Publications by authors named "Brunangelo Falini"

189 Publications

Case Report: Subtotal Lymphoid and Total Marrow Irradiation as Bridge Therapy to CD19-Directed CAR T Cells in a Chemorefractory DLBCL With Leukemic Involvement.

Front Immunol 2022 14;13:934700. Epub 2022 Jul 14.

Radiation Oncology Section, Department of Medicine and Surgery, University of Perugia, Perugia, Italy.

CAR T cell therapy has transformed the salvage approach for relapsed/refractory diffuse large B-cell lymphoma (R/R DLBCL). Maintaining disease control before CAR T cell infusion during product manufacturing (so-called bridging therapy) is an important step to optimizing outcome. Among possible bridging therapies, radiation therapy (RT) represents a valuable option, particularly when the disease is limited. Here, we report for the first time on a patient with chemorefractory-transformed DLBCL showing nodal, extranodal, and massive bone marrow (BM) lymphoma infiltration associated with leukemic involvement, a successful bridge therapy to CD19-directed CAR T cell therapy by subtotal lymphoid/total marrow irradiation plus thiothepa followed by reinfusion of CD34+ autologous hematopoietic stem cells. Such a novel bridging regimen allowed a significant reduction of nodal and BM tumor volume while improving blood cell count before CAR T cell infusion. The PET-CT scan and BM evaluation performed at 1, 3, and 6 months after treatment showed complete remission of the disease. A relapse occurred at almost 1 year in lymph nodes because of CD19 antigen escape while the BM remained free of disease. This extended radiotherapy approach may be an effective bridging therapy for chemorefractory DLBCL patients eligible for CAR T cells who present with a high tumor burden, including massive BM involvement associated with leukemic involvement. This preliminary evidence is worth confirming in additional patients.
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http://dx.doi.org/10.3389/fimmu.2022.934700DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9330448PMC
August 2022

SiCoDEA: A Simple, Fast and Complete App for Analyzing the Effect of Individual Drugs and Their Combinations.

Biomolecules 2022 Jun 28;12(7). Epub 2022 Jun 28.

Department of Medicine and Surgery, Section of Hematology and Clinical Immunology, University of Perugia, 06129 Perugia, Italy.

The administration of combinations of drugs is a method widely used in the treatment of different pathologies as it can lead to an increase in the therapeutic effect and a reduction in the dose compared to the administration of single drugs. For these reasons, it is of interest to study combinations of drugs and to determine whether a specific combination has a synergistic, antagonistic or additive effect. Various mathematical models have been developed, which use different methods to evaluate the synergy of a combination of drugs. We have developed an open access and easy to use app that allows different models to be explored and the most fitting to be chosen for the specific experimental data: SiCoDEA (Single and Combined Drug Effect Analysis). Despite the existence of other tools for drug combination analysis, SiCoDEA remains the most complete and flexible since it offers options such as outlier removal or the ability to choose between different models for analysis. SiCoDEA is an easy to use tool for analyzing drug combination data and to have a view of the various steps and offer different results based on the model chosen.
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http://dx.doi.org/10.3390/biom12070904DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9313187PMC
June 2022

Acute myeloid leukemia risk models: Where do we stand?

Am J Hematol 2022 Jul 20. Epub 2022 Jul 20.

Institute of Hematology and CREO, University and Hospital of Perugia, Perugia, Italy.

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http://dx.doi.org/10.1002/ajh.26666DOI Listing
July 2022

Secondary infections worsen the outcome of COVID-19 in patients with hematological malignancies a report from the ITA-HEMA-COV.

Hematol Oncol 2022 Jul 19. Epub 2022 Jul 19.

Ematologia e terapie cellulari, IRCCS Ospedale Policlinico San Martino, Genova, Italy.

The impact of secondary infections (SI) on COVID-19 outcome in patients with hematological malignancies (HM) is scarcely documented. To evaluate incidence, clinical characteristics, and outcome of SI, we analyzed the microbiologically documented SI in a large multicenter cohort of adult HM patients with COVID-19. Among 1741 HM patients with COVID-19, 134 (7.7%) had 185 SI, with a 1-month cumulative incidence of 5%. Median time between COVID-19 diagnosis and SI was 16 days (IQR: 5-36). Acute myeloid leukemia (AML) and lymphoma/plasma cell neoplasms (PCN) were more frequent diagnoses in SI patients compared to patients without SI (AML: 14.9% vs 7.1%; lymphoma /PCN 71.7% vs 65.3%). Patients with SI were older (median age 70 vs 66 yrs, p=0.002), with more comorbidities (median Charlson Comorbidity Index 5 vs 4, p<0.001), higher frequency of critical COVID-19 (19.5% vs 11.5%, p=0.046), and more frequently not in complete remission (75% vs 64.7% p=0.024). Blood and bronchoalveolar lavage were the main sites of isolation for SI. Etiology of infections was bacterial in 80% (n=148) of cases, mycotic in 9.7% (n=18) and viral in 10.3% (n=19); polymicrobial infections were observed in 24 patients (18%). Escherichia coli represented most of Gram-negative isolates (18.9%), while coagulase-negative Staphylococci were the most frequent among Gram-positive (14.2%). The 30-days mortality of patients with SI was higher when compared to patients without SI (69% vs 15%, p<0.001). The occurrence of SI worsened COVID-19 outcome in HM patients. Timely diagnosis and adequate management should be considered to improve their prognosis. This article is protected by copyright. All rights reserved.
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http://dx.doi.org/10.1002/hon.3048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9349965PMC
July 2022

Long-term follow-up of cladribine treatment in hairy cell leukemia: 30-year experience in a multicentric Italian study.

