Emergence and Cytogenetic Clonal Evolution of Chromosome 7 Abnormalities in Myeloid Malignancies: Investigating the Role of Telomere Dysfunction Carmen Baldazzi, Lorenza Bandini, Valentina Robustelli, Agnese Patuelli, Claudia Venturi, Alessandra Grassi, Giulia Marzocchi, Angela Ielpo, Vincenza Solli, Maria Teresa Bochicchio, Stefania Paolini, Chiara Sartor, Federico Zingarelli, Antonio Curti, Emanuela Ottaviani, Nicoletta Testoni International Journal of Molecular Sciences, 2025 Monosomy 7 and deletion 7q are common chromosomal abnormalities in myeloid malignancies, and they are associated with a poor prognosis. The mechanism underlying their acquisition remains elusive. We identified a cohort of 24 patients exhibiting clones with different chromosome 7 abnormalities, such as deletion 7q, unstable derivatives (ring chromosomes or ‘naked’ centromeres), and monosomy 7. We designated this group as having cytogenetic clonal evolution of chromosome 7 abnormalities (CCE7). In some cases, CCE7 correlated with disease progression, suggesting that deletions or other derivatives involving the q-arm of chromosome 7 may arise early in the disease course. These abnormalities may be transient but can potentially evolve into monosomy 7. Within the CCE7 group, telomere loss or shortening may contribute to chromosomal instability and the emergence of unstable derivatives, as the chromosome 7 derivatives displayed loss or rearrangement of subtelomeric regions. Moreover, we identified variants in genes implicated in telomere biology disorders and observed specific genetic mutation profiles associated with different chromosome 7 abnormalities. These findings shed light on a potential mechanism leading to monosomy 7 through the evolution of chromosome 7q abnormalities. Identifying patients at risk of developing monosomy 7, based on the presence of unstable derivatives with telomere loss or a specific mutation profile, could potentially enhance patient management and guide the development of novel therapeutic strategies.
Tracking Response and Resistance in Acute Myeloid Leukemia through Single-Cell DNA Sequencing Helps Uncover New Therapeutic Targets Samantha Bruno, Enrica Borsi, Agnese Patuelli, Lorenza Bandini, Manuela Mancini, Dorian Forte, Jacopo Nanni, Martina Barone, Alessandra Grassi, Gianluca Cristiano, Claudia Venturi, Valentina Robustelli, Giulia Atzeni, Cristina Mosca, Sara De Santis, Cecilia Monaldi, Andrea Poletti, Carolina Terragna, Antonio Curti, Michele Cavo, Simona Soverini, Emanuela Ottaviani International Journal of Molecular Sciences, 2024 Acute myeloid leukemia (AML) is an aggressive hematologic neoplasia with a complex polyclonal architecture. Among driver lesions, those involving the FLT3 gene represent the most frequent mutations identified at diagnosis. The development of tyrosine kinase inhibitors (TKIs) has improved the clinical outcomes of FLT3-mutated patients (Pt). However, overcoming resistance to these drugs remains a challenge. To unravel the molecular mechanisms underlying therapy resistance and clonal selection, we conducted a longitudinal analysis using a single-cell DNA sequencing approach (MissionBioTapestri® platform, San Francisco, CA, USA) in two patients with FLT3-mutated AML. To this end, samples were collected at the time of diagnosis, during TKI therapy, and at relapse or complete remission. For Pt #1, disease resistance was associated with clonal expansion of minor clones, and 2nd line TKI therapy with gilteritinib provided a proliferative advantage to the clones carrying NRAS and KIT mutations, thereby responsible for relapse. In Pt #2, clonal architecture was less complex, and 1st line TKI therapy with midostaurin was able to eradicate the leukemic clones. Our results corroborate previous findings about clonal selection driven by TKIs, highlighting the importance of a deeper characterization of individual clonal architectures for choosing the best treatment plan for personalized approaches aimed at optimizing outcomes.
