29 results on '"Manuela Gambella"'
Search Results
2. Safety and efficacy of rozanolixizumab in patients with generalised myasthenia gravis (MycarinG): a randomised, double-blind, placebo-controlled, adaptive phase 3 study
- Author
-
Vera Bril, Artur Drużdż, Julian Grosskreutz, Ali A Habib, Renato Mantegazza, Sabrina Sacconi, Kimiaki Utsugisawa, John Vissing, Tuan Vu, Marion Boehnlein, Ali Bozorg, Maryam Gayfieva, Bernhard Greve, Franz Woltering, Henry J Kaminski, Angela Genge, Rami Massie, Maxime Berube, Lubna Daniyal, Shabber Mannan, Eduardo Ng, Ritesh Rohan Raghu Raman, Evelyn Sarpong, Monica Alcantara, Annie Dionne, Zaeem Siddiqi, Derrick Blackmore, Faraz Hussain, Genevieve Matte, Stephan Botez, Michaela Tyblova, Michala Jakubikova, Jana Junkerova, Nanna Witting, Sonja Holm-Yildiz, Mads Stemmerik, Henning Andersen, Izabella Obál, Guilhem Solé, Stéphane Mathis, Marie-Hélène Violleau, Christine Tranchant, Sihame Messai, Jean-Baptiste Chanson, Aleksandra Nadaj-Pakleza, Arnaud Verloes, Leila Zaidi, Manuela Gambella, Michele Cavalli, Tanya Stojkovic, Sophie Demeret, Loic Le Guennec, Giorgia Querin, Nicolas Weiss, Marion Masingue, Laurent Magy, Karima Ghorab, Ia Rukhadze, Alexander Tsiskaridze, Marina Janelidze, Temur Margania, Florian Then Bergh, Eike Hänsel, Andrea Kalb, Bianca Meilick, Mandy Reuschel, Lars-Malte Teußer, Astrid Unterlauft, Clemens Goedel, Tim Hagenacker, Andreas Totzeck, Benjamin Stolte, Franz Blaes, Christine Bindler, Vasilios Tsoutsikas, Annekathrin Roediger, Christian Geis, Jens Schmidt, Jana Zschüntzsch, Margret Schwarz, Stefanie Meyer, Karsten Kummer, Stefanie Glaubitz, Rachel Zeng, Heinz Wiendl, Luisa Klotz, Anna Lammerskitten, Jan Lünemann, Péter Diószeghy, Lorenzo Maggi, Elena Rinaldi, Matteo Gastaldi, Federico Mazzacane, Pietro Businaro, Raffaele Iorio, Giovanni Antonini, Laura Fionda, Rita Rinaldi, Simone Rossi, Francesco Habetswallner, Francesco Tuccillo, Haruna Umehara, Eiko Uenaka, Masanori Takahashi, Keiko Higashi, Makoto Kinoshita, Emika Yoneda, Noriko Nakamura, Saeka Fujita, Tomoya Kubota, Masami Ono, Sana Yamamoto, Taku Hatano, Kazuki Oikoshi, Kazumasa Yokoyama, Yutaka Oji, Yuji Tomizawa, Akiyuki Uzawa, Manato Yasuda, Sachiko Akita, Yukiko Ozawa, Yosuke Onishi, Miki Takaki, Hiromi Yamada, Kanako Minemoto, Miki Sanko, Nanae Izawa, Mayumi Nakayama, Masayuki Masuda, Rune Tsuji, Nobuhiro Ido, Yumi Hyodo, Yoshihiko Okubo, Akiko Minohara, Nana Haraguchi, Makiko Naito, Seiko Yoshida, Yuri Fukushige, Akira Tsujino, Atsushi Nagaoka, Teiichiro Miyazaki, Shunsuke Yoshimura, Takuro Hirayama, Tomoaki Shima, Naoko Okamoto, Riki Matsumoto, Kenji Sekiguchi, Takehiro Ueda, Norio Chihara, Mari Kirimura, Emi Sunagawa, Ayaka Suzuki, Shigeaki Suzuki, Aozora Wada, Kei Ishizuchi, Yasushi Suzuki, Mitsuo Yata, Yuka Komatsu, Kenichi Tsukita, Genya Watanabe, Kazuki Sato, Emiko Kawasaki, Naoki Yamamoto, Hirohiko Ono, Tomoko Tsuda, Shigeki Ohashi, Yuka Fujisawa, Yumiko Yokota, Yuriko Nagane, Kameda Ayumi, Yuka Takematsu, Hiroyuki Naito, Kumiko Kuwada, Konrad Rejdak, Sebastian Szklener, Monika Kitowska, Kandyda Derkacz, Tomasz Berkowicz, Paulina Budzinska, Marek Halas, Leonid Zaslavskiy, Evgeniya Skornyakova, Sergey Kotov, Ekaterina Novikova, Olga Sidorova, Vitalii Goldobin, Tatiana Alekseeva, Patimat Isabekova, Nadezhda Malkova, Denis Korobko, Gordana Djordjevic, Aleksandar Stojanov, Stojan Peric, Dragana Lavrnic, Ivo Bozovic, Aleksa Palibrk, Carlos Casasnovas, Velina Nedkova-Hristova, Nuria Vidal Fernández, Elena Cortés Vicente, Luis Querol Gutiérrez, Maria Salvadó Figueras, Anna Canovas Segura, Raúl Juntas Morales, Daniel Sanchez Tejerina, Albert Saiz, Yolanda Blanco Morgado, Sara Llufriú Durán, María Sepúlveda Gázquez, Eugenia María Martínez Hernández, Gerardo Gutiérrez Gutiérrez, Paqui Iniesta, José Meca Lallana, Yuh-Cherng Guo, Hou-Chang Chiu, Jiann-Horng Yeh, Ya Hui Chen, Mei Fen Lee, Yi-Chung Lee, Kuan Lin Lai, Said Beydoun, Salma Akhter, Lucy Lam, Alisha Thomas, Michael Rivner, Brandy Quarles, Dale Lange, Shara Holzberg, Pantelis Pavlakis, Ashwathy Goutham, Henry Kaminski, Radwa Aly, Lisa Ashworth, Kathryn Bender, Karie Bond, Joanne Buckner, Sara Byerly, James Caress, Jessyca Clemons, Asha Farmer, Catherine Franklin, Summer Harris, Meredith Hiatt, Rachana Gandhi Mehta, Gina Miller, Lynn Smith, Rose Smith, Brian Strittmatter, Tahseen Mozaffar, Isela Hernandez, Kelsey Moulton, Chafic Karam, Pranali Ravikumar, Catherine Lomen-Hoerth, Laura Rosow, Hannah George, Viktoriya Irodenko, Carol Denny, Bart Hanson, Sara Klein, Jennifer Martinez-Thompson, Elie Naddaf, Denny Padgett, Eric Sorenson, Jane L Sultze, Delena Weis, Kourosh Rezania, Jason Thonhoff, Sheetal Shroff, Robert Pascuzzi, Angela Micheels, Cynthia Bodkin, Adam Comer, Gelasio Baras, Renee Wagner, Zabeen Mahuwala, Stephen Ryan, Kai Su, Khema Sharma, Andrew Brown, and Kore Liow
- Subjects
Neurology (clinical) - Published
- 2023
3. Data from Early Relapse Risk in Patients with Newly Diagnosed Multiple Myeloma Characterized by Next-generation Sequencing
- Author
-
Francesca Gay, Francesco Maura, Niccolò Bolli, Mario Boccadoro, Alessandra Larocca, Sara Bringhen, Manuela Gambella, Jonathan J. Keats, Paola Colucci, Jennifer Yesil, Daniel Auclair, Stefania Oliva, Andrea Capra, Elisa Genuardi, Even H. Rustad, Bachisio Ziccheddu, Gian Maria Zaccaria, and Mattia D'Agostino
- Abstract
Purpose:Duration of first remission is important for the survival of patients with multiple myeloma.Experimental Design:From the CoMMpass study (NCT01454297), 926 patients with newly diagnosed multiple myeloma, characterized by next-generation sequencing, were analyzed to evaluate those who experienced early progressive disease (PD; time to progression, TTP ≤18 months).Results:After a median follow-up of 39 months, early PD was detected in 191/926 (20.6%) patients, 228/926 (24.6%) patients had late PD (TTP >18 months), while 507/926 (54.8%) did not have PD at the current follow-up. Compared with patients with late PD, patients with early PD had a lower at least very good partial response rate (47% vs. 82%, P < 0.001) and more frequently acquired double refractoriness to immunomodulatory drugs (IMiD) and proteasome inhibitors (PI; 21% vs. 8%, P < 0.001). Patients with early PD were at higher risk of death compared with patients with late PD and no PD (HR, 3.65; 95% CI, 2.7–4.93; P < 0.001), showing a dismal median overall survival (32.8 months). In a multivariate logistic regression model, independent factors increasing the early PD risk were TP53 mutation (OR, 3.78, P < 0.001), high lactate dehydrogenase levels (OR, 3.15, P = 0.006), λ-chain translocation (OR, 2.25, P = 0.033), and IGLL5 mutation (OR, 2.15, P = 0.007). Carfilzomib-based induction (OR, 0.15, P = 0.014), autologous stem-cell transplantation (OR, 0.27, P < 0.001), and continuous therapy with PIs and IMiDs (OR, 0.34, P = 0.024) mitigated the risk of early PD.Conclusions:Early PD identifies a high-risk multiple myeloma population. Further research is needed to better identify baseline features predicting early PD and the optimal treatment approaches for patients at risk.
- Published
- 2023
4. Supplementary Appendix from Early Relapse Risk in Patients with Newly Diagnosed Multiple Myeloma Characterized by Next-generation Sequencing
- Author
-
Francesca Gay, Francesco Maura, Niccolò Bolli, Mario Boccadoro, Alessandra Larocca, Sara Bringhen, Manuela Gambella, Jonathan J. Keats, Paola Colucci, Jennifer Yesil, Daniel Auclair, Stefania Oliva, Andrea Capra, Elisa Genuardi, Even H. Rustad, Bachisio Ziccheddu, Gian Maria Zaccaria, and Mattia D'Agostino
- Abstract
Supplementary Appendix: Additional methods. Carfilzomib-treated patients - Trial design; Table S1. List and classification method of the analyzed variables; Table S2. Induction treatment classification; Table S3. List of the 21 genes analyzed and mutation frequency; Table S4. Distribution of upfront treatment and ASCT in CT subgroups; Table S5. Distribution of variables in Early PD vs reference population; Figure S1. Multivariate logistic regression model evaluating risk factors associated with death within 24 months; Figure S2. Sub-analysis on patients with or without baseline del(17p) and/or TP53 mutation; Figure S3. TP53 and IGLL5 mutations at diagnosis and at first relapse in available longitudinal samples; Figure S4. Epanechnikov kernel-smoothed estimated hazard rates of progressive disease (PD) over time; Figure S5. Comparison of Seq-FISH and conventional FISH in a subgroup of patients enrolled in clinical trials and analyzed in the same centralized laboratory.