Blood Cancer J 2022 07 19;12(7):109. Epub 2022 Jul 19.

Hematology and Stem Cell Transplantation Unit, Campus Bio-Medico University, Roma, Italy.

Hairy cell leukemia (HCL) is a rare lymphoproliferative disease with an excellent prognosis after treatment with cladribine (2CDA), although relapse may occur during follow-up. The aim of the study is to review the efficacy, safety, long-term remission rate, and overall survival (OS) in those patients who received 2CDA as first-line treatment. We retrospectively reviewed data of HCL patients treated with 2CDA between March 1991 and May 2019 at 18 Italian Hematological centers: 513 patients were evaluable for study purpose. The median age was 54 years (range 24-88) and ECOG was 0 in 84.9% of cases. A total of 330 (64.3%) patients received 2CDA intravenously and 183 (35.7%) subcutaneously. ORR was 91.8%: CR was obtained in 335 patients (65.3%), PR in 96 (18.7%), and hematological response in 40 (7.8%) patients; in 42 (8.2%) no response was observed. Hemoglobin value (p = 0.044), frequency of circulating hairy cells (p = 0.039), recovery of absolute neutrophil count (p = 0.006), and normalization of spleen (p ≤ 0.001) were associated with CR compared to PR in univariable analysis. At a median follow-up of 6.83 years (range 0.04-28.52), the median time to relapse was 12.2 years. A significant difference in duration of response was identified between patients that obtained a CR and PR (19.4 years versus 4.8 years, p < 0.0001). Non-hematological grade 3 or higher early toxicity was reported in 103 (20.1%) patients. Median OS was not reached: 95.3%, 92.4%, and 81.8% of patients were estimated to be alive at 5, 10, and 15 years, respectively. Forty-nine patients died (9.5%), following an infection in 14 cases (2.7%), natural causes in 14 (2.7%), cardiovascular events in 13 (2.5%), a second neoplasm in 6 (1.2%), and progression of HCL in 2 cases (0.4%). Following treatment of HCL with 2CDA, 80% of patients are estimated to be alive 15 years after diagnosis.
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http://dx.doi.org/10.1038/s41408-022-00702-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9296486PMC
July 2022

Defining TCRγδ lymphoproliferative disorders by combined immunophenotypic and molecular evaluation.

Nat Commun 2022 06 8;13(1):3298. Epub 2022 Jun 8.

Department of Medicine (DIMED), Hematology and Clinical Immunology Branch, Padova University School of Medicine, Padova, Italy.

Tγδ large granular lymphocyte leukemia (Tγδ LGLL) is a rare lymphoproliferative disease, scantily described in literature. A deep-analysis, in an initial cohort of 9 Tγδ LGLL compared to 23 healthy controls, shows that Tγδ LGLL dominant clonotypes are mainly public and exhibit different V-(D)-J γ/δ usage between patients with symptomatic and indolent Tγδ neoplasm. Moreover, some clonotypes share the same rearranged sequence. Data obtained in an enlarged cohort (n = 36) indicate the importance of a combined evaluation of immunophenotype and STAT mutational profile for the correct management of patients with Tγδ cell expansions. In fact, we observe an association between Vδ2/Vγ9 clonality and indolent course, while Vδ2/Vγ9 negativity correlates with symptomatic disease. Moreover, the 7 patients with STAT3 mutations have neutropenia and a CD56-/Vδ2- phenotype, and the 3 cases with STAT5B mutations display an asymptomatic clinical course and CD56/Vδ2 expression. All these data indicate that biological characterization is needed for Tγδ-cell neoplasm definition.
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http://dx.doi.org/10.1038/s41467-022-31015-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9177852PMC
June 2022

The International Consensus Classification of Mature Lymphoid Neoplasms: A Report from the Clinical Advisory Committee.

Blood 2022 06 2. Epub 2022 Jun 2.

City of Hope National Medical Center, Duarte, California, United States.

Since the publication of the Revised European-American Classification of mature lymphoid neoplasms in 1994, subsequent updates of the classification of mature lymphoid neoplasms have been generated through iterative international efforts to achieve broad consensus among hematopathologists, geneticists, molecular scientists, and clinicians. Significant progress in the characterization of malignancies of the immune system in the last years, with many new insights provided by genomic studies, have led to the current proposal. We have followed the same process that was successfully used for the 3rd and 4th editions of the WHO classification of hematological neoplasms. The definition, recommended studies, and criteria for the diagnosis of many entities have been extensively refined. Some categories considered provisional are now upgraded to definite entities. Terminology of some diseases has been revised to adapt nomenclature to the current knowledge of their biology, but these modifications have been restricted to well-justified situations. Major findings from recent genomic studies have impacted the conceptual framework and diagnostic criteria for many disease entities. These changes will have an impact on optimal clinical management. The conclusions of this work are summarized in this report as the proposed International Consensus Classification (ICC) of mature lymphoid, histiocytic, and dendritic cell tumors.
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http://dx.doi.org/10.1182/blood.2022015851DOI Listing
June 2022

Identification of novel STAT5B mutations and characterization of TCRβ signatures in CD4+ T-cell large granular lymphocyte leukemia.

Blood Cancer J 2022 02 24;12(2):31. Epub 2022 Feb 24.