Ex vivo characterization of acute myeloid leukemia patients undergoing hypomethylating agents and venetoclax regimen reveals a venetoclax-specific effect on non-suppressive regulatory T cells and bona fide PD-1+TIM3+ exhausted CD8+ T cells Giulia Corradi, Dorian Forte, Gianluca Cristiano, Andrea Polimeno, Marilena Ciciarello, Valentina Salvestrini, Lorenza Bandini, Valentina Robustelli, Emanuela Ottaviani, Michele Cavo, Darina Ocadlikova, Antonio Curti Frontiers in Immunology, 2024 Acute myeloid leukemia (AML) is an aggressive heterogeneous disease characterized by several alterations of the immune system prompting disease progression and treatment response. The therapies available for AML can affect lymphocyte function, limiting the efficacy of immunotherapy while hindering leukemia-specific immune reactions. Recently, the treatment based on Venetoclax (VEN), a specific B-cell lymphoma 2 (BCL-2) inhibitor, in combination with hypomethylating agents (HMAs) or low-dose cytarabine, has emerged as a promising clinical strategy in AML. To better understand the immunological effect of VEN treatment, we characterized the phenotype and immune checkpoint (IC) receptors’ expression on CD4+ and CD8+ T cells from AML patients after the first and second cycle of HMA in combination with VEN. HMA and VEN treatment significantly increased the percentage of naïve CD8+ T cells and TIM-3+ CD4+ and CD8+ T cells and reduced cytokine-secreting non-suppressive T regulatory cells (Tregs). Of note, a comparison between AML patients treated with HMA only and HMA in combination with VEN revealed the specific contribution of VEN in modulating the immune cell repertoire. Indeed, the reduction of cytokine-secreting non-suppressive Tregs, the increased TIM-3 expression on CD8+ T cells, and the reduced co-expression of PD-1 and TIM-3 on both CD4+ and CD8+ T cells are all VEN-specific. Collectively, our study shed light on immune modulation induced by VEN treatment, providing the rationale for a novel therapeutic combination of VEN and IC inhibitors in AML patients.
Exploring the ATR-CHK1 pathway in the response of doxorubicin-induced DNA damages in acute lymphoblastic leukemia cells Andrea Ghelli Luserna Di Rorà, Martina Ghetti, Lorenzo Ledda, Anna Ferrari, Matteo Bocconcelli, Antonella Padella, Roberta Napolitano, Maria Chiara Fontana, Chiara Liverani, Enrica Imbrogno, Maria Teresa Bochicchio, Matteo Paganelli, Valentina Robustelli, Seydou Sanogo, Claudio Cerchione, Monica Fumagalli, Michela Rondoni, Annalisa Imovilli, Gerardo Musuraca, Giovanni Martinelli, Giorgia Simonetti Cell Biology and Toxicology, 2023 Doxorubicin (Dox) is one of the most commonly used anthracyclines for the treatment of solid and hematological tumors such as B−/T cell acute lymphoblastic leukemia (ALL). Dox compromises topoisomerase II enzyme functionality, thus inducing structural damages during DNA replication and causes direct damages intercalating into DNA double helix. Eukaryotic cells respond to DNA damages by activating the ATM-CHK2 and/or ATR-CHK1 pathway, whose function is to regulate cell cycle progression, to promote damage repair, and to control apoptosis. We evaluated the efficacy of a new drug schedule combining Dox and specific ATR (VE-821) or CHK1 (prexasertib, PX) inhibitors in the treatment of human B−/T cell precursor ALL cell lines and primary ALL leukemic cells. We found that ALL cell lines respond to Dox activating the G2/M cell cycle checkpoint. Exposure of Dox-pretreated ALL cell lines to VE-821 or PX enhanced Dox cytotoxic effect. This phenomenon was associated with the abrogation of the G2/M cell cycle checkpoint with changes in the expression pCDK1 and cyclin B1, and cell entry in mitosis, followed by the induction of apoptosis. Indeed, the inhibition of the G2/M checkpoint led to a significant increment of normal and aberrant mitotic cells, including those showing tripolar spindles, metaphases with lagging chromosomes, and massive chromosomes fragmentation. In conclusion, we found that the ATR-CHK1 pathway is involved in the response to Dox-induced DNA damages and we demonstrated that our new in vitro drug schedule that combines Dox followed by ATR/CHK1 inhibitors can increase Dox cytotoxicity against ALL cells, while using lower drug doses. Graphical abstract • Doxorubicin activates the G2/M cell cycle checkpoint in acute lymphoblastic leukemia (ALL) cells. • ALL cells respond to doxorubicin-induced DNA damages by activating the ATR-CHK1 pathway. • The inhibition of the ATR-CHK1 pathway synergizes with doxorubicin in the induction of cytotoxicity in ALL cells. • The inhibition of ATR-CHK1 pathway induces aberrant chromosome segregation and mitotic spindle defects in doxorubicin-pretreated ALL cells.