- Published
- 2023
5. Minimal residual disease by flow cytometry and allelic-specific oligonucleotide real-time quantitative polymerase chain reaction in patients with myeloma receiving lenalidomide maintenance: A pooled analysis
- Author
-
Manuela Gambella, Barbara Gamberi, Pieter Sonneveld, Elona Saraci, Massimo Offidani, Antonio Palumbo, Alberto Rocci, Annalisa Bernardini, Vittorio Emanuele Muccio, Sara Grammatico, Mario Boccadoro, Stefano Spada, Paola Omedè, Rossella Troia, Concetta Conticello, Michele Cavo, Alessandra Larocca, Milena Gilestro, Anna Marina Liberati, Stefania Oliva, Gambella, Manuela, Omedé, Paola, Spada, Stefano, Muccio, Vittorio Emanuele, Gilestro, Milena, Saraci, Elona, Grammatico, Sara, Larocca, Alessandra, Conticello, Concetta, Bernardini, Annalisa, Gamberi, Barbara, Troia, Rossella, Liberati, Anna Marina, Offidani, Massimo, Rocci, Alberto, Palumbo, Antonio, Cavo, Michele, Sonneveld, Pieter, Boccadoro, Mario, Oliva, Stefania, and Hematology
- Subjects
Oncology ,Male ,medicine.medical_specialty ,Cancer Research ,Neoplasm, Residual ,allelic-specific oligonucleotide real-time quantitative polymerase chain reaction (ASO-RQ-PCR) ,maintenance ,minimal residual disease (MRD) ,multiparameter flow cytometry (MFC) ,multiple myeloma (MM) ,new diagnosis ,Real-Time Polymerase Chain Reaction ,Disease-Free Survival ,Maintenance Chemotherapy ,03 medical and health sciences ,0302 clinical medicine ,Maintenance therapy ,Interquartile range ,Risk Factors ,Internal medicine ,hemic and lymphatic diseases ,medicine ,Humans ,Immunologic Factors ,030212 general & internal medicine ,Lenalidomide ,Multiple myeloma ,Very Good Partial Response ,business.industry ,Hazard ratio ,Middle Aged ,medicine.disease ,Flow Cytometry ,Minimal residual disease ,Confidence interval ,body regions ,Treatment Outcome ,Clinical Trials, Phase III as Topic ,030220 oncology & carcinogenesis ,Female ,business ,Multiple Myeloma ,medicine.drug - Abstract
Background: Minimal residual disease (MRD) is one of the most relevant prognostic factors in patients with multiple myeloma (MM); however, the impact of maintenance therapy on MRD levels remains unclear. Among patients with newly diagnosed MM (NDMM) who received lenalidomide maintenance until they developed disease progression, the role of MRD status as a predictor of progression-free survival (PFS) was evaluated by multiparameter flow cytometry (MFC) and allelic-specific oligonucleotide real-time quantitative polymerase chain reaction (ASO-RQ-PCR) analysis. Methods: Seventy-three patients with NDMM enrolled in the RV-MM-EMN-441 (clinical trials.gov identifier, NCT01091831) and RV-MM-COOP-0556 (clinicaltrials.gov identifier, NCT01208766; European Myeloma Network EMN02/HO95 MM Trial) phase 3 trials who achieved at least a very good partial response after intensification/consolidation were included. The median patient age was 57 years (interquartile range, 53-61 years), and all patients received lenalidomide maintenance until they developed progression. MRD was evaluated on bone marrow after intensification/consolidation, after 6 courses of maintenance, and every 6 months thereafter until clinical relapse using both ASO-RQ-PCR (sensitivity, 10 −5 ) and MFC (sensitivity, from 10 −4 to 10 −5 ). Results: After intensification/consolidation, 33 of 72 patients (46%) achieved a molecular complete response (m-CR), and 44 of 70 (63%) achieved a flow complete response (flow-CR). Almost 27% of patients who were MRD-positive after consolidation became MRD-negative during maintenance. After a median follow-up of 38 months, PFS was prolonged in patients who achieved negative MRD status during maintenance according to results from both ASO-RQ-PCR analysis (hazard ratio, 0.29; 95% confidence interval, 0.14-0.62; P =.0013) and MFC (hazard ratio, 0.19; 95% confidence interval, 0.09-0.41; P
- Published
- 2019
6. Early Relapse Risk in Patients with Newly Diagnosed Multiple Myeloma Characterized by Next-generation Sequencing
- Author
-
Mattia D'Agostino, Gian Maria Zaccaria, Bachisio Ziccheddu, Even H. Rustad, Elisa Genuardi, Andrea Capra, Stefania Oliva, Daniel Auclair, Jennifer Yesil, Paola Colucci, Jonathan J. Keats, Manuela Gambella, Sara Bringhen, Alessandra Larocca, Mario Boccadoro, Niccolò Bolli, Francesco Maura, and Francesca Gay
- Subjects
Male ,0301 basic medicine ,Cancer Research ,Time Factors ,Computer science ,MEDLINE ,Risk Assessment ,Disease-Free Survival ,DNA sequencing ,Terminology ,03 medical and health sciences ,0302 clinical medicine ,Antineoplastic Combined Chemotherapy Protocols ,Humans ,Immunologic Factors ,Longitudinal Studies ,Prospective Studies ,Organism ,Aged ,Animal Welfare (journal) ,High-Throughput Nucleotide Sequencing ,Sampling (statistics) ,Middle Aged ,Term (time) ,030104 developmental biology ,Oncology ,Risk analysis (engineering) ,Drug Resistance, Neoplasm ,030220 oncology & carcinogenesis ,Disease Progression ,Female ,Identification (biology) ,Neoplasm Recurrence, Local ,Multiple Myeloma ,Proteasome Inhibitors ,Follow-Up Studies - Abstract
Purpose: Duration of first remission is important for the survival of patients with multiple myeloma. Experimental Design: From the CoMMpass study (NCT01454297), 926 patients with newly diagnosed multiple myeloma, characterized by next-generation sequencing, were analyzed to evaluate those who experienced early progressive disease (PD; time to progression, TTP ≤18 months). Results: After a median follow-up of 39 months, early PD was detected in 191/926 (20.6%) patients, 228/926 (24.6%) patients had late PD (TTP >18 months), while 507/926 (54.8%) did not have PD at the current follow-up. Compared with patients with late PD, patients with early PD had a lower at least very good partial response rate (47% vs. 82%, P < 0.001) and more frequently acquired double refractoriness to immunomodulatory drugs (IMiD) and proteasome inhibitors (PI; 21% vs. 8%, P < 0.001). Patients with early PD were at higher risk of death compared with patients with late PD and no PD (HR, 3.65; 95% CI, 2.7–4.93; P < 0.001), showing a dismal median overall survival (32.8 months). In a multivariate logistic regression model, independent factors increasing the early PD risk were TP53 mutation (OR, 3.78, P < 0.001), high lactate dehydrogenase levels (OR, 3.15, P = 0.006), λ-chain translocation (OR, 2.25, P = 0.033), and IGLL5 mutation (OR, 2.15, P = 0.007). Carfilzomib-based induction (OR, 0.15, P = 0.014), autologous stem-cell transplantation (OR, 0.27, P < 0.001), and continuous therapy with PIs and IMiDs (OR, 0.34, P = 0.024) mitigated the risk of early PD. Conclusions: Early PD identifies a high-risk multiple myeloma population. Further research is needed to better identify baseline features predicting early PD and the optimal treatment approaches for patients at risk.
- Published
- 2020
7. Clinical and Biological Early Relapse Predictors in Multiple Myeloma: An Analysis from the MMRF CoMMpass Study
- Author
-
Mattia D'Agostino, Gian Maria Zaccaria, Bachisio Ziccheddu, Elisa Genuardi, Francesco Maura, Stefania Oliva, Daniel Auclair, Jennifer Yesil, Andrea Capra, Paola Colucci, Marco Poggiu, Jonathan Keats, Alessandra Larocca, Manuela Gambella, Niccolo Bolli, Mario Boccadoro, and Francesca Gay
- Subjects
Oncology ,Cancer Research ,medicine.medical_specialty ,business.industry ,Internal medicine ,medicine ,Early Relapse ,Hematology ,medicine.disease ,business ,Multiple myeloma - Published
- 2019
8. Minimal Residual Disease Detection by Droplet Digital PCR in Multiple Myeloma, Mantle Cell Lymphoma, and Follicular Lymphoma
- Author
-
Simone Ferrero, Manuela Gambella, Paola Ghione, Marco Ladetto, Luigia Monitillo, Davide Barberio, Sergio Cortelazzo, Giorgio Inghirami, Umberto Vitolo, Roman Hájek, Lenka Kubiczkova-Besse, Nadia Dani, Daniela Drandi, Elisa Genuardi, Antonio Palumbo, Daniela Barbero, Paola Omedè, Mario Boccadoro, Roberto Passera, Barbara Mantoan, and Elona Saraci
- Subjects
Immunoglobulin gene ,Follicular lymphoma ,Biology ,medicine.disease ,Minimal residual disease ,Molecular biology ,3. Good health ,Pathology and Forensic Medicine ,Lymphoma ,Real-time polymerase chain reaction ,hemic and lymphatic diseases ,medicine ,Molecular Medicine ,Mantle cell lymphoma ,Digital polymerase chain reaction ,Multiple myeloma - Abstract
Real-time quantitative PCR (qPCR) is a well-established tool for minimal residual disease (MRD) detection in mature lymphoid malignancies. Despite remarkable sensitivity and specificity, qPCR has some limitations, particularly in the need for a reference standard curve, based on target serial dilutions. In this study, we established droplet digital PCR (ddPCR) for MRD monitoring in multiple myeloma, mantle cell lymphoma, and follicular lymphoma and compared it head-to-head with qPCR. We observed that ddPCR has sensitivity, accuracy, and reproducibility comparable with qPCR. We then compared the two approaches in 69 patients with a documented molecular marker at diagnosis (18 multiple myelomas, 21 mantle cell lymphomas assessed with the immunoglobulin gene rearrangement, and 30 follicular lymphomas with the use of the BCL2/immunoglobulin gene major breakpoint region rearrangement). ddPCR was successful in 100% of cases, whereas qPCR failed to provide a reliable standard curve in three patients. Overall, 222 of 225 samples were evaluable by both methods. The comparison highlighted a good concordance (r = 0.94, P
- Published
- 2015
9. Systemic virotherapy for multiple myeloma
- Author
-
Manuela Gambella, Mario Boccadoro, Stefania Oliva, and Sara Bringhen
- Subjects
0301 basic medicine ,viruses ,Clinical Biochemistry ,Drug Evaluation, Preclinical ,Myxoma virus ,Coxsackievirus ,medicine.disease_cause ,Virus ,Measles virus ,Multiple myeloma ,oncolytic viruses ,treatment ,virotherapy ,Clinical Trials as Topic ,Humans ,Interferons ,Multiple Myeloma ,Oncolytic Viruses ,ras Proteins ,Oncolytic Virotherapy ,Pharmacology ,Drug Discovery3003 Pharmaceutical Science ,03 medical and health sciences ,Drug Discovery ,Medicine ,Virotherapy ,biology ,business.industry ,biology.organism_classification ,Virology ,Preclinical ,Oncolytic virus ,030104 developmental biology ,Herpes simplex virus ,Vesicular stomatitis virus ,Drug Evaluation ,business - Abstract
The multiple myeloma (MM) treatment scenario has changed considerably over the past few years. Several novel targeted therapies are currently under consideration including oncolytic virotherapy. Areas covered: This review provides an analysis of the mechanisms of action of virotherapy, and summarizes the preclinical and clinical studies of systemic virotherapy developed for the treatment of MM. Different types of viruses have been identified, including: adenovirus, vaccinia virus, herpes simplex virus 1, myxoma virus, reovirus, measles virus, vesicular stomatitis virus and coxsackievirus A21. Expert opinion: The above-mentioned viruses can do more than simply infect and kill malignant plasma cells alone or in combination with chemo and/or radiotherapy. In fact, some of them can also be used to purge myeloma cells from an autologous bone marrow (BM) transplant. Further investigations are required to better explore the best therapeutic combinations for MM and to also overcome antiviral response immunity that can limit the efficacy of this therapeutic strategy.