Hematology Research Unit Helsinki, University of Helsinki and Helsinki University Hospital Comprehensive Cancer Center, Helsinki, Finland.

CD4+ T-cell large granular lymphocyte leukemia (T-LGLL) is a rare subtype of T-LGLL with unknown etiology. In this study, we molecularly characterized a cohort of patients (n = 35) by studying their T-cell receptor (TCR) repertoire and the presence of somatic STAT5B mutations. In addition to the previously described gain-of-function mutations (N642H, Y665F, Q706L, S715F), we discovered six novel STAT5B mutations (Q220H, E433K, T628S, P658R, P702A, and V712E). Multiple STAT5B mutations were present in 22% (5/23) of STAT5B mutated CD4+ T-LGLL cases, either coexisting in one clone or in distinct clones. Patients with STAT5B mutations had increased lymphocyte and LGL counts when compared to STAT5B wild-type patients. TCRβ sequencing showed that, in addition to large LGL expansions, non-leukemic T cell repertoires were more clonal in CD4+ T-LGLL compared to healthy. Interestingly, 25% (15/59) of CD4+ T-LGLL clonotypes were found, albeit in much lower frequencies, in the non-leukemic CD4+ T cell repertoires of the CD4+ T-LGLL patients. Additionally, we further confirmed the previously reported clonal dominance of TRBV6-expressing clones in CD4+ T-LGLL. In conclusion, CD4+ T-LGLL patients have a typical TCR and mutation profile suggestive of aberrant antigen response underlying the disease.
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http://dx.doi.org/10.1038/s41408-022-00630-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8873566PMC
February 2022

Choroidal vasculature analysis in MEK inhibitor-associated retinopathy.

Eur J Ophthalmol 2022 Feb 23:11206721221081471. Epub 2022 Feb 23.

Department of Medicine and Surgery, Section of Ophthalmology, 9309University of Perugia, Perugia, Italy.

Purpose: To evaluate choroidal vascularity index (CVI) in patients developing mitogen-activated protein kinase kinase (MEK) inhibitor-associated retinopathy (MEKAR).

Methods: In this prospective observational study, extensive ophthalmic examination was performed, including enhanced-depth-imaging-optical coherence tomography (EDI-OCT). EDI-OCT scans of patients receiving Cobimetinib, taken at baseline and at MEKAR manifestation, were considered for choroid analysis. Choroidal thickness (CT) was measured on high-resolution b-scans passing through the fovea at three different locations. Same scans were therefore imported for binarization into a previously reported software and CVI was calculated as the ratio of luminal area (LA) to total choroid area (TCA).

Results: When compared to baseline, eyes with MEKAR (14 eyes) did not show significative CT variation in subfoveal region (p = 0,57), 750-µm-nasal to the fovea (p = 0,08) and 750-µm-temporal to the fovea (p = 0,07). Similarly, there were no statistically significant differences for TCA (p = 0.54), LA (p = 0.85), stromal area (SA) (p = 0.13), LA/SA (p = 0.34) and CVI (p = 0.47). Best-corrected visual acuity was significantly reduced at fluid accumulation when compared to baseline values (p = 0.03), with complete recovery after fluid resolution (p = 0.73).

Conclusion: Multiple parameters reflecting the status of the choroid seemed not influenced by Cobimetinib administration. Retinal pigment epithelium toxic disfunction likely represents the crucial step in MEKAR pathogenesis.
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http://dx.doi.org/10.1177/11206721221081471DOI Listing
February 2022

Metalloproteinase inhibition reduces AML growth, prevents stem cell loss, and improves chemotherapy effectiveness.

Blood Adv 2022 05;6(10):3126-3141

Department of Life Sciences, Sir Alexander Fleming Building, Imperial College London, London, United Kingdom.

Acute myeloid leukemia (AML) is a blood cancer of the myeloid lineage. Its prognosis remains poor, highlighting the need for new therapeutic and precision medicine approaches. AML symptoms often include cytopenias linked to loss of healthy hematopoietic stem and progenitor cells (HSPCs). The mechanisms behind HSPC decline are complex and still poorly understood. Here, intravital microscopy (IVM) of a well-established experimental model of AML allows direct observation of the interactions between healthy and malignant cells in the bone marrow (BM), suggesting that physical dislodgment of healthy cells by AML through damaged vasculature may play an important role. Multiple matrix metalloproteinases (MMPs), known to remodel extracellular matrix, are expressed by AML cells and the BM microenvironment. We reason MMPs could be involved in cell displacement and vascular leakiness; therefore, we evaluate the therapeutic potential of MMP pharmacological inhibition using the broad-spectrum inhibitor prinomastat. IVM analyses of prinomastat-treated mice reveal reduced vascular permeability and healthy cell clusters in circulation and lower AML infiltration, proliferation, and cell migration. Furthermore, treated mice have increased retention of healthy HSPCs in the BM and increased survival following chemotherapy. Analysis of a human AML transcriptomic database reveals widespread MMP deregulation, and human AML cells show susceptibility to MMP inhibition. Overall, our results suggest that MMP inhibition could be a promising complementary therapy to reduce AML growth and limit HSPC loss and BM vascular damage caused by MLL-AF9 and possibly other AML subtypes.
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http://dx.doi.org/10.1182/bloodadvances.2021004321DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9131921PMC
May 2022

How I treat refractory/relapsed hairy cell leukemia with BRAF inhibitors.

