Carfilzomib, lenalidomide, dexamethasone (KRD) in BTKi relapsed or refractory mantle cell lymphoma: A phase II study from Fondazione Italiana Linfomi.

Mantle cell lymphoma (MCL) is a rare lymphoproliferative neoplasm considered incurable, with a median survival of 3-5 years. In recent years, Bruton's tyrosine kinase inhibitors (BTKi) have been introduced, demonstrating high therapeutic activity. However, the prognosis for MCL patients failing ibrutinib therapy is particularly poor, with a survival expectation of a few months. In this phase II trial, we assessed the efficacy and safety of the carfilzomib-lenalidomide-dexamethasone (KRD) combination in MCL patients who were relapsed/refractory (R/R) or intolerant to BTKi and in need of treatment. The primary objective of the study was to evaluate the antitumor efficacy of the KRD combination in terms of 12-month overall survival (12-month OS). From September 2019 to December 2020, 16 patients were enrolled from 11 Italian centers. After a median follow-up of 2.37 months (95% CI 0.92-6.47), the 12-month OS was 13%. The rate of grade 3-4 adverse events (AEs) was 35%, and the overall response rate (ORR) was 19%. These results led to the premature termination of enrollment, as defined in the protocol stopping rules. The efficacy of the KRD combination in advanced-stage MCL patients who are R/R to BTKi is unsatisfactory and too toxic.

Secondary primary malignancies after CD-19 directed CAR-T-cell therapy in lymphomas: A report from the Italian CART-SIE study.

Secondary primary malignancies (SPM) have been reported after anti-BCMA or anti-CD19 chimeric antigen receptor (CAR)-T-cell therapies. While the cytotoxic effect of antecedent therapies, including chemotherapy and radiotherapy, has been well established, few data are available on risk related to CAR-T immunotherapies. The study aimed to analyse the incidence of SPM in 651 patients enrolled in the Italian prospective observational CART-SIE study. SPMs were documented in 4.3% (28/651), and the most frequent SPMs were haematological malignancies. In conclusion, the frequency of SPMs in our cohort of heavily pretreated patients receiving CAR-T was relatively low and consistent with previous studies.

XPO1 mutations identify early-stage CLL characterized by shorter time to first treatment and enhanced BCR signalling.

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.

CD49d promotes disease progression in chronic lymphocytic leukemia: new insights from CD49d bimodal expression.

CD49d is a remarkable prognostic biomarker of chronic lymphocytic leukemia (CLL). The cutoff value for the extensively validated 30% of positive CLL cells is able to separate CLL patients into 2 subgroups with different prognoses, but it does not consider the pattern of CD49d expression. In the present study, we analyzed a cohort of 1630 CLL samples and identified the presence of ∼20% of CLL cases (n = 313) characterized by a bimodal expression of CD49d, that is, concomitant presence of a CD49d+ subpopulation and a CD49d- subpopulation. At variance with the highly stable CD49d expression observed in CLL patients with a homogeneous pattern of CD49d expression, CD49d bimodal CLL showed a higher level of variability in sequential samples, and an increase in the CD49d+ subpopulation over time after therapy. The CD49d+ subpopulation from CD49d bimodal CLL displayed higher levels of proliferation compared with the CD49d- cells; and was more highly represented in the bone marrow compared with peripheral blood (PB), and in PB CLL subsets expressing the CXCR4dim/CD5bright phenotype, known to be enriched in proliferative cells. From a clinical standpoint, CLL patients with CD49d bimodal expression, regardless of whether the CD49d+ subpopulation exceeded the 30% cutoff or not, experienced clinical behavior similar to CD49d+ CLL, both in chemoimmunotherapy (n = 1522) and in ibrutinib (n = 158) settings. Altogether, these results suggest that CD49d can drive disease progression in CLL, and that the pattern of CD49d expression should also be considered to improve the prognostic impact of this biomarker in CLL.