- Published
- 2017
10. Ficoll-hypaque separation vs whole blood lysis: Comparison of efficiency and impact on minimal residual disease analysis
- Author
-
Irene Dogliotti, Manuela Gambella, Marzia Cavalli, Claudio Agostinelli, Elena Ciabatti, I. Del Giudice, Claudia Mannu, I. Della Starza, Pier Paolo Piccaluga, Elisa Genuardi, Daniela Barbero, Simone Ferrero, Anna Gazzola, S Grassi, Luigia Monitillo, Gian Maria Zaccaria, L.A. De Novi, Marco Ladetto, Daniela Drandi, Barbara Mantoan, S Galimberti, Genuardi, E., Barbero, D., Dogliotti, I., Mantoan, B., Drandi, D., Gambella, M., Zaccaria, G.M., Monitillo, L., Della Starza, I., Cavalli, M., De Novi, L.A., Ciabatti, E., Grassi, S., Gazzola, A., Mannu, C., Del Giudice, I., Galimberti, S., Agostinelli, C., Piccaluga, P.P., Ladetto, M., and Ferrero, S.
- Subjects
medicine.medical_specialty ,Neoplasm, Residual ,cell recovering ,Mononuclear ,Clinical Biochemistry ,Follicular lymphoma ,Ficoll ,Urology ,Diatrizoate ,Peripheral blood mononuclear cell ,Hemolysis ,03 medical and health sciences ,0302 clinical medicine ,hemic and lymphatic diseases ,Internal medicine ,medicine ,red blood cell lysis ,Leukocytes ,Methods ,Humans ,Whole blood ,Clinical Trials as Topic ,Hematology ,business.industry ,Biochemistry (medical) ,General Medicine ,medicine.disease ,Minimal residual disease ,medicine.anatomical_structure ,minimal residual disease ,Residual ,030220 oncology & carcinogenesis ,Leukocytes, Mononuclear ,Neoplasm ,Mantle cell lymphoma ,Bone marrow ,business ,red blood cell lysi ,030215 immunology - Abstract
Introduction The high-throughput era remarkably changed molecular laboratory practice. Actually, the increasing number of processed samples requires to reduce the risk of operator biases, by automating or simplifying as much as possible both the analytical and the pre-analytical phases. Minimal residual disease (MRD) studies in hematology often require a simultaneous processing of many bone marrow and peripheral blood samples from patients enrolled in prospective, multicenter, clinical trials, monitored at several planned time points. Methods In this study, we demonstrate that red blood cell lysis (RBL) pre-analytical procedure can replace the time-consuming Ficoll stratification as cell recovering step. Here, we show a MRD comparison study using both total white blood cells and mononuclear cells recovered by the 2 procedures from 46 follicular lymphoma (FL), 15 multiple myeloma (MM), and 11 mantle cell lymphoma (MCL) patients enrolled in prospective clinical trials. Results The experiments were performed in the 4 laboratories of the Fondazione Italiana Linfomi (FIL) MRD Network and showed superimposable results, in terms of good correlation (R = 0.87) of the MRD data obtained by recovering blood cells by the 2 approaches. Conclusion Based on these results, the FIL MRD Network suggests to optimize the pre-analytical phases introducing RBL approach for cell recovery in the clinical trials including MRD analysis.
- Published
- 2017
11. Minimal residual disease after transplantation or lenalidomide-based consolidation in myeloma patients: a prospective analysis
- Author
-
Stefania Oliva, Manuela Gambella, Milena Gilestro, Vittorio Emanuele Muccio, Francesca Gay, Daniela Drandi, Simone Ferrero, Roberto Passera, Chiara Pautasso, Annalisa Bernardini, Mariella Genuardi, Francesca Patriarca, Elona Saraci, Maria Teresa Petrucci, Norbert Pescosta, Anna Marina Liberati, Tommaso Caravita, Concetta Conticello, Alberto Rocci, Pellegrino Musto, Mario Boccadoro, Antonio Palumbo, and Paola Omedè
- Subjects
Male ,medicine.medical_specialty ,Neoplasm, Residual ,Novel agents ,Tumor burden ,Myeloma ,Transplantation, Autologous ,Maintenance Chemotherapy ,03 medical and health sciences ,Prospective analysis ,0302 clinical medicine ,ASO-RQ-PCR ,Flow cytometry ,MRD ,Oncology ,Antineoplastic Combined Chemotherapy Protocols ,Biomarkers, Tumor ,medicine ,Humans ,In patient ,Prospective Studies ,Lenalidomide ,Complete response ,business.industry ,General surgery ,Middle Aged ,Minimal residual disease ,Thalidomide ,Consolidation Chemotherapy ,Transplantation ,Treatment Outcome ,030220 oncology & carcinogenesis ,Disease Progression ,Female ,Multiple Myeloma ,business ,Research Paper ,030215 immunology ,medicine.drug - Abstract
// Stefania Oliva 1, * , Manuela Gambella 1, * , Milena Gilestro 1 , Vittorio Emanuele Muccio 1 , Francesca Gay 1 , Daniela Drandi 2 , Simone Ferrero 2 , Roberto Passera 3 , Chiara Pautasso 1 , Annalisa Bernardini 1 , Mariella Genuardi 1 , Francesca Patriarca 4 , Elona Saraci 1 , Maria Teresa Petrucci 5 , Norbert Pescosta 6 , Anna Marina Liberati 7 , Tommaso Caravita 8 , Concetta Conticello 9 , Alberto Rocci 10 , Pellegrino Musto 11 , Mario Boccadoro 1 , Antonio Palumbo 1 , Paola Omede 1 1 Myeloma Unit, Division of Hematology, University of Torino, Azienda Ospeadliero-Universitaria Citta della Salute e della Scienza di Torino, Torino, Italy 2 Division of Hematology, Department of Molecular Biotechnologies and Health Sciences, University of Torino, Torino, Italy 3 Division of Nuclear Medicine, University of Torino, Azienda Ospedaliero-Universitaria Citta della Salute e della Scienza di Torino, Torino, Italy 4 Azienda Ospedaliera-Universitaria di Udine, DISM Universita di Udine, Udine, Italy 5 Division of Hematology, Department of Cellular Biotechnologies and Hematology, Sapienza University of Rome, Rome, Italy 6 Ematologia e Centro TMO, Ospedale Centrale Bolzano, Bozen, Italy 7 AO S.Maria di Terni, SC Oncoematologia, Terni, Italy 8 UOC Ematologia S.Eugenio ASL RM2 Roma, Rome, Italy 9 Divisione di Ematologia, Azienda Policlinico-OVE, Universita di Catania, Catania, Italy 10 Department of Haematology, Manchester Royal Infirmary, Central Manchester University Hospitals NHS Foundation Trust, Manchester, UK 11 Scientific Direction, IRCCS-CROB, Referral Cancer Center of Basilicata, Rionero in Vulture (Pz), Italy * Both authors share first authorship of the manuscript Correspondence to: Paola Omede, email: paolaomede@yahoo.com Keywords: myeloma, MRD, ASO-RQ-PCR, novel agents, flow cytometry Received: June 11, 2016 Accepted: September 21, 2016 Published: October 13, 2016 ABSTRACT We analyzed 50 patients who achieved at least a very good partial response in the RV-MM-EMN-441 study. Patients received consolidation with autologous stem-cell transplantation (ASCT) or cyclophosphamide-lenalidomide-dexamethasone (CRD), followed by Lenalidomide-based maintenance. We assessed minimal residual disease (MRD) by multi-parameter flow cytometry (MFC) and allelic-specific oligonucleotide real-time quantitative polymerase chain reaction (ASO-RQ-PCR) after consolidation, after 3 and 6 courses of maintenance, and thereafter every 6 months until progression. By MFC analysis, 19/50 patients achieved complete response (CR) after consolidation, and 7 additional patients during maintenance. A molecular marker was identified in 25/50 patients, 4/25 achieved molecular-CR after consolidation, and 3 additional patients during maintenance. A lower MRD value by MFC was found in ASCT patients compared with CRD patients (p=0.0134). Tumor burden reduction was different in patients with high-risk vs standard-risk cytogenetics (3.4 vs 5.2, ln-MFC; 3 vs 6 ln-PCR, respectively) and in patients who relapsed vs those who did not (4 vs 5, ln-MFC; 4.4 vs 7.8 ln-PCR). MRD progression anticipated clinical relapse by a median of 9 months while biochemical relapse by a median of 4 months. MRD allows the identification of a low-risk group, independently of response, and a better characterization of the activity of treatments.
