = 3; 3%), and acute coronary syndrome (n = 1; 1%). Mild bleeding events were observed in 27 patients (34.2%). Ibrutinib was permanently discontinued in 26 patients due to progressive disease (n = 11, including 5 Richter's syndromes), secondary malignancies (n = 6), infections (n = 3), cardiac failure (n = 3), severe bleeding (n = 2), and sudden death (n = 1). In conclusion, our analyses confirmed the overall effectiveness and favorable safety profile of the ibrutinib-single agent therapeutic approach in CLL patients ≥80 years.

Patients' preferences for chronic lymphocytic leukemia treatment: The CHOICE study.

Chronic lymphocytic leukemia (CLL) therapies differ in efficacy, side effects, route, frequency, and duration of administration. We assessed patient preferences for treatment attributes and evaluated associations with disease stage, treatment line, and socio-demographic characteristics in a cross sectional, observational study conducted at 16 Italian hematology centers. Study visits occurred between February and July 2020; 401 adult patients with CLL (201 Watch and Wait (W&W), 200 treated) participated in a discrete choice experiment (DCE), composed of 8 choices between pairs of treatment profiles with different levels of 5 attributes of currently available CLL treatments (length of response, route and duration of administration, risk of side effects including diarrhea, infections, or organ damage). Health-related quality of life was assessed with the EQ-5D-5L, EORTC QLQ-C30 and QLQ CLL-16. Previously treated patients had longer disease duration (7 vs. 5 years), higher prevalence of serious comorbidities (45.5% vs. 36.2%) and high-risk molecular markers (unmutated IGHV 55.6% vs. 17.1%; TP53 mutation 15.2% vs. 4.0%). Health-related quality of life scores were similar between groups. In the DCE, W&W patients rated "possible occurrence of infections" highest (relative importance [RI] = 36.2%), followed by "treatment and relevant duration" (RI = 28.0%) and "progression-free survival (PFS)" (RI = 16.9%). Previously treated patients rated "treatment and relevant duration" highest (RI = 33.3%), followed by "possible occurrence of infections" (RI = 28.8%), "possible occurrence of organ damage" (RI = 19.4%), and "PFS" (RI = 9.8%). Concern over infection was rated highest overall; unexpectedly PFS was not among the most important criteria in either group, suggesting that the first COVID-19 pandemic wave may have influenced patient preferences and concerns about CLL therapy options.

Pro-inflammatory cells sustain leukemic clonal expansion in T-cell large granular lymphocyte leukemia.

T-cell large granular lymphocyte leukemia (T-LGLL) is a chronic lymphoproliferative disorder characterized by the clonal expansion of T-cell large granular lymphocytes (T-LGL). Immunophenotypic and genotypic features contribute to discriminate symptomatic (CD8+ STAT3-mutated T-LGLL) from clinically indolent patients, this latter group including CD8+ wildtype (wt), CD4+ STAT5B-mutated and wt cases. T-LGL lymphoproliferation is sustained both by somatic gain-offunction mutations (i.e., STAT3 and STAT5B) and by pro-inflammatory cytokines, but little information is available on the activity of T-LGLL non-leukemic cells. In this study, we characterized pro-inflammatory cells in the peripheral blood of T-LGLL patients and analyzed their role in supporting the leukemic growth. In symptomatic patients we found that cell populations not belonging to the leukemic component showed a discrete pro-inflammatory pattern. In particular, CD8+ STAT3-mutated cases showed a skewed Th17/Treg ratio and an abnormal distribution of monocyte populations characterized by increased intermediate and non-classical monocytes. We also demonstrated that monocytes released high levels of interleukin-6 after CCL5 stimulation, a chemokine specifically expressed only by leukemic LGL. Conversely, in asymptomatic cases an altered distribution of monocyte populations was not detected. Moreover, T-LGLL patients' monocytes showed abnormal activation of signaling pathways, further supporting the different pathogenic role of monocytes in patients in discrete clinical settings. Altogether, our data contribute to deepening the knowledge on the different cell subtypes in T-LGLL, focusing particularly on non-leukemic cell populations and thus offering the rationale for new therapeutic strategies.

CK2ß Regulates Hematopoietic Stem Cell Biology and Erythropoiesis.

The Ser-Thr kinase CK2 plays important roles in sustaining cell survival and resistance to stress and these functions are exploited by different types of blood tumors. Yet, the physiological involvement of CK2 in normal blood cell development is poorly known. Here, we discovered that the ß regulatory subunit of CK2 is critical for normal hematopoiesis in the mouse. Fetal livers of conditional CK2ß knockout embryos showed increased numbers of hematopoietic stem cells associated to a higher proliferation rate compared to control animals. Both hematopoietic stem and progenitor cells (HSPCs) displayed alterations in the expression of transcription factors involved in cell quiescence, self-renewal, and lineage commitment. HSPCs lacking CK2ß were functionally impaired in supporting both in vitro and in vivo hematopoiesis as demonstrated by transplantation assays. Furthermore, KO mice developed anemia due to a reduced number of mature erythroid cells. This compartment was characterized by dysplasia, proliferative defects at early precursor stage, and apoptosis at late-stage erythroblasts. Erythroid cells exhibited a marked compromise of signaling cascades downstream of the cKit and erythropoietin receptor, with a defective activation of ERK/JNK, JAK/STAT5, and PI3K/AKT pathways and perturbations of several transcriptional programs as demonstrated by RNA-Seq analysis. Moreover, we unraveled an unforeseen molecular mechanism whereby CK2 sustains GATA1 stability and transcriptional proficiency. Thus, our work demonstrates new and crucial functions of CK2 in HSPC biology and in erythropoiesis.