SF3B1-mutated chronic lymphocytic leukemia shows evidence of NOTCH1 pathway activation including CD20 downregulation

Chronic lymphocytic leukemia (CLL) is characterized by a low CD20 expression, in part explained by an epigenetic-driven downregulation triggered by mutations of the NOTCH1 gene. In the present study, by taking advantage of a wide and well-characterized CLL cohort (n=537), we demonstrate that CD20 expression is downregulated in SF3B1-mutated CLL in an extent similar to NOTCH1-mutated CLL. In fact, SF3B1-mutated CLL cells show common features with NOTCH1-mutated CLL cells, including a gene expression profile enriched of NOTCH1-related gene sets and elevated expression of the active intracytoplasmic NOTCH1. Activation of the NOTCH1 signaling and down-regulation of surface CD20 in SF3B1-mutated CLL cells correlate with over-expression of an alternatively spliced form of DVL2, a component of the Wnt pathway and negative regulator of the NOTCH1 pathway. These findings are confirmed by separately analyzing the CD20-dim and CD20-bright cell fractions from SF3B1-mutated cases as well as by DVL2 knock-out experiments in CLL-like cell models. Altogether, the clinical and biological features that characterize NOTCH1-mutated CLL may also be recapitulated in SF3B1-mutated CLL, contributing to explain the poor prognosis of this CLL subset and providing the rationale for expanding novel agents-based therapies to SF3B1-mutated CLL.

Nilotinib Treatment of Patients Affected by Chronic Graft-versus-Host Disease Reduces Collagen Production and Skin Fibrosis by Downmodulating the TGF-ß and p-SMAD Pathway.

The present study was conducted to investigate cellular and molecular features of chronic graft-versus-host disease fibroblasts (GVHD-Fbs) and to assess the effectiveness of nilotinib as a fibrosis modulator. Growth kinetics, phenotype, and differentiation of cultured skin biopsy-derived GVHD-Fbs were compared with normal fibroblasts from both a dermal cell line (n-Fbs) and healthy individuals undergoing cosmetic surgery (n-skin-Fbs). Collagen genes (COL1α1/COL1α2) and p-SMAD2 expression were assessed by real-time PCR and immunofluorescence. The in vivo effects of nilotinib on chronic GVHD (cGVHD)-affected skin were investigated by immunohistochemistry; the relationship to TGF-ß plasma levels was assessed. Although the morphology, phenotype, and differentiation of cultured GVHD-Fbs were comparable to normal fibroblasts, growth was slower and senescence was reached earlier. The expression of COL1α1 and COL1α2 mRNAs was respectively 4 and 1.6 times higher in cGVHD-Fbs (P = .02); the addition of TGF-ß increased n-Fbs, but not GVHD-Fbs, collagen gene expression. Compared with the baseline, the addition of 1 µM nilotinib induced 86.5% and 49% reduction in COL1α1 and COL1α2 expression in cultured GVHD-Fbs, respectively (P< .01). In vivo immunohistochemistry analysis of skin biopsy specimens from patients with cGVHD showed strong baseline staining for COL1α1 and COL1α2, which decreased sharply after 180 days of nilotinib; immunofluorescence revealed TGF-ß inhibition and p-Smad2 reduction at the intracellular level. Of note, nilotinib treatment was associated with normalization of TGF-ß levels both in culture supernatants and in plasma. In general, the data show that cGVHD fibroblasts promote fibrosis through abnormal collagen production induced by hyperactive TGF-ß signaling. TGF-ß inhibition at the intracellular and systemic level represents an essential antifibrotic mechanism of nilotinib in a clinical setting.

A novel method to evaluate prethawing viability of cryopreserved CD34+ hematopoietic stem cells for autologous transplantation.