- Published
- 2017
12. Prognostic Impact of Minimal Residual Disease By ASO-RQ-PCR in Multiple Myeloma: A Pooled Analysis of 2 Phase III Studies in Patients Treated with Lenalidomide after Front-Line Therapy
- Author
-
Chiara Pautasso, Barbara Mantoan, Eleonora Marzanati, Sara Grammatico, Stefania Oliva, Massimo Offidani, Mario Boccadoro, Stefano Spada, Alessandra Larocca, Anna Marina Liberati, Manuela Gambella, Concetta Conticello, Barbara Gamberi, Antonio Palumbo, and Paola Omedè
- Subjects
Oncology ,Melphalan ,medicine.medical_specialty ,Immunology ,Biochemistry ,03 medical and health sciences ,0302 clinical medicine ,Autologous stem-cell transplantation ,Maintenance therapy ,Internal medicine ,0502 economics and business ,medicine ,Multiple myeloma ,Lenalidomide ,business.industry ,05 social sciences ,Hazard ratio ,Cell Biology ,Hematology ,medicine.disease ,Minimal residual disease ,Surgery ,Log-rank test ,030220 oncology & carcinogenesis ,050211 marketing ,business ,medicine.drug - Abstract
Background: The prognostic utility of minimal residual disease (MRD) analysis in multiple myeloma (MM) patients has been well described in the last few years. The role of prolonged maintenance therapy even in persistent MRD negative patients is still unclear. The aim of this study is to evaluate the role of MRD by allelic-specific oligonucleotide real-time quantitative polymerase chain reaction (ASO-RQ-PCR) as predictor of progression-free survival (PFS) in newly diagnosed MM (NDMM) patients receiving Lenalidomide maintenance after frontline treatment. Patients and Methods: NDMM patients enrolled in the RV-MM-EMN-441 (NCT01091831) and the RV-MM-COOP-0556 (EMN02/HO95 MM) phase III trials achieving ≥ very good partial response (VGPR) after consolidation/intensification were included in the pooled MRD molecular analysis. After induction therapy, patients in the RV-MM-EMN-441 study were randomized to Cyclophosphamide-Lenalidomide-Dexamethasone (CRD) or Autologous Stem Cell Transplantation (ASCT); patients in the RV-MM-COOP-0556 were randomized to Bortezomib-Melphalan-Prednisone (VMP) vs ASCT (Gay F et al Lancet Oncol 2016, Cavo M et al J Clin Oncol 34, 2016 abstr 8000). All patients received Lenalidomide maintenance until progression or intolerance. MRD analysis was performed on bone marrow (BM) aspirates after intensification/consolidation, after 6 courses of maintenance and then every 6 months until clinical relapse. Patient-specific IgH rearrangements were amplified and directly sequenced from genomic DNA at diagnosis. IgH-based MRD detection by ASO-RQ-PCR was performed using an AbiPrism7900HT.MRD analysis was interpreted following the Euro-MRD guidelines(van der Velden VH et al. Leukemia 2007). Molecular-CR (m-CR) was defined as two consecutive negative MRD results by ASO-RQ-PCR with minimal sensitivity of 10−5. PFS was analyzed using the Kaplan-Meier method, curves were compared with the log-rank test. Multivariate Cox model was used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs). Results: a total of 105 patients entered the molecular MRD pooled study: a specific IgH molecular marker was identified in 73 patients (70%), 32 (30%) did not obtain a successful sequencing. Median age was 57 years (37-65); 30 (41%) patients had International Staging System (ISS) stage I, 33 (45%) stage II and 10 (14%) stage III. FISH risk profile was standard in 43 (59%) patients, high in 24 (33%) and not available in 6 (8%). Thirty-eight (52%) patients did not receive ASCT consolidation and 35 (48%) underwent ASCT. After consolidation/intensification 33/73 (45%) patients achieved m-CR: 19/35 (54%) ASCT patients and 14/38 (37%) no ASCT patients. The impact of m-CR on outcome after consolidation was explored: after a median follow-up of 44 months, median PFS was 48.8 months versus not reached in no m-CR vs m-CR patients, respectively (p=0.01). Lenalidomide maintenance further improved depth of MRD response: 11/40 (27%) MRD positive patients after consolidation obtained a m-CR during maintenance and a median of 2 natural logarithms of tumor burden reduction was recorded. In multivariable Cox analysis the risk of progression/death was higher for ISS stage II/III versus I (HR, 2.91, CI: 1.01-8.41, p=0.048), high-risk FISH versus standard-risk (HR, 2.23 CI: 0.81-6.10, p=0.12), age > 60 years versus ≤60 years (HR: 3.55, CI: 1.26-10.04, p=0.017) and patients who did not achieve m-CR during treatment versus patients who did (HR, 7.65 CI: 2.77-21.11, p We identified a very high risk group defined by high risk FISH at diagnosis and persistent MRD positivity, with a median PFS of 29.4 months (figure1). Conclusions: MRD status by ASO-RQ-PCR is a predictor of outcome significantly superior to standard risk factors in NDMM patients and the achievement of m-CR seems to overcome the high risk FISH status in PFS analysis. Molecular CR rate and reduction of tumor burden obtained after consolidation can be enhanced with Lenalidomide maintenance. Based on these preliminary results, the assessment and monitoring of MRD should be suggested as a better prognostic indicator than CR, and the potential role of a MRD-guided therapy should be investigated in future prospective trials. Figure 1 PFS of patients stratified by MRD status (molecular-CR vs no molecular-CR) and FISH (high risk vs standard risk) Figure 1. PFS of patients stratified by MRD status (molecular-CR vs no molecular-CR) and FISH (high risk vs standard risk) Disclosures Oliva: Celgene: Honoraria; Takeda: Honoraria; Amgen: Honoraria. Larocca:Amgen, Celgene, BMS, Janssen-Cilag: Honoraria. Offidani:Janssen: Honoraria; Celgene: Honoraria, Research Funding. Palumbo:Janssen Cilag: Honoraria; Takeda: Employment, Honoraria. Boccadoro:Sanofi, Celgene, Amgen, Janssen, Novartis, Abbivie, BMS: Honoraria; Celgene, Janssen, Amgen, BMS, Mundipharma, Novartis, Sanofi: Research Funding.
- Published
- 2016
13. Long Term Outcome of Lenalidomide-Dexamethasone (Rd) Vs Melphalan-Lenalidomide-Prednisone (MPR) Vs Cyclophosphamide-Prednisone-Lenalidomide (CPR) As Induction Followed By Lenalidomide-Prednisone (RP) Vs Lenalidomide (R) As Maintenance in a Community-Based Newly Diagnosed Myeloma Population: Updated Analysis of EMN01 Phase III Study
- Author
-
Chiara Pautasso, Gianluca Gaidano, Nicola Cascavilla, Mariella Genuardi, Anna Marina Liberati, Giorgio La Nasa, Daniele Derudas, Alida Dominietto, Laura Maracci, Giulia Benevolo, Giovanni De Sabbata, Manuela Gambella, Sara Bringhen, Giovanni Pizzolo, Renato Zambello, Caterina Musolino, Roman Hájek, Pellegrino Musto, Antonio Palumbo, Massimo Offidani, Paola Ferrando, Donatella Zamagni, Mario Boccadoro, Ombretta Annibali, and Attilio Gabbas
- Subjects
0301 basic medicine ,Community based ,Melphalan ,medicine.medical_specialty ,education.field_of_study ,business.industry ,Immunology ,Population ,Cell Biology ,Hematology ,Newly diagnosed ,Biochemistry ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Prednisone ,030220 oncology & carcinogenesis ,Internal medicine ,Cyclophosphamide/prednisone ,medicine ,education ,business ,Dexamethasone ,medicine.drug ,Lenalidomide - Abstract
Introduction : Rd and MPR showed to be effective combinations in elderly newly diagnosed multiple myeloma (NDMM) patients (pts). Cyclophosphamide is a less toxic alkylating alternative agent. EMN01 is the first trial to formally compare these three different Lenalidomide-based combinations. Maintenance with Lenalidomide has been recently approved in patients eligible for autologous stem cell transplant (ASCT). Few data are available about the best combination as maintenance in patients not eligible for ASCT. Methods : 662 pts with NDMM were randomized to receive 9 28-day cycles of Rd (lenalidomide 25 mg/day for 21 days; dexamethasone 40 mg on days 1,8,15 and 22 in pts 65-75 years old and 20 mg in those >75 years), MPR (lenalidomide 10 mg/day for 21 days; melphalan orally 0.18 mg/Kg for 4 days in pts 65-75 years old and 0.13 mg/Kg in >75 years pts; prednisone 1.5 mg/Kg for 4 days) or CPR (lenalidomide 25 mg/day for 21 days; cyclophosphamide orally 50 mg/day for 21 days in pts 65-75 years old and 50 mg every other day in >75 years pts; prednisone 25 mg every other day). After induction, pts were randomized to receive maintenance with lenalidomide alone (R; 10 mg/day for 21 days) or with prednisone (RP; R, 10 mg/day for 21 days and P, 25 mg every other day), until disease progression. Results : Pts characteristics were well balanced in all groups; 217 pts in Rd, 217 in MPR and 220 in CPR arms could be evaluated. After a median follow-up of 63.7 months, median PFS was 23.2 months in MPR, 18.9 months in CPR and 18.6 months in Rd (MPR vs CPR p=0.02; MPR vs Rd p=0.08). Median overall survival (OS) was 79.9 months in MPR, 69.4 months in CPR and 68.1 months in Rd (MPR vs CPR p=0.98; MPR vs Rd p=0.64). The most common grade ≥3 adverse event (AEs) was neutropenia: 64% in MPR, 29% in CPR and 25% in Rd pts (p Conclusion : This phase III trial compared 2 different Lenalidomide-containing induction regimens and 2 different Lenalidomide-containing maintenance regimens in an elderly community-based NDMM population. MPR prolonged PFS by approximately 5 months, yet the higher incidence of hematologic toxicity should be carefully considered. The addition of low-dose prednisone to standard lenalidomide maintenance reduced the risk of death/progression by 20%, with a good safety profile. Updated results will be presented at the meeting. Disclosures Bringhen: Mundipharma: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria; Celgene: Honoraria; Bristol Myers Squibb: Honoraria; Karyipharm: Membership on an entity's Board of Directors or advisory committees. Offidani: celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Musto: Celgene: Honoraria; Janssen: Honoraria. Gaidano: Gilead: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Roche: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria. De Sabbata: Celgene: Membership on an entity's Board of Directors or advisory committees. Palumbo: Sanofi: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Binding Site: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Merck: Consultancy, Honoraria, Research Funding; Genmab A/S: Consultancy, Honoraria, Research Funding; Janssen-Cilag: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding, Speakers Bureau; Takeda: Consultancy, Employment, Equity Ownership, Honoraria, Research Funding. Hájek: Amgen, Takeda, BMS, Celgene, Novartis, Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Consultancy, Honoraria; Pharma MAR: Consultancy, Honoraria. Boccadoro: Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; AbbVie: Honoraria; Mundipharma: Research Funding; Sanofi: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding.
- Published
- 2017
14. Minimal Residual Disease Detection by Droplet Digital PCR in Multiple Myeloma, Mantle Cell Lymphoma, and Follicular Lymphoma: A Comparison with Real-Time PCR
- Author
-
Daniela, Drandi, Lenka, Kubiczkova-Besse, Simone, Ferrero, Nadia, Dani, Roberto, Passera, Barbara, Mantoan, Manuela, Gambella, Luigia, Monitillo, Elona, Saraci, Paola, Ghione, Elisa, Genuardi, Daniela, Barbero, Paola, Omedè, Davide, Barberio, Roman, Hajek, Umberto, Vitolo, Antonio, Palumbo, Sergio, Cortelazzo, Mario, Boccadoro, Giorgio, Inghirami, and Marco, Ladetto
- Subjects
Neoplasm, Residual ,Humans ,Reproducibility of Results ,Molecular Medicine ,Lymphoma, Mantle-Cell ,Reference Standards ,Multiple Myeloma ,Real-Time Polymerase Chain Reaction ,Lymphoma, Follicular ,Sensitivity and Specificity ,2734 - Abstract
Real-time quantitative PCR (qPCR) is a well-established tool for minimal residual disease (MRD) detection in mature lymphoid malignancies. Despite remarkable sensitivity and specificity, qPCR has some limitations, particularly in the need for a reference standard curve, based on target serial dilutions. In this study, we established droplet digital PCR (ddPCR) for MRD monitoring in multiple myeloma, mantle cell lymphoma, and follicular lymphoma and compared it head-to-head with qPCR. We observed that ddPCR has sensitivity, accuracy, and reproducibility comparable with qPCR. We then compared the two approaches in 69 patients with a documented molecular marker at diagnosis (18 multiple myelomas, 21 mantle cell lymphomas assessed with the immunoglobulin gene rearrangement, and 30 follicular lymphomas with the use of the BCL2/immunoglobulin gene major breakpoint region rearrangement). ddPCR was successful in 100% of cases, whereas qPCR failed to provide a reliable standard curve in three patients. Overall, 222 of 225 samples were evaluable by both methods. The comparison highlighted a good concordance (r = 0.94, P 0.0001) with 189 of 222 samples (85.1%; 95% CI, 80.4%-89.8%) being fully concordant. We found that ddPCR is a reliable tool for MRD detection with greater applicability and reduced labor intensiveness than qPCR. It will be necessary to authorize ddPCR as an outcome predictor tool in controlled clinical settings and multilaboratory standardization programs.