Blood 2022 04;139(15):2294-2305

Section of Hematology and Center for Hemato-Oncological Research (CREO), Department of Medicine and Surgery, University of Perugia and Hospital Santa Maria della Misericordia, Perugia, Italy.

Hairy cell leukemia (HCL) responds very well to frontline chemotherapy with purine analogs (cladribine and pentostatine). However, approximately half of patients experience 1 or more relapses, which become progressively resistant to these myelotoxic and immunosuppressive agents. At progression, standard therapeutic options include a second course of purine analogs alone or in combination with rituximab and, upon second relapse, therapy with the anti-CD22 immunotoxin moxetumomab pasudotox. Furthermore, blockade of the mutant BRAF-V600E kinase (the pathogenetic hallmark of HCL) through orally available specific inhibitors (vemurafenib or dabrafenib) effaces the peculiar morphologic, phenotypic, and molecular identity of this disease and its typical antiapoptotic behavior and is emerging as an attractive chemotherapy-free strategy in various clinical scenarios. These include patients with, or at risk of, severe infections and, in a highly effective combination with rituximab, patients with relapsed or refractory HCL. Other treatments explored in clinical trials are BTK inhibition with ibrutinib and co-inhibition of BRAF (through dabrafenib or vemurafenib) and its downstream target MEK (through trametinib or cobimetinib). Here, we focus on our experience with BRAF inhibitors in clinical trials and as off-label use in routine practice by presenting 3 challenging clinical cases to illustrate their management in the context of all available treatment options.
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http://dx.doi.org/10.1182/blood.2021013502DOI Listing
April 2022

A Curious Novel Combination of () Gene Mutations Leading to Aberrant Cytoplasmic Dislocation of in Acute Myeloid Leukemia (AML).

Genes (Basel) 2021 09 16;12(9). Epub 2021 Sep 16.

Hematology and Clinical Immunology, Centro di Ricerche Emato-Oncologiche (CREO), University of Perugia, 06132 Perugia, Italy.

mutations occurring in acute myeloid leukemia (AML) (about 50 so far identified) cluster almost exclusively in exon 12 and lead to common changes at the mutants C-terminus, i.e., loss of tryptophans 288 and 290 (or 290 alone) and creation of a new nuclear export signal (NES), at the bases of exportin-1(XPO1)-mediated aberrant cytoplasmic . Immunohistochemistry (IHC) detects cytoplasmic and is predictive of the molecular alteration. Besides IHC and molecular sequencing, Western blotting (WB) with anti- mutant specific antibodies is another approach to identify -mutated AML. Here, we show that among 382 AML cases with exon 12 mutations, one was not recognized by WB, and describe the discovery of a novel combination of two mutations involving exon 12. This appeared as a conventional mutation A with the known TCTG nucleotides insertion/duplication accompanied by a second event (i.e., an 8-nucleotide deletion occurring 15 nucleotides downstream of the TCTG insertion), resulting in a new C-terminal protein sequence. Strikingly, the sequence included a functional NES ensuring cytoplasmic relocation of the new mutant supporting the role of cytoplasmic as critical in AML leukemogenesis.
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http://dx.doi.org/10.3390/genes12091426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8468273PMC
September 2021

Novel NPM1 exon 5 mutations and gene fusions leading to aberrant cytoplasmic nucleophosmin in AML.

Blood 2021 12;138(25):2696-2701

Munich Leukemia Laboratory, Munich, Germany.

Nucleophosmin (NPM1) mutations in acute myeloid leukemia (AML) affect exon 12, but also sporadically affect exons 9 and 11, causing changes at the protein C-terminal end (tryptophan loss, nuclear export signal [NES] motif creation) that lead to aberrant cytoplasmic NPM1 (NPM1c+), detectable by immunohistochemistry. Combining immunohistochemistry and molecular analyses in 929 patients with AML, we found non-exon 12 NPM1 mutations in 5 (1.3%) of 387 NPM1c+ cases. Besides mutations in exons 9 (n = 1) and 11 (n = 1), novel exon 5 mutations were discovered (n = 3). Another exon 5 mutation was identified in an additional 141 patients with AML selected for wild-type NPM1 exon 12. Three NPM1 rearrangements (NPM1/RPP30, NPM1/SETBP1, NPM1/CCDC28A) were detected and characterized among 13 979 AML samples screened by cytogenetic/fluorescence in situ hybridization and RNA sequencing. Functional studies demonstrated that in AML cases, new NPM1 proteins harbored an efficient extra NES, either newly created or already present in the fusion partner, ensuring its cytoplasmic accumulation. Our findings support NPM1 cytoplasmic relocation as critical for leukemogenesis and reinforce the role of immunohistochemistry in predicting AML-associated NPM1 genetic lesions. This study highlights the need to develop new assays for molecular diagnosis and monitoring of NPM1-mutated AML.
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http://dx.doi.org/10.1182/blood.2021012732DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9037756PMC
December 2021

Actinomycin D Targets NPM1c-Primed Mitochondria to Restore PML-Driven Senescence in AML Therapy.

Cancer Discov 2021 12;11(12):3198-3213

Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.