BACKGROUND: Cryopreservation of CD34+ hematopoietic stem cells (HSCs) is associated with variable loss of viability. Although postfreezing CD34+ cell viability can be assessed on the sampling tube (bag tail) directly connected to the main bag (mother bag), results often underestimate the actual viability observed when the mother bag is thawed and reinfused. We assessed a novel method to measure postfreezing CD34+ cell viability, based on small bag (minibag) samples; results were compared with those obtained on the corresponding mother bags and bag tails. STUDY DESIGN AND METHODS: Sixty-one apheresis procedures of 42 patients undergoing autologous HSC transplant were analyzed. Viable CD34+ cells were quantified with flow cytometry before controlled rate freezing (ICE-CUBE14M system, SY-LAB- IceCube, SIAD), after 10 days of storage (mini-bag and bag tail), and before reinfusion (aliquot from a thawed mother bag). Results were compared using Student's t test and Spearman's rho correlation test. RESULTS: The mean CD34+ cell viability before cryopreservation was 99.3% (confidence interval [CI], 98.94-99.65%); the mean amount of CD34+ cells, white blood cells and neutrophils in the mother bag was 0.8 ± 1.1 × 109 /L, 63.4 ± 23.5 × 109 /L, and 25.7 ± 15.5 × 109 /L, respectively. Mother bags postthawing CD34+ cell viability was 72.3% (CI, 67.74-76.85%; p < 0.01 compared to prefreezing); no difference was observed with respect to minibags (73.7%; CI, 69.80-77.59%; p = NS), whereas significantly lower values were found for bag tails (58.6%; CI, 54.19-63.00%; p < 0.01 vs. both mini- and mother bags). CONCLUSION: Compared to bag tails, minibags represent a more accurate tool to measure the CD34+ cell viability of the apheresis mother bag prior to reinfusion; in addition, minibags may could be of help for case-by-case calculation of the amount of apheresis to be infused to patients undergoing autologous HSC transplant.

Antiemetic prophylaxis in patients undergoing hematopoietic stem cell transplantation: a multicenter survey of the Gruppo Italiano Trapianto Midollo Osseo (GITMO) transplant programs.

A survey within hematopoietic stem cell transplant (HSCT) centers of the Gruppo Italiano Trapianto Midollo Osseo (GITMO) was performed in order to describe current antiemetic prophylaxis in patients undergoing HSCT. The multicenter survey was performed by a questionnaire, covering the main areas on chemotherapy-induced nausea and vomiting (CINV): antiemetic prophylaxis guidelines used, antiemetic prophylaxis in different conditioning regimens, and methods of CINV evaluation. The survey was carried out in November 2016, and it was repeated 6 months after the publication of the Multinational Association of Supportive Care in Cancer (MASCC)/European Society for Medical Oncology (ESMO) specific guidelines on antiemetic prophylaxis in HSCT. The results show a remarkable heterogeneity of prophylaxis among the various centers and a significant difference between the guidelines and the clinical practice. In the main conditioning regimens, the combination of a serotonin3 receptor antagonist (5-HT3-RA) with dexamethasone and neurokin1 receptor antagonist (NK1-RA), as recommended by MASCC/ESMO guidelines, increased from 0 to 15% (before the publication of the guidelines) to 9-30% (after the publication of the guidelines). This study shows a lack of compliance with specific antiemetic guidelines, resulting mainly in under-prophylaxis. Concerted strategies are required to improve the current CINV prophylaxis, to draft shared common guidelines, and to increase the knowledge and the adherence to the current recommendations for CINV prophylaxis in the specific field of HSCT.

Outcome of Allogeneic Hematopoietic Stem Cell Transplantation in Adult Patients with Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia in the Era of Tyrosine Kinase Inhibitors: A Registry-Based Study of the Italian Blood and Marrow Transplantation Society (GITMO).