- Published
- 2015
15. MET/HGF pathway in multiple myeloma: From diagnosis to targeted therapy?
- Author
-
Antonio Palumbo, Manuela Gambella, and Alberto Rocci
- Subjects
Angiogenesis ,medicine.medical_treatment ,Motility ,Drug resistance ,Plasma cell ,Pathology and Forensic Medicine ,Targeted therapy ,Pathogenesis ,Biomarkers, Tumor ,Tumor Microenvironment ,medicine ,Genetics ,Animals ,Humans ,Molecular Targeted Therapy ,HGF ,MET ,myeloma ,prognosis ,target therapy ,Molecular Medicine ,Molecular Biology ,2734 ,Multiple myeloma ,Hepatocyte Growth Factor ,business.industry ,Proto-Oncogene Proteins c-met ,medicine.disease ,Molecular medicine ,Cell Transformation, Neoplastic ,medicine.anatomical_structure ,Molecular Diagnostic Techniques ,Drug Resistance, Neoplasm ,Immunology ,Cancer research ,Multiple Myeloma ,business ,Signal Transduction - Abstract
The interaction between neoplastic cells and the microenvironment is critical in several cancers and plays a central role in multiple myeloma. Microenvironmental stimuli support plasma cell proliferation, survival, motility and can determine drug resistance. The network between plasma cells and surrounding cells is also responsible for increasing angiogenesis, unbalancing bone formation and bony lesions. The MET/HGF pathway is a key player in this interaction and has been found to be abnormally active in both malignant plasma cells and surrounding cells. Patients with abnormal MET and/or HGF levels usually have a poor outcome even when treated with novel drugs. This review addresses the role of MET/HGF in the pathogenesis of myeloma and describes the role of MET/HGF signaling as a prognostic factor. The different techniques to detect MET/HGF abnormalities are examined and a description of compounds targeting MET/HGF is also provided.
- Published
- 2015
16. Indoleamine 2,3-dioxygenase 1 (IDO1) activity correlates with immune system abnormalities in multiple myeloma
- Author
-
Franco Locatelli, Alberto Rocci, Valentina Folgiero, Antonio Palumbo, Giuseppina Bonanno, Manuela Gambella, Raimondo De Cristofaro, Sergio Rutella, Linda Novarese, Giovanni Scambia, Andrea Mariotti, Marilena Ciciarello, Annabella Procoli, Luca De Rosa, Daniela Natale, Ignazio Majolino, Giuseppina Bonanno, Andrea Mariotti, Annabella Procoli, Valentina Folgiero, Daniela Natale, Luca De Rosa, Ignazio Majolino, Linda Novarese, Alberto Rocci, Manuela Gambella, Marilena Ciciarello, Giovanni Scambia, Antonio Palumbo, Franco Locatelli, Raimondo De Cristofaro, and Sergio Rutella
- Subjects
Cellular differentiation ,T-Lymphocytes ,lcsh:Medicine ,Antigens, Neoplasm ,CD8-Positive T-Lymphocytes ,Cell Differentiation ,Cell Line, Tumor ,Cell Proliferation ,Hepatocyte Growth Factor ,Humans ,Immune System ,Indoleamine-Pyrrole 2,3,-Dioxygenase ,Interleukin-10 ,Membrane Proteins ,Multiple Myeloma ,Plasma Cells ,STAT3 Transcription Factor ,Signal Transduction ,T-Lymphocytes, Regulatory ,Transforming Growth Factor beta ,Tumor Burden ,Plasma cell ,chemistry.chemical_compound ,IDO1 ,Indoleamine 2,3-dioxygenase ,Multiple myeloma ,Medicine(all) ,Tumor ,General Medicine ,Regulatory ,Interleukin 10 ,medicine.anatomical_structure ,Indoleamine ,Biology ,Indoleamine-Pyrrole 2 ,General Biochemistry, Genetics and Molecular Biology ,Cell Line ,Immune system ,Antigen ,medicine ,Antigens ,Biochemistry, Genetics and Molecular Biology(all) ,Research ,Settore MED/09 - MEDICINA INTERNA ,lcsh:R ,medicine.disease ,Settore MED/40 - GINECOLOGIA E OSTETRICIA ,chemistry ,Immunology ,Dioxygenase ,Neoplasm ,Kynurenine - Abstract
Background Multiple myeloma (MM) is a plasma cell malignancy with a multifaceted immune dysfunction. Indoleamine 2,3-dioxygenase 1 (IDO1) degrades tryptophan into kynurenine (KYN), which inhibits effector T cells and promote regulatory T-cell (Treg) differentiation. It is presently unknown whether MM cells express IDO1 and whether IDO1 activity correlates with immune system impairment. Methods We investigated IDO1 expression in 25 consecutive patients with symptomatic MM and in 7 patients with either monoclonal gammopathy of unknown significance (MGUS; n=3) or smoldering MM (SMM; n=4). IDO1-driven tryptophan breakdown was correlated with the release of hepatocyte growth factor (HGF) and with the frequency of Treg cells and NY-ESO-1-specific CD8+ T cells. Results KYN was increased in 75% of patients with symptomatic MM and correlated with the expansion of CD4+CD25+FoxP3+ Treg cells and the contraction of NY-ESO-1-specific CD8+ T cells. In vitro, primary MM cells promoted the differentiation of allogeneic CD4+ T cells into bona fide CD4+CD25hiFoxP3hi Treg cells and suppressed IFN-γ/IL-2 secretion, while preserving IL-4 and IL-10 production. Both Treg expansion and inhibition of Th1 differentiation by MM cells were reverted, at least in part, by d,l-1-methyl-tryptophan, a chemical inhibitor of IDO. Notably, HGF levels were higher within the BM microenvironment of patients with IDO+ myeloma disease compared with patients having IDO- MM. Mechanistically, the antagonism of MET receptor for HGF with SU11274, a MET inhibitor, prevented HGF-induced AKT phosphorylation in MM cells and translated into reduced IDO protein levels and functional activity. Conclusions These data suggest that IDO1 expression may contribute to immune suppression in patients with MM and possibly other HGF-producing cancers.
- Published
- 2012
17. Minimal residual disease (MRD) monitoring by multiparameter flow cytometry (MFC) in newly diagnosed transplant eligible multiple myeloma (MM) patients: Results from the EMN02/HO95 phase 3 trial
- Author
-
Pieter Sonneveld, Manuela Gambella, Mario Boccadoro, Monica Galli, Stefania Oliva, Rossella Ribolla, Lucie Říhová, Rossella Troia, Antonio Palumbo, Milena Gilestro, Roman Hájek, Massimo Offidani, Michele Cavo, Stefano Spada, Bronno van der Holt, Vincent H.J. van der Velden, Sara Grammatico, Paola Omedè, Lucia Pantani, and Davine Hofste op Bruinink
- Subjects
Cancer Research ,medicine.medical_specialty ,business.industry ,Newly diagnosed ,medicine.disease ,Minimal residual disease ,3. Good health ,03 medical and health sciences ,0302 clinical medicine ,Oncology ,030220 oncology & carcinogenesis ,Medicine ,Radiology ,Multiparameter flow cytometry ,business ,Multiple myeloma ,030215 immunology - Abstract
8011 Background: MRD detection is a sensitive tool to measure response in MM. We assessed MRD by MFC in newly diagnosed MM patients (pts) enrolled in the EMN02/HO95 phase 3 trial. Methods: Pts were ≤65 years old and received Bortezomib-Cyclophosphamide-Dexamethasone (VCD) induction, intensification with Bortezomib-Melphalan-Prednisone (VMP) vs High-Dose-Melphalan (HDM) followed by stem cell transplant, consolidation with Bortezomib-Lenalidomide-Dexamethasone (VRD) vs no consolidation, and Lenalidomide maintenance. MRD analysis was performed in pts achieving at least a very good partial response (VGPR) before starting maintenance (after HDM, VMP or VRD) and during maintenance every 6-12 months; samples were centralized to 3 European labs. MFC was performed on bone marrow according to Euroflow-based methods (8 colors, 2 tubes) with a sensitivity of 10-5. Quality checks were performed to compare sensitivity and to show correlation between protocols (Hofste op Bruinink D ASH 2016 abstract 2072). Results: 316 pts were evaluable before maintenance: median age was 57 years, 18% (57/316) pts had ISS III and 22% (70/316) had high risk cytogenetic (HR-C) defined as having at least one among del17, t(4;14) or t(14;16); 63% (199/316) had received HDM and 37% (117/316) VMP; thereafter 51% (160/316) had received VRD. 76% (239/316) were MRD negative (MRD-) of whom 64% (153/239) received HDM vs 36% (86/239) VMP, with a median follow-up time of 30 months from MRD enrolment. 3-year PFS was 50% in MRD positive (MRD+) vs 77% in MRD- pts (HR: 2.87, p < 0.001). Subgroup analyses were performed to evaluate the risk factors for MRD+ according to baseline characteristics and therapies: HR-C was the most important risk factor (HR 9.87, interaction-p = 0.001). Finally, 48% of MRD+ pts at pre-maintenance who had a second MRD evaluation after at least 1 year of lenalidomide became MRD-. Conclusions: MRD by MFC is a strong prognostic factor in MM pts receiving intensification with novel agents or transplant; lenalidomide maintenance further improved depth of response; HR-C is the most important prognostic factor in MRD+ pts. Clinical trial information: NCT01208766.
- Published
- 2017
18. Circulating miRNA markers show promise as new prognosticators for multiple myeloma
- Author
-
Gianluca Isaia, Tiffany Talabere, Silvia Gentili, Andrew Stiff, Flavia Pichiorri, L. De Luca, Manuela Gambella, Alfredo Ferro, Luciano Cascione, Valeria Magarotto, Roberto Ria, Vincenzo Callea, G Marcucci, Jingwen Guan, Antonio Palumbo, Paola Omedè, Emily Smith, Hansjuerg Alder, Davide Rossi, Giulia Benevolo, Susan Geyer, Craig C. Hofmeister, Don M. Benson, Jessica Consiglio, Yvonne A. Efebera, Alberto Rocci, Vijaya R. Dirisala, Brendan M. Weiss, M. Boccadoro, Sara Bringhen, Giuseppina Uccello, and John C. Byrd
- Subjects
Circulating mirnas ,Cancer Research ,Disease free survival ,Proportional hazards model ,business.industry ,MEDLINE ,Hematology ,medicine.disease ,Bioinformatics ,Prognosis ,Article ,Disease-Free Survival ,MicroRNAs ,Text mining ,Oncology ,microRNA ,medicine ,Humans ,business ,Multiple Myeloma ,Multiple myeloma ,Biomarkers ,Proportional Hazards Models - Published
- 2014
19. MET dysregulation is a hallmark of aggressive disease in multiple myeloma patients
- Author
-
Alberto Rocci, Manuela Gambella, Simona Aschero, Ileana Baldi, Livio Trusolino, Federica Cavallo, Francesca Gay, Alessandra Larocca, Valeria Magarotto, Paola Omedè, Gianluca Isaia, Andrea Bertotti, Anna M. Liberati, Lucio Catalano, Luca De Rosa, Pellegrino Musto, Roberto Vallone, Antonietta Falcone, Daniela Drandi, Marco Ladetto, Paolo M. Comoglio, Mario Boccadoro, and Antonio Palumbo
- Subjects
Male ,medicine.medical_specialty ,Pathology ,multiple myeloma ,MET ,hepatocyte growth factor ,biomarker ,prognostic factor ,Aggressive disease ,Gastroenterology ,Internal medicine ,Gene expression ,Biomarkers, Tumor ,medicine ,Humans ,In patient ,RNA, Messenger ,Gene ,Multiple myeloma ,Aged ,Aged, 80 and over ,Very Good Partial Response ,business.industry ,Hematology ,Middle Aged ,Prognosis ,medicine.disease ,Biomarker (medicine) ,Female ,Hepatocyte growth factor ,business ,medicine.drug - Abstract
Abnormal activation of MET/HGF (Hepatocyte Growth Factor) pathway has been described in several tumours and increased HGF plasmatic levels have been detected in patients with aggressive multiple myeloma (MM). MET and HGF mRNA expression was investigated in 105 samples of purified plasma cells derived from newly diagnosed MM patients treated with bortezomib-based induction therapy. Gene expression was compared with response to therapy and clinical outcome. MET gene copy number was also evaluated. MET mRNA expression was higher in CD138(+) than in CD138(-) cells (median 76·90 vs. 11·24; P = 0·0009). Low MET mRNA expression characterized patients with better response (complete response or very good partial response) compared to other patients (median 56·10 vs. 134·83; P = 0·0006). After a median follow-up of 50 months, patients with high MET mRNA expression displayed a worse progression-free survival (PFS; P = 0·0029) and overall survival (OS; P = 0·0023) compared to those with low MET mRNA levels. Patients with both high MET mRNA expression and high β2-microglobulin level (>5·5 mg/l) had further worse median PFS (P < 0·0001) and OS (P < 0·0001). Patients carrying 4 MET gene copies (8 out of 82, 9·8%) also had a short PFS. High MET mRNA expression identifies patients with dismal PFS and OS and the combination with high β2-microglobulin further characterizes patients with worse outcome.