Circulating cell-free DNA for target quantification in hematologic malignancies: Validation of a protocol to overcome pre-analytical biases Roberta Soscia, Irene Della Starza, Lucia Anna De Novi, Caterina Ilari, Michela Ansuinelli, Marzia Cavalli, Vittorio Bellomarino, Luciana Cafforio, Mariangela Di Trani, Giovanni Cazzaniga, Grazia Fazio, Alessandra Santoro, Domenico Salemi, Orietta Spinelli, Manuela Tosi, Carolina Terragna, Valentina Robustelli, Teresa Bellissimo, Gioia Colafigli, Massimo Breccia, Sabina Chiaretti, Alice Di Rocco, Maurizio Martelli, Anna Guarini, Ilaria Del Giudice, Robin Foà Hematological Oncology, 2023 Circulating tumor DNA (ctDNA) has become the most investigated analyte in blood. It is shed from the tumor into the circulation and represents a subset of the total cell‐free DNA (cfDNA) pool released into the peripheral blood. In order to define if ctDNA could represent a useful tool to monitor hematologic malignancies, we analyzed 81 plasma samples from patients affected by different diseases. The results showed that: (i) the comparison between two different extraction methods Qiagen (Hilden, Germany) and Promega (Madison, WI) showed no significant differences in cfDNA yield, though the first recovered higher amounts of larger DNA fragments; (ii) cfDNA concentrations showed a notable inter‐patient variability and differed among diseases: acute lymphoblastic leukemia and chronic myeloid leukemia released higher amounts of cfDNA than chronic lymphocytic leukemia, and diffuse large B‐cell lymphoma released higher cfDNA quantities than localized and advanced follicular lymphoma; (iii) focusing on the tumor fraction of cfDNA, the quantity of ctDNA released was insufficient for an adequate target quantification for minimal residual disease monitoring; (iv) an amplification system proved to be free of analytical biases and efficient in increasing ctDNA amounts at diagnosis and in follow‐up samples as shown by droplet digital PCR target quantification. The protocol has been validated by quality control rounds involving external laboratories. To conclusively document the feasibility of a ctDNA‐based monitoring of patients with hematologic malignancies, more post‐treatment samples need to be evaluated. This will open new possibilities for ctDNA use in the clinical practice.