We performed a nationwide registry-based analysis to describe the clinical outcome of adult patients with Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) who underwent an allogeneic hematopoietic stem cell transplantation (HSCT) after a tyrosine kinase inhibitor (TKI)-based treatment A total of 441 patients were included in the study. The median age at HSCT was 44 years (range, 18 to 70 years). All 441 patients (100%) received TKI before HSCT (performed between 2005 and 2016). Of these 441 patients, 404 (92%) were in cytologic complete remission (CR), whereas the remaining 37 (8%) had active disease at the time of HSCT. Molecular minimal residual disease (MRD) was negative in 147 patients (36%) at the time of HSCT. The donor was unrelated in 46% of patients. The most prevalent source of stem cells was peripheral blood (70%). The conditioning regimen was myeloablative in 82% of cases (total body irradiation-based in 50%) and included antithymocyte globulin in 51% of patients. With a median follow-up after HSCT of 39.4 months (range, 1 to 145 months), the probability of overall survival (OS) at 1, 2, and 5 years was 69.6%, 61.1% and 50.3%, respectively, with a median OS of 62 months. Progression-free survival (PFS) at 1, 2, and 5 years was 60.2%, 52.1% and 43.7%, respectively. OS and PFS were significantly better in patients who were in CR and MRD-negative at the time of HSCT compared with patients who were in CR but MRD-positive (50% OS not reached versus 36 months; P = .015; 50% PFS not reached versus 26 months, P = .003). The subgroup of MRD-negative patients both at HSCT and at 3 months after HSCT had a better outcome (5-year OS, 70%). Conversely, the 37 patients who underwent a HSCT with active Ph+ ALL had a median OS of 7 months and a median PFS of 5 months. The 5-year cumulative incidence of relapse was significantly lower in MRD-negative patients (19.5% versus 35.4%; P = .001). Nonrelapse mortality (NRM) after 1, 2, and 5 years was 19.1% (95% confidence interval [CI], 15.5% to 22.9%), 20.7% (95% CI, 17% to 24.7%), and 24.1% (95% CI, 20% to 28.5%), respectively. NRM was significantly lower with a modified European Society for Blood and Marrow Transplantation (mEBMT) risk score of 0 to 2 compared with ≥3 (15% versus 25%; P = .016). The median OS for Ph+ ALL patients who underwent a TKI-based treatment followed by an allogeneic HSCT, in recent years at the GITMO centers, was 62 months. Evaluation of the mEBMT risk score can be useful to predict NRM. Our data confirm that HSCT is a potentially curative treatment for Ph+ ALL with an excellent outcome for the subgroup of MRD-negative patients both at HSCT and at 3 months after HSCT (5-year OS, 70%).

Immunomodulatory Effects of Tyrosine Kinase Inhibitor In Vitro and In Vivo Study.

Pathogenesis of chronic graft-versus-host disease (cGVHD) is incompletely defined, involving donor-derived CD4 and CD8-positive T lymphocytes as well as B cells. Standard treatment is lacking for steroid-dependent/refractory cases; therefore, the potential usefulness of tyrosine kinase inhibitors (TKIs) has been suggested, based on their potent antifibrotic effect. However, TKIs seem to have pleiotropic activity. We sought to evaluate the in vitro and in vivo impact of different TKIs on lymphocyte phenotype and function. Peripheral blood mononuclear cells (PBMCs) from healthy donors were cultured in the presence of increasing concentrations of nilotinib, imatinib, dasatinib, and ponatinib; in parallel, 44 PBMC samples from 15 patients with steroid-dependent/refractory cGVHD treated with nilotinib in the setting of a phase I/II trial were analyzed at baseline, after 90, and after 180 days of therapy. Flow cytometry was performed after labeling lymphocytes with a panel of monoclonal antibodies (CD3, CD4, CD16, CD56, CD25, CD19, CD45RA, FoxP3, CD127, and 7-amino actinomycin D). Cytokine production was assessed in supernatants of purified CD3+ T cells and in plasma samples from nilotinib-treated patients. Main T lymphocyte subpopulations were not significantly affected by therapeutic concentrations of TKIs in vitro, whereas proinflammatory cytokine (in particular, IL-2, IFN-γ, tumor necrosis factor-α, and IL-10) and IL-17 production showed a sharp decline. Frequency of T regulatory, B, and natural killer (NK) cells decreased progressively in presence of therapeutic concentrations of all TKIs tested in vitro, except for nilotinib, which showed little effect on these subsets. Of note, naive T regulatory cell (Treg) subset accumulated after exposure to TKIs. Results obtained in vivo on nilotinib-treated patients were largely comparable, both on lymphocyte subset kinetics and on cytokine production by CD3-positive cells. This study underlines the anti-inflammatory and immunomodulatory effects of TKIs and supports their potential usefulness as treatment for patients with steroid-dependent/refractory cGVHD. In addition, both in vitro and in vivo data point out that compared with other TKIs, nilotinib could better preserve the integrity of some important regulatory subsets, such as Treg and NK cells.