- Published
- 2014
20. Prognostic Implication of Somatic Mutations By Next Generation Sequencing: An Analysis from the Mmrf Commpass Study in Newly Diagnosed Multiple Myeloma Patients
- Author
-
Mattia D'Agostino, Stefania Oliva, Stefano Spada, Manuela Gambella, Mary DeRome, Michele Cavo, Massimo Aglietta, Massimo Offidani, Jennifer Yesil, Daniela Oddolo, Chiara Cerrato, Giuseppe Rossi, Antonio Ledda, Letizia Canepa, Mariella Grasso, Foà Roberto, Daniel Auclair, Pellegrino Musto, Andrea Evangelista, Francesca Gay, Antonio Palumbo, Mario Boccadoro, and Alessandra Larocca
- Subjects
Oncology ,Cancer Research ,medicine.medical_specialty ,Intention-to-treat analysis ,business.industry ,Proportional hazards model ,Hazard ratio ,Immunology ,Cell Biology ,Hematology ,medicine.disease_cause ,medicine.disease ,Interim analysis ,Biochemistry ,Germline mutation ,Internal medicine ,Medicine ,KRAS ,Progression-free survival ,business ,Multiple myeloma - Abstract
Introduction: High throughput techniques, such as massively parallel sequencing, are becoming an attractive approach to characterize multiple myeloma (MM) genomic profiles. However, the clinical relevance and contribution to risk assessment of such approaches need to be established. The Multiple Myeloma Research Foundation (MMRF)CoMMpass trial (NCT01454297) has collected data from 1000 newly-diagnosed MM patients enrolled worldwide and observed through an expected follow-up of up to eight-years. Comprehensive analysis of somatic mutations detected in purified MM cells could reveal disease features with prognostic value, which may not have been detected using traditional approaches. Materials and methods: We analyzed data from the interim analysis 8 cohort. CD138+ purified MM specimens from bone marrow aspirates and mononuclear cells from peripheral blood were collected at diagnosis. Whole exome libraries from both tumor and constitutional DNA samples were created. Somatic single nucleotide variants (SNV) were identified using three different variant callers (Seurat,Strelka andMutect), only nonsynonymous SNV calls made from at least two callers were included in the analysis. Patients were analyzed on an intention-to-treat basis. We evaluated the impact on progression free survival (PFS) of recurrently mutated genes (with at least a nonsynonymous SNV in more than 10 patients) in a Cox model adjusted for international staging system (ISS) and cytogenetic profile (high risk, standard risk and missing). An additive score related to mutated genes was calculated on the basis the level of each coefficient estimated using a Cox Model with backward selection based on theAkaikeInformation Criterion (AIC). Results: 517 patients with baseline somatic mutation data were included in the analysis. Median age at diagnosis was 64 years (range 27-93). All patients received novel agents as first line treatment; 236 (45.6%) received autologous stem cell transplantation (ASCT). Each patient showed a median number of 55 nonsynonymous somatic SNV (range 8-1970) in a median number of 47 genes (range 5-1741). Excluding immunoglobulin genes, the most recurrent mutated genes were KRAS (25%), NRAS (19.5%), TTN (12.1%), MUC16 (9.1%) and DIS3 (9.1%). Based on the results of a multivariable Cox model corrected for ISS and cytogeneticprofile, we created a scoring system determined by the mutational status of 9 genes identified in a nonbiased manner (Table 1). Three groups were identified: group I (score 0-2, 17%); group II (score=3, 51%), and group III (score >3, 32%). After a median follow-up of 371 days, the 18-month PFS rate was 93% for group I, 85% for group II, and 67% for group III. In a Cox model adjusted for ISS and cytogeneticprofile, the hazard ratio was 2.34 (p=0.112) for group II versus group I, and 5.96 (p Conclusion: The use of a prognostic model based on the mutational status of 9 recurrently mutated genes could improve risk assessment of newly-diagnosed MM patients. To our knowledge, this is the largest study correlating nonsynonymous somatic SNV with MM patients' outcome. Longer follow-up and validation in independent cohorts are needed to confirm our findings. Disclosures Larocca: Amgen, Celgene, BMS, Janssen-Cilag: Honoraria. Gay:Amgen: Honoraria; Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Mundipharma: Membership on an entity's Board of Directors or advisory committees. Palumbo:Janssen Cilag: Honoraria; Takeda: Employment, Honoraria.
- Published
- 2016
21. Minimal Residual Disease Detection By Multiparametric Flow Cytometry in Newly Diagnosed Multiple Myeloma Patients: A Preliminary Analysis of the EMN02/HO95 MM Study
- Author
-
Stefania Oliva, Manuela Gambella, Milena Gilestro, Francesca Gay, Alessandra Larocca, Anna Marina Liberati, Francesca Bonello, Giulia Benevolo, Giovannino Ciccone, Tommaso Caravita, Vittorio Emanuele Muccio, Simona Caltagirone, Stefano Spada, Barbara Gamberi, Simone Ferrero, Marina Ruggeri, Giuseppe Rossi, Elona Saraci, Massimo Offidani, Maria Teresa Petrucci, Mario Boccadoro, Pieter Sonneveld, Antonio Palumbo, and Paola Omedè
- Subjects
medicine.medical_specialty ,Cyclophosphamide ,business.industry ,Immunology ,Cell Biology ,Hematology ,medicine.disease ,Off-label use ,Biochemistry ,Minimal residual disease ,Surgery ,Autologous stem-cell transplantation ,Maintenance therapy ,Prednisone ,Internal medicine ,medicine ,business ,Multiple myeloma ,Lenalidomide ,medicine.drug - Abstract
Introduction: Multiple myeloma (MM) is still an incurable disease and patients may relapse despite achievement of complete remission (CR). Minimal residual disease (MRD) assessment by multiparameter flow cytometry (MFC) on bone marrow (BM) is a sensitive diagnostic tool to measure response and is highly predictive of outcome in MM as previously reported. The aim of this study is to evaluate the role of MRD monitoring by MFC in MM patients receiving novel agents with or without autologous stem cell transplantation (ASCT) and to investigate the efficacy of treatments by using MRD-negativity as a deeper response criteria. Methods: The RV-MM-COOP-0556 (EMN02/HO95 MM) study is a phase III, randomized, trial including newly diagnosed MM patients ≤ 65 years. All subjects received 4 cycles of Bortezomib-Cyclophosphamide-Dexamethasone (VCD) induction, followed by Cyclophosphamide chemotherapy and subsequent stem cell mobilization and collection. Afterward, patients were randomized to receive 4 cycles of Bortezomib-Melphalan-Prednisone (VMP) vs one or two cycles of High-Dose-Melphalan (HDM) followed by ASCT. After intensification patients were secondly randomized to receive two cycles of consolidation with Bortezomib-Lenalidomide-Dexamethasone (VRD) vs no consolidation, followed by Lenalidomide maintenance in both arms. Patients who achieved CR/sCR according to IMWG criteria (Rajkumar et al. Blood 2011) after intensification/consolidation treatment, were eligible for the MRD sub-study. MRD analysis by MFC was performed on BM samples after intensification/consolidation, after 6 courses of maintenance, and thereafter every 6 months until progression, to detect monoclonal plasma cells (PCs). Here, we used a double antibody combination (CD138Fitc/CD20PerCp-Cy5.5/CD117APC/CD45APC-H7/CD38PE-Cy7; cyKappaFitc/cyLambdaPE/CD19PerCp-Cy5.5/CD56APC/CD45APC-H7/CD38 PE-Cy7): one tube was employed to obtain PCs quantification, the other one to validate PCs clonality. MRD-negativity was defined when Results: One hundred-eleven Italian patients (58 male/53 female) with a median age of 56 years (IQR 52-62) entered MRD sub-study. Sixteen (14%) were ISS stage III, 24 (22%) had high-risk cytogenetic profile and 10 (9%) had LDH levels higher than the upper normal limit. Forty-five patients (40%) received VMP as intensification and 66 (60%) underwent ASCT, thereafter 65 (58%) received VRD consolidation, 24 after VMP and 41 after ASCT. The median follow-up were 28.7 and 17 months from starting treatment and from MRD enrollment, respectively. After intensification/consolidation, 4 patients were not evaluable for MRD due to unsuitable samples, MRD negativity rate was 79% (85 out of 107 evaluable patients) and was independent from the intensification therapy: actually 50/63 patients who received ASCT and 35/44 patients who received VMP achieved MRD negativity. Within MRD-negative patients, 48/85 (56%) received VRD consolidation without major differences between VMP and ASCT. With the limitation related to the shorter follow-up, depth of response further improved during maintenance: 11/22 (50%) of MRD-positive patients became MRD-negative, independently from previous intensification therapy. Conclusions: MRD detection by MFC is a feasible technique in MM and allows to detect residual tumor cells among CR and sCR patients. Preliminary MRD results show that, in patients achieving CR, intensification with VMP or ASCT induced comparable rates of MRD-negativity and maintenance with Lenalidomide further improved depth of response in both arms. Longer follow-up is needed to correlate MRD status to prognosis and clinical outcome and to evaluate the role of maintenance therapy in increasing the quality of response. Disclosures Off Label Use: Use off-label of drugs for the dose and/or schedule and/or association . Gay:Sanofi: Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Honoraria. Larocca:Janssen-Cilag, Celgene: Honoraria. Caravita:Celgene: Honoraria. Gamberi:Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Mundipharma: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees. Rossi:Celgene: Research Funding. Offidani:Janssen-Cilag, Celgene, Sanofi, Amgen, Mundipharma: Honoraria. Boccadoro:Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Onyx Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees. Sonneveld:Janssen-Cilag, Celgene, Onyx, Karyopharm: Honoraria, Research Funding; novartis: Honoraria. Palumbo:Novartis, Sanofi Aventis: Honoraria; Celgene, Millennium Pharmaceuticals, Amgen, Bristol-Myers Squibb, Genmab, Janssen-Cilag, Onyx Pharmaceuticals: Consultancy, Honoraria.