Acute myeloid leukemia (AML) pathogenesis often involves a mutation in the NPM1 nucleolar chaperone, but the bases for its transforming properties and overall association with favorable therapeutic responses remain incompletely understood. Here we demonstrate that an oncogenic mutant form of NPM1 (NPM1c) impairs mitochondrial function. NPM1c also hampers formation of promyelocytic leukemia (PML) nuclear bodies (NB), which are regulators of mitochondrial fitness and key senescence effectors. Actinomycin D (ActD), an antibiotic with unambiguous clinical efficacy in relapsed/refractory NPM1c-AMLs, targets these primed mitochondria, releasing mitochondrial DNA, activating cyclic GMP-AMP synthase signaling, and boosting reactive oxygen species (ROS) production. The latter restore PML NB formation to drive TP53 activation and senescence of NPM1c-AML cells. In several models, dual targeting of mitochondria by venetoclax and ActD synergized to clear AML and prolong survival through targeting of PML. Our studies reveal an unexpected role for mitochondria downstream of NPM1c and implicate a mitochondrial/ROS/PML/TP53 senescence pathway as an effector of ActD-based therapies.

Significance: ActD induces complete remissions in NPM1-mutant AMLs. We found that NPM1c affects mitochondrial biogenesis and PML NBs. ActD targets mitochondria, yielding ROS which enforce PML NB biogenesis and restore senescence. Dual targeting of mitochondria with ActD and venetoclax sharply potentiates their anti-AML activities in vivo. This article is highlighted in the In This Issue feature, p. 2945.
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http://dx.doi.org/10.1158/2159-8290.CD-21-0177DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7612574PMC
December 2021

Rifaximin use favoured micafungin-resistant Candida spp. infections in recipients of allogeneic hematopoietic cell transplantation.

Ann Hematol 2021 Sep 28;100(9):2375-2380. Epub 2021 Jun 28.

Division of Hematology and Clinical Immunology, University of Perugia, Perugia, Italy.

Damage to gut mucosa following conditioning regimens may favour bacterial infections that can trigger graft versus host disease (GvHD) in patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT). Rifaximin, an oral and non-absorbable antibiotic, has been recently proposed as effective prophylaxis to reduce bacterial infections in the gut and consequently acute GvHD in this setting. The present study evaluated safety and outcomes of HSCT patients that were treated with rifaximin prophylaxis at Perugia University Hospital. Rifaximin prophylaxis was introduced as standard of care in HSCT patients in May 2018. We retrieved data from 118 consecutive transplants, and we compared the outcomes of rifaximin-treated patients with historical controls that did not receive antibiotic prophylaxis. While incidences of neutropenic fever, documented bacterial infections, and aGvHD were similar in the two groups, we found an increased frequency of invasive candidiasis and clinically relevant Candida spp. infections in rifaximin-treated patients (5 patients vs 1 patient, 25% [± 0.99%] vs 1% [± 0.01%], p < .0001). Three rifaximin-treated patients experienced life-threating candidemia (2 C. krusei, 1 C. orthopsilosis). Rifaximin was the only factor that increased the risk of Candida spp. infections. Rifaximin could have contributed to microbiome disruption which favoured an outbreak of life-threatening Candida infections. This important complication forced us to halt its use. Larger, prospective studies are needed to assess the impact of rifaximin prophylaxis on incidence of bacterial infections, aGvHD, and survival of HSCT patients.
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http://dx.doi.org/10.1007/s00277-021-04569-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8357665PMC
September 2021

Dissecting Clonal Hematopoiesis in Tissues of Classical Hodgkin Lymphoma Patients.

Blood Cancer Discov 2021 05 10;2(3):216-225. Epub 2021 Apr 10.

Institute of Hematology and Center for Hemato-Oncology Research, University and Hospital of Perugia, Perugia, Italy.

Clonal hematopoiesis predisposes to hematological malignancies. However, clonal hematopoiesis is understudied in classical Hodgkin lymphoma (cHL), a mature B-cell neoplasm exhibiting the most abundant microenvironment. We analyzed clonal hematopoiesis in 40 cHL cases by sequencing microdissected tumor cells and matched normal cells from blood and/or lymph nodes. Five patients had blood and/or tissue clonal hematopoiesis. In three of five patients (all failing first-line therapy), clonal hematopoiesis spread through the tissue microenvironment extensively, and featured mutant , and + * in 33%, 92% and 60% of non-neoplastic cells, respectively. In the latter case, mutant clonal hematopoiesis seeded the neoplastic clone, which was infected by the Epstein-Barr virus and showed almost no other somatic mutations exome-wide. In the former case, -mutant clonal hematopoiesis did not originate the neoplastic clone despite dominating the blood and B-cell lineage (~94% leukocytes; ~96% mature blood B cells), yet led to -mutated acute myeloid leukemia 6 years after therapy for cHL. Our results expand to cHL the spectrum of hematologic malignancies associated with clonal hematopoiesis.
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http://dx.doi.org/10.1158/2643-3230.BCD-20-0203DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7611041PMC
May 2021

Vemurafenib plus Rituximab in Refractory or Relapsed Hairy-Cell Leukemia.