Low-Dose Anti-T Lymphoglobulin as Prophylaxis for Graft-versus-Host Disease in Unrelated Donor Transplantations for Acute Leukemias and Myelodysplastic Syndromes.

Chronic graft-versus-host disease (cGVHD) is a major complication after stem cell transplantation (HSCT). Several randomized studies already demonstrated that anti-T lymphoglobulin (ATLG) is effective in preventing GVHD after myeloablative unrelated and HLA-identical sibling transplants. However, the issue of doses and the potential increase of relapses still remain unsolved. Here we report data on 190 patients with acute leukemia and myelodysplastic syndrome who underwent an unrelated HSCT with low-dose ATLG (15 to 30 mg/kg) given at an earlier timing (days -6 to -2). HSCT was performed from HLA 10/10 (n = 62, 33%), 9/10 (n = 91, 48%), 8/10 (n = 30, 16%), and <8/10 (n = 7, 4%) identical unrelated donor. Peripheral blood was the stem cell source in 42% (n = 80). Median follow-up was 51 months. Grades II to IV and III to IV acute GVHD were 26% and 9%, respectively, and 2-year overall and moderate to severe cGVHD were 23% and 14%, respectively. The 3-year incidences of relapse and nonrelapse mortality were 26% and 18%, respectively. The rates of 3-year overall survival (OS), disease-free survival (DFS), and GVHD-free and relapse-free survival (GRFS) were 60%, 56% and 44%, respectively. Factors such as younger donor, good performance status, and early disease were associated with better outcome in terms of OS, DFS, and GRFS. Our data indicate that doses of ATLG lower that those used in randomized clinical trials can be used for GVHD prevention, even in the adult setting, without clear increases in relapse and infections; these findings need to be further validated by a prospective randomized study.

A Comparison of the Conditioning Regimens BEAM and FEAM for Autologous Hematopoietic Stem Cell Transplantation in Lymphoma: An Observational Study on 1038 Patients From Fondazione Italiana Linfomi.

BEAM (carmustine [bis-chloroethylnitrosourea (BCNU)]-etoposide-cytarabine-melphalan) chemotherapy is the standard conditioning regimen for autologous stem cell transplantation (ASCT) in lymphomas. Owing to BCNU shortages, many centers switched to fotemustine-substituted BEAM (FEAM), lacking proof of equivalence. We conducted a retrospective cohort study in 18 Italian centers to compare the safety and efficacy of BEAM and FEAM regimens for ASCT in lymphomas performed from 2008 to 2015. We enrolled 1038 patients (BEAM = 607, FEAM = 431), of which 27% had Hodgkin lymphoma (HL), 14% indolent non-Hodgkin lymphoma (NHL), and 59% aggressive NHL. Baseline characteristics including age, sex, stage, B-symptoms, extranodal involvement, previous treatments, response before ASCT, and overall conditioning intensity were well balanced between BEAM and FEAM; notable exceptions were median ASCT year (BEAM = 2011 versus FEAM = 2013, P < .001), Sorror score ≥3 (BEAM = 15% versus FEAM = 10%, P = .017), and radiotherapy use (BEAM = 18% versus FEAM = 10%, P < .001). FEAM conditioning resulted in higher rates of gastrointestinal and infectious toxicities, including severe oral mucositis grade ≥3 (BEAM = 31% versus FEAM = 44%, P < .001), and sepsis from Gram-negative bacteria (mean isolates/patient: BEAM = .1 versus FEAM = .19, P < .001). Response status at day 100 post-ASCT (overall response: BEAM = 91% versus FEAM = 88%, P = .42), 2-year overall survival (83.9%; 95% confidence interval [CI], 81.5% to 86.1%) and progression-free survival (70.3%; 95% CI, 67.4% to 73.1%) were not different in the two groups. Mortality from infection was higher in the FEAM group (subhazard ratio, 1.99; 95% CI, 1.02 to 3.88; P = .04). BEAM and FEAM do not appear different in terms of survival and disease control. However, due to concerns of higher toxicity, fotemustine substitution in BEAM does not seem justified, if not for easier supply.