- Published
- 2015
22. High Expression of mRNA and Gene Amplification of Met In Myeloma Plasma Cells Characterize a More Aggressive Disease
- Author
-
S. Aschero, Gianluca Isaia, Manuela Gambella, Giovannino Ciccone, Livio Trusolino, Alessandra Larocca, Paola Omedè, Claudia Crippa, Antonietta Falcone, Paolo M. Comoglio, Antonio Palumbo, Francesca Patriarca, Andrea Bertotti, Mario Boccadoro, Daniela Drandi, Federica Cavallo, Ileana Baldi, Alberto Rocci, Anna Marina Liberati, Vittorio Montefusco, and Marco Ladetto
- Subjects
Oncology ,medicine.medical_specialty ,education.field_of_study ,Angiogenesis ,Bortezomib ,Immunology ,Population ,Cell Biology ,Hematology ,Biology ,medicine.disease ,Biochemistry ,medicine.anatomical_structure ,Median follow-up ,Internal medicine ,medicine ,Cancer research ,Hepatocyte growth factor ,Bone marrow ,Progression-free survival ,education ,Multiple myeloma ,medicine.drug - Abstract
Abstract 1898 Background: Multiple myeloma (MM) cells growth is sustained by several stimuli derived from surrounding cells of bone marrow (BM) microenvironment. Besides the increase in growth factors production, a constitutive activation of several pathways determining protection from apoptosis and growth advantage have been reported too. Aberrant activation of Met/HGF (Hepatocyte Growth Factor) pathway has been described in several tumors causing cell proliferation, cell migration and neoplastic angiogenesis. A qualitative analysis demonstrated that Met transcription is present in MM plasma cells and increase plasmatic levels of HGF has been related to a bad prognosis subgroup of patients. However a comprehensive investigation of quantitative analysis on Met expression, plasmatic HGF values and correlation with clinical outcome on a large amount of MM patients treated with novel agents is still missing. Aim: : to investigate the role of Met mRNA expression and HGF levels in a large panel of myeloma patients treated with novel drugs. Patients and Methods: one hundred and five samples of purified plasma cells derived from newly diagnosed myeloma patients have been included in this study. Fifty two patients received 9 courses of Velcade-Melphalan-Prednisone (VMP) as part of the VMP-VMPT trial (Palumbo A, 2009 ASH Meeting, abs 128) while fifty three patients have been included in the PAD-MEL100-LP-L trial (Palumbo A, JCO 2010). Met and HGF mRNA quantitative expression have been investigated on both purified plasma cells (CD138+ bone marrow fraction) and on bone marrow CD138 negative fraction. mRNA expression has been evaluated using a quantitative Real-Time PCR (qRT-PCR) on Abi Prism 7900 (Applied Biosystems) with a relative quantification based on ΔΔCt approach. JUM2 cell line was used as calibrator and Gus as housekeeping gene. HGF serum level has been evaluated on 76 of those patients too. ELISA assay has been employed to determine HGF serum value. On 51 samples with higher levels of mRNA Met expression, a FISH analysis reaching for Met amplification has been performed. Purified plasma cells were treated with a dual-color FISH assay using a MET/CEP7 probe cocktail (Cytocell, Cambridge, UK). Results: Met mRNA expression was higher in CD138+ cells than in CD138- fractions (median 76,90 range 0,81-916,51 vs 11,03 range 0–243,88 respectively; p=0,0001). Similarly HGF mRNA expression was higher in CD138+ cells than in CD138- population (median 2,07 range 0–65,34 vs 0,49 range 0,11-3,22 respectively; p=0,03). Patients with high and low Met levels were divided using the median value of Met expression as cutoff. At a median follow up of 30 months, patients with low Met mRNA expression displayed a higher progression free survival (PFS) and overall survival (OS) compared with those with high Met levels (PFS 73% vs 42% respectively, p Conclusions: 1) high Met mRNA expression levels identify a group of patients with worse prognosis even when treated with novel drugs containing regiments; 2) Met amplification has been described for the first time in myeloma cells and can determine high Met mRNA levels characterizing a more aggressive disease. Disclosures: Cavallo: Celgene: Honoraria. Patriarca:Roche: Honoraria; Janseen-Cilag: Honoraria; Celgene: Honoraria; Merck: Membership on an entity's Board of Directors or advisory committees. Boccadoro:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janseen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Palumbo:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janseen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees.
- Published
- 2010
23. In Multiple Myeloma, Minimal Residual Disease (MRD) Is an Early Predictor of Progression and Is Modulated By Maintenance Therapy with Lenalidomide
- Author
-
Manuela Gambella, Paola Omedè, Stefania Oliva, Milena Gilestro, Vittorio Emanuele Muccio, Daniela Drandi, Simone Ferrero, Francesca Gay, Francesca Patriarca, Carmela Palladino, Maria Teresa Petrucci, Norbert Pescosta, Anna Marina Liberati, Tommaso Caravita, Francesco Di Raimondo, Alberto Rocci, Pellegrino Musto, Mario Boccadoro, and Antonio Palumbo
- Subjects
medicine.medical_specialty ,business.industry ,Immunology ,Complete remission ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,Minimal residual disease ,Maintenance therapy ,Partial response ,Internal medicine ,Medicine ,business ,Bristol-Myers ,Multiple myeloma ,Lenalidomide ,medicine.drug - Abstract
Background. Minimal residual disease (MRD) detection by multi-parameter flow cytometry (MFC) and real time quantitative PCR (RQ-PCR) is highly predictive of outcome in multiple myeloma (MM) patients (pts). Less is known on the ability of maintenance therapy to modulate MRD levels. The primary end-point of this study was to monitor MRD during maintenance therapy and to evaluate the impact on outcome. Patients and Methods. In the RV-MM-EMN-441 study (NCT01091831), after induction and consolidation pts received maintenance therapy with either Lenalidomide alone (R) or Lenalidomide-Dexamethasone (RD) until progression. Pts achieving at least very good partial response (VGPR) after induction/consolidation treatment were eligible for the MRD sub-study. MRD analysis was performed on bone marrow (BM) samples at diagnosis, after consolidation, after 3 and 6 courses of maintenance, and thereafter every 6 months until progression. MFC complete remission (CR) was defined by Results. MRD sub-study included 50 pts with a median age of 57 years (range 40-65). After consolidation 34/50 (68%) pts achieved VGPR, 16/48 (32%) achieved CR according to IMWG criteria (Rajkumar et al. Blood 2011). After a median follow-up of 44 months, 22/50 (44%) progressed and 7/50 (14%) deaths were recorded, with a 4-year PFS of 49% and 4-year OS of 87%. In the MFC analysis 19/50 pts (38%) achieved MFC-CR after consolidation, while other additional 7/50 pts (14%) achieved MFC-CR during maintenance. Among pts who achieved MFC–CR (< 1E-04), 7/26 (27%) pts progressed after a median of 30 months; among those who did not reach MFC–CR, 15/24 (62%) pts progressed after a median of 26 months (p=0.009). In 18/19 pts MFC-progression anticipated clinical relapse of a median of 9 months. In 1/19 pts, clinical extra-medullary progression anticipated MFC-progression. A molecular marker was identified in 25/50 pts (50%); 4/25 pts (16%) achieved molecular-CR after consolidation, while other additional 3/25 pts (20%) achieved molecular-CR during maintenance. Among pts who achieved molecular–CR ( Conclusions. Lower MRD values, both with MFC and RQ-PCR procedures, predict better outcome. 30% of patients achieved MFC-CR (7/26) or molecular-CR (3/7) during maintenance therapy suggesting that MRD levels can be modified by maintenance. MRD progression anticipates clinical relapse of approximately 8 months. Disclosures Off Label Use: lenalidomide used as off-label. Ferrero:MUNDIPHARMA: Honoraria. Gay:Sanofi: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Patriarca:Merck Sharp & Dohme: Honoraria; Janssen and Cilag: Honoraria; Celgene: Honoraria. Petrucci:CELGENE: Honoraria; JANSSEN-CILAG: Honoraria; SANOFI: Honoraria; BRISTOL MYERS SQUIBB: Honoraria. Caravita:Celgene: Honoraria. Di Raimondo:CELGENE: Honoraria; JANSSEN-CILAG: Honoraria. Musto:CELGENE: Honoraria; JANSSEN-CILAG: Honoraria. Boccadoro:Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees; Onyx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees. Palumbo:Array BioPharma: Honoraria; Onyx Pharmaceuticals: Consultancy, Honoraria; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Genmab A/S: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Sanofi: Honoraria.
- Published
- 2014
24. Proteomic Characterization of the Multiple Myeloma Bone Marrow Extracellular Matrix
- Author
-
Antonio Palumbo, Alberto Rocci, Michele Moschetta, Siobhan Glavey, Manuela Gambella, Irene M. Ghobrial, Yawara Kawano, John M. Asara, Richard O. Hynes, Yuji Mishima, Salomon Manier, Antonio Sacco, Michaela R. Reagan, Alexandra Naba, and Aldo M. Roccaro
- Subjects
Tumor microenvironment ,Stromal cell ,Immunology ,Cell Biology ,Hematology ,Biology ,medicine.disease ,Proteomics ,Biochemistry ,Metastasis ,Extracellular matrix ,medicine.anatomical_structure ,medicine ,Cancer research ,Bone marrow ,Multiple myeloma ,Whole Bone Marrow - Abstract
Background The extracellular matrix (ECM) is a major component of the tumor microenvironment, contributing to the regulation of cell survival, proliferation, differentiation and metastasis. In multiple myeloma (MM), interactions between MM cells and the bone marrow (BM) microenvironment, of which the ECM forms a major component, are critical to the pathogenesis of the disease and the development of drug resistance. To date, despite some knowledge of the composition of the ECM in tumors, detailed profiling of the composition of the ECM in MM has not been carried out. Until recently ECM proteins have proven difficult to characterize due to their biochemical properties and large size. Recent advances in proteomics have led to the characterization of the ECM and ECM-associated proteins (“matrisome”) in normal human tissues and tumors using a systematic and comprehensive approach. Methods Tumor Xenograft models; MM1S-GFP-Luc+ cells (5x106) were injected intravenously into SCID-Bg mice (n=4/group) and animals underwent weekly bioluminescent imaging (BLI). Mice were sacrificed after 2 weeks in order to mimic early tumor development (luminescence = 1x105 p/sec/cm2/sr) or 5 weeks (1x108 p/sec/cm2/sr) to model more advanced MM. Human bone marrow aspirates; Whole bone marrow was obtained from newly diagnosed MM patients (n=9) and healthy human donors (ND) (n=4) following written informed consent. ECM proteins were enriched from bone marrow samples obtained from MM patients, NDs and mice according to previously published methods.Tandem Mass Spectrometry (LC-MS/MS): Peptides were run using reversed-phase microcapillary liquid chromatography – tandem mass spectrometry (LC-MS/MS) on a high resolution hybrid Orbitrap Elite mass spectrometer. MS/MS data were searched against the UniProt Human database using MASCOT to identify proteins. Spectral counts were used as a semi-quantitative measure of abundance. ECM proteins were defined according to the in-silico definition of the matrisome. Validation of expression of ECM mRNA in MM cell lines (MM1s, RPMI-8226 and U266) and in CD138+ cells and bone marrow stromal cells (BMSC’s) from MM patients in comparison to NDs was performed using qRT-PCR. Results Primary myeloma sample ECM; Using a spectral count of 2 as a cutoff of peptide abundance we identified a total of 536 unique proteins in ND bone marrow of which 35 are defined as matrisome proteins. 982 unique proteins were enriched from whole bone marrow samples of newly diagnosed MM patients of which 26 are defined as matrisome proteins, 7 unique proteins were identified as ECM or ECM-associated in newly diagnosed patients which were not detected in the ND samples including PRG3, FGG, LEG10, TLN1 and PLEC. Critical ECM components such as laminins, matrix metalloproteinases and collagens were also found to be significantly altered in newly diagnosed MM with evidence of destruction of ECM components in active disease. Tumor Xenograft ECM; In mice with an earlier phase of human MM1s cell tumor burden we detected a total of 329 unique proteins of which 48 were defined as matrisome proteins, 23 of these proteins were unique to the earlier phase of MM in these mice. Mice with more advanced tumor development had unique ECM proteins which were not detected in the earlier disease stage including collagens, laminins and matrix metalloproteinases, indicating that these ECM components may be critical for re-modelling the ECM in MM. Interestingly, in our xenograft model of MM we were able to detect both human and mouse ECM components indicating that the tumor ECM is secreted from both the murine stroma and the human MM cells and allowing delineation of the source of individual ECM components. This indicates that as MM progresses certain ECM components, including FBN1, which were initially derived from stroma are later derived from MM cells. Differential expression of ECM components, including FBN1 between normal and malignant plasma cells was confirmed using qRT-PCR. Conclusions We have performed proteomic profiling of the unique tumor ECM in MM using mass spectrometry with a view to determining the specific components that may be altered with disease progression. Through this approach plasma-cell-derived ECM can be identified with a view to developing therapeutic strategies in this disease. Disclosures Glavey: BMS: Consultancy, Research Funding. Palumbo:Bristol-Myers Squibb: Consultancy, Honoraria; Genmab A/S: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria; Onyx Pharmaceuticals: Consultancy, Honoraria; Array BioPharma: Honoraria; Amgen: Consultancy, Honoraria; Sanofi: Honoraria. Ghobrial:Onyx: Advisory board Other; BMS: Advisory board, Advisory board Other, Research Funding; Noxxon: Research Funding; Sanofi: Research Funding.