N Engl J Med 2021 05;384(19):1810-1823

From the Institute of Hematology, Ospedale S. Maria della Misericordia, and the Department of Medicine, University of Perugia, Perugia (E.T., L.D.C., E.S., M.C., S.A., V.M.P., B.F.), the Department of Medicine, Section of Hematology, University of Verona, Verona (A. Ambrosetti), the Hematology Unit, Ospedale di Cosenza, Cosenza (E.L.), the Hematology Unit, Department of Transfusional Medicine-SIMMT, Maria Paternò-Arezzo Hospital, Ragusa (A. Antolino), the Hematology Unit, Department of Translational and Precision Medicine, Sapienza University of Rome (A.P., R.F.), and the Hematology and Stem Cell Transplant Unit, A.O. San Camillo Forlanini (L.R.), Rome, the Department of Hematology, Spedali Civili di Brescia, Brescia (S.F.), IRCCS Azienda Ospedaliero-Universitaria di Bologna, Istituto di Ematologia "Seràgnoli" and Dipartimento di Medicina Specialistica, Diagnostica e Sperimentale, Università di Bologna, Bologna (P.L.Z.), the Department of Translational Medicine, Division of Hematology, Amedeo Avogadro University of Eastern Piedmont, Novara (G.G.), the Hematology Unit, Ospedale di Carrara, Carrara (R.D.S.), the Hematology Unit, Ospedale di Frosinone, Frosinone (N.F.), the Hematology Unit, Ospedale di Ronciglione, Viterbo (P.F.), the Hematology Unit, Ospedale di Pesaro, Pesaro (G.V.), and the Hematology Unit, Azienda Sanitaria Universitaria Integrata-Ospedale Maggiore, Trieste (F.Z.) - all in Italy.

Background: Hairy-cell leukemia (HCL) is a CD20+ indolent B-cell cancer in which a BRAF V600E kinase-activating mutation plays a pathogenetic role. In clinical trials involving patients with refractory or relapsed HCL, the targeting of BRAF V600E with the oral BRAF inhibitor vemurafenib led to a response in 91% of the patients; 35% of the patients had a complete response. However, the median relapse-free survival was only 9 months after treatment was stopped.

Methods: In a phase 2, single-center, academic trial involving patients with refractory or relapsed HCL, we assessed the safety and efficacy of vemurafenib (960 mg, administered twice daily for 8 weeks) plus concurrent and sequential rituximab (375 mg per square meter of body-surface area, administered for 8 doses over a period of 18 weeks). The primary end point was a complete response at the end of planned treatment.

Results: Among the 30 enrolled patients with HCL, the median number of previous therapies was 3. A complete response was observed in 26 patients (87%) in the intention-to-treat population. All the patients who had HCL that had been refractory to chemotherapy (10 patients) or rituximab (5) and all those who had previously been treated with BRAF inhibitors (7) had a complete response. Thrombocytopenia resolved after a median of 2 weeks, and neutropenia after a median of 4 weeks. Of the 26 patients with a complete response, 17 (65%) were cleared of minimal residual disease (MRD). Progression-free survival among all 30 patients was 78% at a median follow-up of 37 months; relapse-free survival among the 26 patients with a response was 85% at a median follow-up of 34 months. In post hoc analyses, MRD negativity and no previous BRAF inhibitor treatment correlated with longer relapse-free survival. Toxic effects, mostly of grade 1 or 2, were those that had previously been noted for these agents.

Conclusions: In this small study, a short, chemotherapy-free, nonmyelotoxic regimen of vemurafenib plus rituximab was associated with a durable complete response in most patients with refractory or relapsed HCL. (Funded by the European Research Council and others; HCL-PG03 EudraCT number, 2014-003046-27.).
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http://dx.doi.org/10.1056/NEJMoa2031298DOI Listing
May 2021

Hairy cell leukemia and COVID-19 adaptation of treatment guidelines.

Leukemia 2021 07 4;35(7):1864-1872. Epub 2021 May 4.

Haematology Department, Peter MacCallum Cancer Centre and Royal Melbourne Hospital, University of Melbourne, Melbourne, VIC, Australia.

Standard treatment options in classic HCL (cHCL) result in high response rates and near normal life expectancy. However, the disease itself and the recommended standard treatment are associated with profound and prolonged immunosuppression, increasing susceptibility to infections and the risk for a severe course of COVID-19. The Hairy Cell Leukemia Foundation (HCLF) has recently convened experts and discussed different clinical strategies for the management of these patients. The new recommendations adapt the 2017 consensus for the diagnosis and management with cHCL to the current COVID-19 pandemic. They underline the option of active surveillance in patients with low but stable blood counts, consider the use of targeted and non-immunosuppressive agents as first-line treatment for cHCL, and give recommendations on preventive measures against COVID-19.
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http://dx.doi.org/10.1038/s41375-021-01257-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8093591PMC
July 2021

BCOR gene alterations in hematologic diseases.

Blood 2021 12;138(24):2455-2468

Institute of Hematology, Center for Hemato-Oncological Research (CREO), University of Perugia, Perugia, Italy.

The BCL6 corepressor (BCOR) is a transcription factor involved in the control of embryogenesis, mesenchymal stem cells function, hematopoiesis, and lymphoid development. Recurrent somatic clonal mutations of the BCOR gene and its homolog BCORL1 have been detected in several hematologic malignancies and aplastic anemia. They are scattered across the whole gene length and mostly represent frameshifts (deletions, insertions), nonsense, and missence mutations. These disruptive events lead to the loss of full-length BCOR protein and to the lack or low expression of a truncated form of the protein, both consistent with the tumor suppressor role of BCOR.BCOR and BCORL1 mutations are similar to those causing 2 rare X-linked diseases: oculofaciocardiodental (OFCD) and Shukla-Vernon syndromes, respectively. Here, we focus on the structure and function of normal BCOR and BCORL1 in normal hematopoietic and lymphoid tissues and review the frequency and clinical significance of the mutations of these genes in malignant and nonmalignant hematologic diseases. Moreover, we discuss the importance of mouse models to better understand the role of Bcor loss, alone and combined with alterations of other genes (eg, Dnmt3a and Tet2), in promoting hematologic malignancies and in providing a useful platform for the development of new targeted therapies.
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http://dx.doi.org/10.1182/blood.2021010958DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8887995PMC
December 2021

Diagnostic and therapeutic pitfalls in NPM1-mutated AML: notes from the field.