Modern Management of Anthracycline-Induced Cardiotoxicity in Lymphoma Patients: Low Occurrence of Cardiotoxicity with Comprehensive Assessment and Tailored Substitution by Nonpegylated Liposomal Doxorubicin.

BACKGROUND: Anthracyclines (AC) are still undeniable drugs in lymphoma treatment, despite occasionally causing cardiotoxicity. Liposomal AC may reduce cardiotoxicity while retaining clinical efficacy; also, biomarker monitoring during chemotherapy allows early detection of cardiac damage, enabling strategies to prevent left ventricular ejection fraction (LVEF) deterioration. MATERIALS AND METHODS: We conducted a prospective observational trial in a real-life population of lymphoma patients, combining advanced echocardiography and biomarkers (Troponin I [TnI]) for early detection of cardiotoxicity; we applied a prespecified policy to minimize cardiotoxicity, selecting patients with higher baseline risk to replace doxorubicin with nonpegylated liposomal doxorubicin (NPLD) and starting cardioprotective treatment when subclinical cardiotoxicity was detected. RESULTS: Ninety-nine patients received ≥1 cycle of chemotherapy (39 with NPLD): 38 (NPLD = 34) were older than 65 years. At baseline, the NPLD subgroup had more cardiovascular risk factors and comorbidities than the doxorubicin subgroup. After treatment, echocardiographic parameters did not worsen in the NPLD subgroup; significant LVEF reduction occurred in two patients treated with doxorubicin. Over treatment course, TnI rises increased linearly in the doxorubicin subgroup but modestly in the NPLD subgroup. At doxorubicin doses >200 mg/m2 the difference was statistically significant, with more TnI rises in the doxorubicin subgroup. NPLD-treated patients did not experience higher rates of grade 3-4 adverse events. Within the diffuse large B-cell lymphomas category, we observed similar rates of complete and overall responses between doxorubicin- and NPLD-treated patients. CONCLUSION: A comprehensive strategy to prevent, detect, and treat cardiotoxicity allows an optimal management of the lymphoma with low incidence of cardiac complications. The Oncologist 2017;22:422-431 IMPLICATIONS FOR PRACTICE: Despite the recent advances of targeted therapy in cancer, old cytotoxic drugs such as anthracyclines (AC) still play a fundamental role in the treatment of many lymphoma patients. We tested and validated in a real-life setting a personalized approach to prevent, detect, and treat AC-induced cardiotoxicity; biomarker monitoring was accomplished by Troponin I measurements before and after chemotherapy infusions, allowing detection of early subclinical cardiotoxicity, which was preemptively treated with cardio-protectants (beta blockers and angiotensin-converting-enzyme inhibitors). A telemedicine system allowed interdisciplinary management of the patients with an expert cardiologist. Furthermore, tailored use of liposomal AC following a prespecified policy appeared to prevent the excess cardiotoxicity expected in high-risk patients.