Baseline cluster of differentiation 22 fluorescent intensity correlates with patient outcome after Inotuzumab Ozogamicin treatment Chiara Sartor, Mario Arpinati, Gabriella Chirumbolo, Luca Dozza, Gianluca Cristiano, Jacopo Nanni, Giovanni Marconi, Valentina Robustelli, Ilaria Vigliotta, Sarah Parisi, Carolina Terragna, Nicoletta Testoni, Stefania Paolini, Giovanni Martinelli, Antonio Curti, Michele Cavo, Cristina Papayannidis Hematological Oncology, 2022 Antigen‐directed target therapy for B‐cell acute lymphoblastic leukemia (B‐ALL) is now the standard of care for relapsed/refractory (R/R) disease. A comprehensive determination of the target itself is mandatory to aid physician's choice. We determined baseline Cluster of differentiation 22 (CD22) expression percentage and fluorescent intensity on lymphoblasts of 30 patients with R/R B‐ALL treated with anti‐CD22 immunoconjugate drug Inotuzumab Ozogamicin (INO) and analyzed the impact of both parameters on patient outcome. Most patients (24/30, 80%) had a high leukemic blast CD22‐positivity defined as ≥90%. We did not observe a benefit in terms of complete remission, overall survival (OS) and duration of response (DoR) for patients with CD22 ≥ 90% versus CD22 < 90%. Concerning CD22‐FI quartile analysis we appreciated a trend for superior response rates in higher quartiles (Q2‐Q4) compared to Q1 and a significant benefit in terms of OS and DoR for patients with higher CD22‐FI. INO demonstrates to be effective also in patients with lower CD22 expression, but therapeutical benefits are more evident in patients with higher CD22‐FI. The evaluation of both CD22 percentage and CD22‐FI of the leukemic blast may help physicians in therapeutic choices for R/R B‐ALL patients when multiple treatment options are available, although no CD22 expression threshold can currently be identified below which INO should be considered not effective.
INCB84344-201: Ponatinib and steroids in frontline therapy for unfit patients with Ph1 acute lymphoblastic leukemia Giovanni Martinelli, Cristina Papayannidis, Alfonso Piciocchi, Valentina Robustelli, Simona Soverini, Carolina Terragna, Giovanni Marconi, Roberto M. Lemoli, Fabio Guolo, Antonella Fornaro, Monia Lunghi, Paolo de Fabritiis, Anna Candoni, Carmine Selleri, Federico Simonetti, Monica Bocchia, Antonella Vitale, Luca Frison, Alessandra Tedeschi, Antonio Cuneo, Massimiliano Bonifacio, Maria Paola Martelli, Stefano D’Ardia, Silvia Trappolini, Patrizia Tosi, Piero Galieni, Francesco Fabbiano, Maria Chiara Abbenante, Muriel Granier, Zhaoyin Zhu, Mingyue Wang, Chiara Sartor, Stefania Paolini, Michele Cavo, Robin Foà, Paola Fazi, Marco Vignetti, Michele Baccarani Blood Advances, 2022 Tyrosine kinase inhibitors have improved survival for patients with Philadelphia chromosome–positive (Ph+) acute lymphoblastic leukemia (ALL). However, prognosis for old or unfit patients remains poor. In the INCB84344-201 (formerly GIMEMA LAL 1811) prospective, multicenter, phase 2 trial, we tested the efficacy and safety of ponatinib plus prednisone in newly diagnosed patients with Ph+ ALL ≥60 years, or unfit for intensive chemotherapy and stem cell transplantation. Forty-four patients received oral ponatinib 45 mg/d for 48 weeks (core phase), with prednisone tapered to 60 mg/m2/d from days-14-29. Prophylactic intrathecal chemotherapy was administered monthly. Median age was 66.5 years (range, 26-85). The primary endpoint (complete hematologic response [CHR] at 24 weeks) was reached in 38/44 patients (86.4%); complete molecular response (CMR) in 18/44 patients (40.9%) at 24 weeks. 61.4% of patients completed the core phase. As of 24 April 2020, median event-free survival was 14.31 months (95% CI 9.30-22.31). Median overall survival and duration of CHR were not reached; median duration of CMR was 11.6 months. Most common treatment-emergent adverse events (TEAEs) were rash (36.4%), asthenia (22.7%), alanine transaminase increase (15.9%), erythema (15.9%), and γ-glutamyltransferase increase (15.9%). Cardiac and vascular TEAEs occurred in 29.5% (grade ≥3, 18.2%) and 27.3% (grade ≥3, 15.9%), respectively. Dose reductions, interruptions, and discontinuations due to TEAEs occurred in 43.2%, 43.2%, and 27.3% of patients, respectively; 5 patients had fatal TEAEs. Ponatinib and prednisone showed efficacy in unfit patients with Ph+ ALL; however, a lower ponatinib dose may be more appropriate in this population. This trial was registered at www.clinicaltrials.gov as #NCT01641107.