Leukemia 2021 11 20;35(11):3113-3126. Epub 2021 Apr 20.

Institute of Haematology, Centro Ricerche Emato-Oncologiche (CREO), Ospedale S. Maria della Misericordia, University of Perugia, Perugia, Italy.

Mutations of Nucleophosmin (NPM1) are the most common genetic abnormalities in adult acute myeloid leukaemia (AML), accounting for about 30% of cases. NPM1-mutated AML has been recognized as distinct entity in the 2017 World Health Organization (WHO) classification of lympho-haematopoietic neoplasms. WHO criteria allow recognition of this leukaemia entity and its distinction from AML with myelodysplasia-related changes, AML with BCR-ABL1 rearrangement and AML with RUNX1 mutations. Nevertheless, controversial issues include the percentage of blasts required for the diagnosis of NPM1-mutated AML and whether cases of NPM1-mutated myelodysplasia and chronic myelomonocytic leukaemia do exist. Evaluation of NPM1 and FLT3 status represents a major pillar of the European LeukemiaNet (ELN) genetic-based risk stratification model. Moreover, NPM1 mutations are particularly suitable for assessing measurable residual disease (MRD) since they are frequent, stable at relapse and do not drive clonal haematopoiesis. Ideally, combining monitoring of MRD with the ELN prognostication model can help to guide therapeutic decisions. Here, we provide examples of instructive cases of NPM1-mutated AML, in order to provide criteria for the appropriate diagnosis and therapy of this frequent leukaemia entity.
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http://dx.doi.org/10.1038/s41375-021-01222-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8056374PMC
November 2021

Dactinomycin induces complete remission associated with nucleolar stress response in relapsed/refractory NPM1-mutated AML.

Leukemia 2021 09 2;35(9):2552-2562. Epub 2021 Mar 2.

Hematology, Center for Research in Hemato-Oncology (CREO), University of Perugia, Perugia, Italy.

Acute myeloid leukemia (AML) with mutated NPM1 accounts for one-third of newly diagnosed AML. Despite recent advances, treatment of relapsed/refractory NPM1-mutated AML remains challenging, with the majority of patients eventually dying due to disease progression. Moreover, the prognosis is particularly poor in elderly and unfit patients, mainly because they cannot receive intensive treatment. Therefore, alternative treatment strategies are needed. Dactinomycin is a low-cost chemotherapeutic agent, which has been anecdotally reported to induce remission in NPM1-mutated patients, although its mechanism of action remains unclear. Here, we describe the results of a single-center phase 2 pilot study investigating the safety and efficacy of single-agent dactinomycin in relapsed/refractory NPM1-mutated adult AML patients, demonstrating that this drug can induce complete responses and is relatively well tolerated. We also provide evidence that the activity of dactinomycin associates with nucleolar stress both in vitro and in vivo in patients. Finally, we show that low-dose dactinomycin generates more efficient stress response in cells expressing NPM1 mutant compared to wild-type cells, suggesting that NPM1-mutated AML may be more sensitive to nucleolar stress. In conclusion, we establish that dactinomycin is a potential therapeutic alternative in relapsed/refractory NPM1-mutated AML that deserves further investigation in larger clinical studies.
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http://dx.doi.org/10.1038/s41375-021-01192-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8410589PMC
September 2021

Haploidentical age-adapted myeloablative transplant and regulatory and effector T cells for acute myeloid leukemia.

Blood Adv 2021 03;5(5):1199-1208

Division of Hematology and Clinical Immunology, Department of Medicine, University of Perugia, Perugia, Italy.

Allogeneic hematopoietic stem cell transplantation (HSCT) is the most effective treatment in eradicating high-risk acute myeloid leukemia (AML). Here, we present data from a novel HLA-haploidentical HSCT protocol that addressed the 2 remaining major unmet medical needs: leukemia relapse and chronic graft-versus-host disease (cGVHD). Fifty AML patients were enrolled in the study. The conditioning regimen included total body irradiation for patients up to age 50 years and total marrow/lymphoid irradiation for patients age 51 to 65 years. Irradiation was followed by thiotepa, fludarabine, and cyclophosphamide. Patients received an infusion of 2 × 106/kg donor regulatory T cells on day -4 followed by 1 × 106/kg donor conventional T cells on day -1 and a mean of 10.7 × 106 ± 3.4 × 106/kgpurified CD34+ hematopoietic progenitor cells on day 0. No pharmacological GVHD prophylaxis was administered posttransplantation. Patients achieved full donor-type engraftment. Fifteen patients developed grade ≥2 acute GVHD (aGVHD). Twelve of the 15 patients with aGVHD were alive and no longer receiving immunosuppressive therapy. Moderate/severe cGVHD occurred in only 1 patient. Nonrelapse mortality occurred in 10 patients. Only 2 patients relapsed. Consequently, at a median follow-up of 29 months, the probability of moderate/severe cGVHD/relapse-free survival was 75% (95% confidence interval, 71%-78%). A novel HLA-haploidentical HSCT strategy that combines an age-adapted myeloablative conditioning regimen with regulatory and conventional T-cell adoptive immunotherapy resulted in an unprecedented cGVHD/relapse-free survival rate in 50 AML patients with a median age of 53 years. This trial was registered with the Umbria Region Institutional Review Board Public Registry as identification code 02/14 and public registry #2384/14 and at www.clinicaltrials.gov as #NCT03977103.
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http://dx.doi.org/10.1182/bloodadvances.2020003739DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7948281PMC
March 2021

CD123 Is Consistently Expressed on -Mutated AML Cells.