Severe Fatal Mucormycosis in a Patient with Chronic Lymphocytic Leukaemia Treated with Zanubrutinib: A Case Report and Review of the Literature.

Severe mucormycosis is a fatal disease rarely complicating chronic lymphoproliferative disorders. We present a fulminant and fatal case of a 74-year-old Caucasian woman suffering from CLL treated with second-generation BTK inhibitor zanubrutinib. After a first septic episode a month prior, originating from the lung with later systemic involvement by an unidentified agent and treated with large-spectrum antibiotics and fluconazonle, a slow-onset enlarging tender warm and erythematous nodular swollen cutaneous lesion appeared in her lower limbs and spread subsequently to her upper limbs, progressing towards central ulceration with a necrotic core. Suspecting a mycotic dissemination from an unknown agent, a skin punch biopsy was performed, and intraconazole was started. Due to spread of the skin lesions, the patient was hospitalized and intravenous liposomal ampthotericin B was started. Histopathology showed an atypical sporangium-rich mycotic angioinvasion of the small vessels. Only the increase of BDG and GM could corroborate the hypothesis of mycotic infection. However, long-term CLL, immunosuppressive therapies, neutropenia, and prior use of azoles and other antimycotic agents were risk factors for mucormycosis; BTK inhibitor could also be added as another novel risk factor. Despite all therapeutic efforts, the patient died. Post-mortem molecular exams confirmed the diagnosis of disseminated mucormycosis.

Lymphadenopathy as a predictor of progression during venetoclax treatment in chronic lymphocytic leukemia. A campus chronic lymphocytic leukemia study.

Clinical or biological parameters useful to predict progression during treatment in real-life setting with ibrutinib, idelalisib and venetoclax in relapsed/refractory chronic lymphocytic leukemia (CLL) are still debated. We conducted a multi-center retrospective study on CLL patients treated with ibrutinib and/or idelalisib who were switched to venetoclax for progression or due to adverse events to identify any clinical and/or biological parameters useful to predict progression during treatment with venetoclax. Of all the 128 evaluable patients, 81 had received ibrutinib prior to switching to venetoclax, 35 had received idelalisib and 12 both. When comparing the three subgroups, we did not notice any statistical difference in terms of clinical or biological features. No variable at baseline and at different time points during the follow-up (at 6, 12, 18 and 24 months) was found to predict progression nor to have significance for Progression Free Survival (PFS) in the ibrutinib group and in the idelalisib group and in subgroups according to the line of treatment. Analyzing the data of the venetoclax treatment, after a median follow up of 14.3 months, median PFS was not reached and estimated 3-year PFS was 54%. Of the 128 patients treated with venetoclax, 28 (22%) experienced progressive disease. At multivariate analysis for predictive factors for progression, lymph node diameter >56.5 mm before starting treatment emerged as an independent risk factor for progression. The lymph node predictive role for progression during venetoclax treatment could be a new parameter that deserves to be investigate in future studies.

Focal adhesion kinase activation by calcium-dependent calpain is involved in chronic lymphocytic leukaemia cell aggressiveness.

Signalling events downstream the B-cell receptor (BCR) are central for the survival and progression of chronic lymphocytic leukaemia (CLL) cells. Focal adhesion kinase (FAK), regulated through calpain, interacts with molecules of BCR signalling, cytoskeletal modelling and disease progression, such as Src/Lyn, cortactin and HS1. Hypothesizing that FAK might play a key role in CLL pathogenesis, we observed a down-modulation of FAK whole form, associated with FAK cleavage due to calpain activity upon BCR stimulation. Patients, whose cells were able to release Ca++ after BCR stimulation, had less amount of full-length FAK, which translated into a higher presence of cleaved/activated form of the protein phosphorylated at Y397, these features being mostly shown by immunoglobulin heavy chain (IGHV)-unmutated poor-prognosis patients. Moreover, we found that cortactin and HS1 proteins were overexpressed in those cells, suggesting a possible interplay with FAK. Treatment with the FAK inhibitor Defactinib was able to induce apoptosis in CLL cells. In conclusion, the malignant phenotype in unfavourable-prognosis patients seems to be encouraged by the overexpression of cortactin and HS1, that, together with FAK, may be involved in a druggable pathogenetic pathway in CLL.

Tixagevimab/Cilgavimab as pre-exposure prophylaxis against SARS-CoV-2 in patients with hematological malignancies.

The approved combination of Tixagevimab/Cilgavimab has been shown to decrease the rate of symptomatic SARS-CoV-2 infection in patients at increased risk of inadequate response to vaccination. However, Tixagevimab/Cilgavimab was tested in a few studies that included patients with hematological malignancies, even if this population has shown an increased risk of unfavorable outcomes following infection (with high rates of hospitalization, intensive care unit admission, and mortality) and poor significant immunization following vaccines. We performed a real-life prospective cohort study to evaluate the rate of SARS-CoV-2 infection following pre-exposure prophylaxis with Tixagevimab/Cilgavimab in anti-spike seronegative patients compared to a cohort of seropositive patients who were observed or received a fourth vaccine dose. We recruited 103 patients with a mean age of 67 years: 35 (34%) received Tixagevimab/Cilgavimab and were followed from March 17, 2022, until November 15, 2022. After a median follow-up of 4.24 months, the 3-month cumulative incidence of infection was 20% versus 12% in the Tixagevimab/Cilgavimab and observation/vaccine groups respectively (HR 1.57; 95% CI: 0.65-3.56; p = 0.34). In this study, we report our experience with Tixagevimab/Cilgavimab and a tailored approach to SARS-CoV-2 infection prevention in patients with hematological malignancies during the SARS-CoV-2 omicron surge.