Case Report: A Novel Activating FLT3 Mutation in Acute Myeloid Leukemia Samantha Bruno, Lorenza Bandini, Agnese Patuelli, Valentina Robustelli, Claudia Venturi, Manuela Mancini, Dorian Forte, Sara De Santis, Cecilia Monaldi, Alessandra Grassi, Gabriella Chiurumbolo, Stefania Paolini, Gianluca Cristiano, Cristina Papayannidis, Chiara Sartor, Jacopo Nanni, Emanuela Ottaviani, Antonio Curti, Michele Cavo, Simona Soverini Frontiers in Oncology, 2021 FMS-like tyrosine kinase 3 (FLT3) is among the most common driver genes recurrently mutated in acute myeloid leukemia (AML), accounting for approximately 30% of cases. Activating mutations of the FLT3 receptor include internal tandem duplications (ITD) that map to the auto-inhibitory juxtamembrane (JM) domain or point mutations within the tyrosine kinase domain (TKD). Several FLT3 tyrosine kinase inhibitors have been developed in the last few years, but midostaurin is currently the only one approved for the treatment of newly diagnosed patients harboring FLT3 mutations. Here we describe for the first time a novel in-frame deletion in exon 14 (JM domain) of the FLT3 gene, that we identified in a young woman with CBFb-MYH11-positive AML. We demonstrated that this novel FLT3 variant is pathogenic, since it is responsible for constitutive activation of FLT3 receptor. Finally, ex-vivo studies demonstrated that this novel mutation is sensitive to midostaurin.
Novel and rare fusion transcripts involving transcription factors and tumor suppressor genes in acute myeloid leukemia Antonella Padella, Giorgia Simonetti, Giulia Paciello, George Giotopoulos, Carmen Baldazzi, Simona Righi, Martina Ghetti, Anna Stengel, Viviana Guadagnuolo, Rossella De Tommaso, Cristina Papayannidis, Valentina Robustelli, Eugenia Franchini, Andrea Ghelli Luserna di Rorà, Anna Ferrari, Maria Chiara Fontana, Samantha Bruno, Emanuela Ottaviani, Simona Soverini, Clelia Tiziana Storlazzi, Claudia Haferlach, Elena Sabattini, Nicoletta Testoni, Ilaria Iacobucci, Brian J. P. Huntly, Elisa Ficarra, Giovanni Martinelli Cancers, 2019
Identification of Two DNMT3A Mutations Compromising Protein Stability and Methylation Capacity in Acute Myeloid Leukemia Samantha Bruno, Maria Teresa Bochicchio, Eugenia Franchini, Antonella Padella, Giovanni Marconi, Andrea Ghelli Luserna di Rorà, Claudia Venturi, Maddalena Raffini, Giovanna Prisinzano, Anna Ferrari, Lorenza Bandini, Valentina Robustelli, Martina Pazzaglia, Maria Chiara Fontana, Chiara Sartor, Maria Chiara Abbenante, Cristina Papayannidis, Simona Soverini, Emanuela Ottaviani, Giorgia Simonetti, Giovanni Martinelli Journal of Oncology, 2019
Targeting WEE1 to enhance conventional therapies for acute lymphoblastic leukemia Andrea Ghelli Luserna Di Rorà, Neil Beeharry, Enrica Imbrogno, Anna Ferrari, Valentina Robustelli, Simona Righi, Elena Sabattini, Maria Vittoria Verga Falzacappa, Chiara Ronchini, Nicoletta Testoni, Carmen Baldazzi, Cristina Papayannidis, Maria Chiara Abbenante, Giovanni Marconi, Stefania Paolini, Sarah Parisi, Chiara Sartor, Maria Chiara Fontana, Serena De Matteis, Ilaria Iacobucci, Pier Giuseppe Pelicci, Michele Cavo, Timothy J. Yen, Giovanni Martinelli Journal of Hematology and Oncology, 2018