Cancers (Basel) 2021 Jan 28;13(3). Epub 2021 Jan 28.

Department of Medicine and Surgery, University of Perugia, 06131 Perugia, Italy.

-mutated (mut) acute myeloid leukemia (AML) comprises about 30% of newly diagnosed AML in adults. Despite notable advances in the treatment of this frequent AML subtype, about 50% of mut AML patients treated with conventional treatment die due to disease progression. CD123 has been identified as potential target for immunotherapy in AML, and several anti-CD123 therapeutic approaches have been developed for AML resistant to conventional therapies. As this antigen has been previously reported to be expressed by mut cells, we performed a deep flow cytometry analysis of CD123 expression in a large cohort of mut and wild-type samples, examining the whole blastic population, as well as CD34CD38 leukemic cells. We demonstrate that CD123 is highly expressed on mut cells, with particularly high expression levels showed by CD34CD38 leukemic cells. Additionally, CD123 expression was further enhanced by mutations, which frequently co-occur with mutations. Our results identify -mutated and particularly double-mutated AML as disease subsets that may benefit from anti-CD123 targeted therapies.
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http://dx.doi.org/10.3390/cancers13030496DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7865228PMC
January 2021

ARPIR: automatic RNA-Seq pipelines with interactive report.

BMC Bioinformatics 2020 Dec 21;21(Suppl 19):574. Epub 2020 Dec 21.

Department of Medicine, Section of Hematology, University of Perugia, Perugia, Italy.

Background: RNA-Seq is an increasing used methodology to study either coding and non-coding RNA expression. There are many software tools available for each phase of the RNA-Seq analysis and each of them uses different algorithms. Furthermore, the analysis consists of several steps regarding alignment (primary-analysis), quantification, differential analysis (secondary-analysis) and any tertiary-analysis and can therefore be time-consuming to deal with each step separately, in addition to requiring a computer knowledge. For this reason, the development of an automated pipeline that allows the entire analysis to be managed through a single initial command and that is easy to use even for those without computer skills can be useful. Faced with the vast availability of RNA-Seq analysis tools, it is first of all necessary to select a limited number of pipelines to include. For this purpose, we compared eight pipelines obtained by combining the most used tools and for each one we evaluated peak of RAM, time, sensitivity and specificity.

Results: The pipeline with shorter times, lower consumption of RAM and higher sensitivity is the one consisting in HISAT2 for alignment, featureCounts for quantification and edgeR for differential analysis. Here, we developed ARPIR, an automated pipeline that recurs by default to the cited pipeline, but it also allows to choose, between different tools, those of the pipelines having the best performances.

Conclusions: ARPIR allows the analysis of RNA-Seq data from groups undergoing different treatment allowing multiple comparisons in a single launch and can be used either for paired-end or single-end analysis. All the required prerequisites can be installed via a configuration script and the analysis can be launched via a graphical interface or by a template script. In addition, ARPIR makes a final tertiary-analysis that includes a Gene Ontology and Pathway analysis. The results can be viewed in an interactive Shiny App and exported in a report (pdf, word or html formats). ARPIR is an efficient and easy-to-use tool for RNA-Seq analysis from quality control to Pathway analysis that allows you to choose between different pipelines.
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http://dx.doi.org/10.1186/s12859-020-03846-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7751108PMC
December 2020

How I diagnose and treat NPM1-mutated AML.

Blood 2021 02;137(5):589-599

Institute of Hematology, Centro Ricerche Emato-Oncologiche, Ospedale S. Maria della Misericordia, University of Perugia, Perugia, Italy.

Mutations of the nucleophosmin (NPM1) gene, encoding for a nucleolar multifunctional protein, occur in approximately one-third of adult acute myeloid leukemia (AML). NPM1-mutated AML exhibits unique molecular, pathological, and clinical features, which led to its recognition as distinct entity in the 2017 World Health Organization (WHO) classification of myeloid neoplasms. Although WHO criteria for the diagnosis of NPM1-mutated AML are well established, its distinction from other AML entities may be difficult. Moreover, the percentage of blasts required to diagnose NPM1-mutated AML remains controversial. According to the European LeukemiaNet (ELN), determining the mutational status of NPM1 (together with FLT3) is mandatory for accurate relapse-risk assessment. NPM1 mutations are ideal targets for measurable residual disease (MRD) monitoring, since they are AML specific, frequent, very stable at relapse, and do not drive clonal hematopoiesis of undetermined significance. MRD monitoring by quantitative polymerase chain reaction of NPM1-mutant transcripts, possibly combined with ELN genetic-based risk stratification, can guide therapeutic decisions after remission. Furthermore, immunohistochemistry can be very useful in selected situations, such as diagnosis of NPM1-mutated myeloid sarcoma. Herein, we present 4 illustrative cases of NPM1-mutated AML that address important issues surrounding the biology, diagnosis, and therapy of this common form of leukemia.
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http://dx.doi.org/10.1182/blood.2020008211DOI Listing
February 2021
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