- Published
- 2014
25. Cell-Free DNA for Minimal Residual Disease Monitoring in Multiple Myeloma Patients
- Author
-
Lenka Kubiczkova-Besse, Paola Omedè, Ludek Pour, Lenka Sedlarikova, Daniela Drandi, Stefania Oliva, Manuela Gambella, Sabina Ševčíková, Antonio Palumbo, Roman Hájek, Zdenek Adam, and Mario Boccadoro
- Subjects
Immunoglobulin A ,0303 health sciences ,Pathology ,medicine.medical_specialty ,biology ,Immunology ,Cancer ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,Minimal residual disease ,3. Good health ,Cell-Free Nucleic Acids ,03 medical and health sciences ,0302 clinical medicine ,Cell-free fetal DNA ,030220 oncology & carcinogenesis ,biology.protein ,medicine ,Cancer research ,Antibody ,Multiple myeloma ,030304 developmental biology ,Tumor marker - Abstract
Background Circulating nucleic acids, such as cell-free DNA (cf-DNA), are becoming a promising minimally-invasive diagnostic tool for cancer detection. Recent studies demonstrated that tumor-derived cf-DNA can be used to monitor tumor burden and response to treatment in patients (pts) with solid tumors as well as hematological malignancies (Dawson et al, 2013, Armand et al, 2013). In this study we investigated the clinical utility of cf-DNA in the monitoring of minimal residual disease (MRD) of pts with multiple myeloma (MM) carrying the tumor specific immunoglobulin (IGH) rearrangement. Methods Cf-DNA was extracted from 1 ml of serum sample from 13 MM patients enrolled in Italian CRD/MEL-200 and EMN-02 protocols. The total amount of cf-DNA was estimated by fluorometric measurement (median 560 ng, range 15-5158 ng) and the length of fragments was evaluated by high sensitivity dsDNA chips (Agilent). Patient specific clonal IGH rearrangement was identified at the time of diagnosis from bone marrow (BM) genomic DNA (gDNA) as previously reported (Ladetto et al, 2000). For each patient, MRD in BM and peripheral blood (PB) was estimated by real time quantitative PCR (qPCR) using ASO-specific primers and the quantification was based on serial 10-fold dilution standard curves from plasmid carrying the patient specific IGH rearrangement. The amount of IGH rearrangement in cf-DNA (cf-IGH) was estimated by qPCR and droplet digital PCR (ddPCR) (Bio-Rad) on diagnostic and follow up samples and was expressed as the amount of copies per 1 µg of total cf-DNA. qPCR and ddPCR results were interpreted according to the Euro-MRD guidelines (van der Velden et al, 2003). Results Overall, 54 cf-DNA samples from MM serum (13 diagnostic, 41 follow-up samples) were analyzed for the presence of patient specific IGH rearrangement. The most abundant fraction of cf-DNA was 180-220bp, than 350-400bp and 700-10000bp (in 100%, 85% and 68% of samples respectively), whereas longer fragments more often appeared in follow-up samples. By qPCR, cf-IGH at diagnosis were observed in 11/13 diagnostic samples. Only 3/13 pts were quantifiable (116, 85, 187 copies/1 µg of cfDNA) and 8/13 pts were positive but not quantifiable (PNQ) cf-IGH. By ddPCR, levels of cf-IGH at diagnosis were observed in 9/13 pts. 6/13 pts were quantifiable (246, 195, 96, 88, 184, 25 copies/1µg of cfDNA), and only 3/13 pts were PNQ. In follow-up samples, levels of cf-IGH were undetectable by qRT-PCR; however in 5 samples they were PNQ by ddPCR. Interestingly, in one available relapse sample, cf-IGH reappeared again to quantifiable level (61 copies by qRT-PCR and 190 copies by ddPCR). The levels of cf-IGH are quantifiable in samples with higher amount of tumor specific IGH rearrangements in BM or PB; however, no association was observed between cf-IGH level at diagnosis and disease burden estimated by the PCs infiltration in BM or the monoclonal immunoglobulin concentration in blood/urine. Conclusions These data show the potential utility of cf-IGH monitoring in MM pts. Although by qPCR, cf-IGH were detected in 11/13 pts, they were quantifiable only in 3/13 pts and ddPCR was more precise as it was able to quantify cf-IGH in 6/13 pts. Since cf-IGH copies were quantifiable only in diagnostic samples and in 1 available sample at the relapse, we conclude that higher amounts of serum are necessary to overcome the limitation of assay sensitivity. Potential advantages and predictive value, for monitoring tumor marker in a non-invasive manner, need to be further validated on larger cohort of samples using increased amount of cf-DNA. Work was supported by IGA grants NT12130, NT14575. This work is funded by a Black Swan Research Initiative grant by the International Myeloma Foundation "Dynamics of microRNA and cell-free DNA profiles during multiple myeloma progression“. Disclosures Boccadoro: Celgene: Honoraria; Janssen: Honoraria; Onyx: Honoraria. Palumbo:Amgen: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Array BioPharma: Honoraria; Genmab A/S: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria; Onyx Pharmaceuticals: Consultancy, Honoraria; Sanofi Aventis: Honoraria.
- Published
- 2014
26. Abstract 4855: Proteomic characterization of the extracellular matrix in multiple myeloma
- Author
-
Antonio Sacco, John M. Asara, Richard O. Hynes, Aldo M. Roccaro, Alberto Rocci, Alexandra Naba, Irene M. Ghobrial, Manuela Gambella, Antonio Palumbo, and Siobhan Glavey
- Subjects
Cancer Research ,Tumor microenvironment ,Pathology ,medicine.medical_specialty ,biology ,business.industry ,Integrin ,medicine.disease ,Metastasis ,Fibronectin ,Extracellular matrix ,medicine.anatomical_structure ,Oncology ,Cancer research ,biology.protein ,medicine ,Bone marrow ,business ,Whole Bone Marrow ,Multiple myeloma - Abstract
Background: The extracellular matrix is a major component of the tumor microenvironment, contributing to the regulation of cell survival, proliferation, differentiation and metastasis. In multiple myeloma (MM) ECM components, such as integrins, fibronectin and collagens, have been shown are critical to the pathogenesis of MM and the development of drug resistance. To date, despite some knowledge of the composition of the ECM in tumors, detailed profiling of the composition of the ECM in MM has not been carried out. Recent advances in proteomics have led to the characterization of the ECM and ECM-related proteins (“matrisome”) in normal human tissues and tumors in a systematic and comprehensive approach. Methods: Tumor Xenograft models; MM1S-GFP-Luc+ cells (5x106) were injected intravenously into SCID-Bg mice (n=4/group) and animals underwent weekly bioluminescent imaging (BLI). Mice were sacrificed after two weeks in order to mimic early tumor development (luminescence= 1x105 p/sec/cm2/sr). and compared to mice demonstrating high tumor burden (1x108 p/sec/cm2/sr) 5 weeks post injection. Human bone marrow aspirates; Whole bone marrow was obtained from MM patients (newly diagnosed n=9, relapsed n=9) and healthy human donors (n=9) following written informed consent. Sequential extractions of whole pooled bone marrow from mice and individual bone marrow humans was performed using the CNMCS(Cytosol/Nucleus/Membrane/Cytoskeleton) Compartmental Protein Extraction Kit (Cytomol, CA). Following this, proteins underwent reversed-phase high performance liquid chromatography followed by tandem mass spectrometry (MS). Identified peptide spectra were counted as a semi quantitative measure of abundance. Results: We detected a total of 1202, 982 and 329 unique proteins from enriched whole bone marrow samples from relapsed patients, newly diagnosed patients and mice, respectively. Of these, critical ECM components such as laminins, matrix metalloproteinases and collagens were found to be enriched in human MM ECM in comparison to healthy donors with increased abundance apparent with disease progression in mice. Specifically Bone Marrow Proteoglycan and Proteoglycan 3 were amongst the ECM proteins significantly enriched in the ECM of newly diagnosed patients. PRG3 is a p53 responsive gene that has been demonstrated to be upregulated in apoptotic cells in several cancers, including CLL. The ECM protein Elastin, the expression of which is closely associated with the invasive/metastatic potential of various cancer types, was also upregulated in the MM ECM where it may play an important role in the tumor-ECM interaction. Conclusions: We have profiled the ECM in MM using mass spectrometry with a view to determining the specific components that may be important in MM disease biology. Through this approach molecular mechanisms that influence MM development and progression can be uncovered and potential targets for therapy identified. Citation Format: Siobhan V. Glavey, Alexandra Naba, Manuela Gambella, Alberto Rocci, Antonio Sacco, John Asara, Antonio Palumbo, Richard O. Hynes, Aldo M. Roccaro, Irene M. Ghobrial. Proteomic characterization of the extracellular matrix in multiple myeloma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4855. doi:10.1158/1538-7445.AM2014-4855
- Published
- 2014
27. Minimal Residual Disease Monitoring During Maintenance In Multiple Myeloma Patients
- Author
-
Alberto Rocci, Manuela Gambella, Paola Omedè, Daniela Drandi, Francesca Gay, Francesca Patriarca, Federica Cavallo, Maria Teresa Petrucci, Caravita Tommaso, Giulia Benevolo, Norbert Pescosta, Stefania Oliva, Massimo Offidani, Vittorio Montefusco, Anna Marina Liberati, Antonietta Pia Falcone, Stelvio Ballanti, Tonino Spadano, Tommasina Guglielmelli, Pellegrino Musto, Renato Zambello, Francesco Di Raimondo, Marco Ladetto, Mario Boccadoro, and Antonio Palumbo
- Subjects
Melphalan ,medicine.medical_specialty ,Cyclophosphamide ,business.industry ,Immunology ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,Minimal residual disease ,Surgery ,Transplantation ,Autologous stem-cell transplantation ,Maintenance therapy ,Internal medicine ,Medicine ,business ,Multiple myeloma ,medicine.drug ,Lenalidomide - Abstract
Background The quality of response and the residual disease after treatment are important prognostic factors in several hematological diseases including multiple myeloma (MM). Several papers demonstrated that the deeper the response after treatment, the longer the survival. However few data are available on the monitoring of minimal residual disease (MRD) during the maintenance therapy in transplant eligible MM patients. Aims to evaluate the role of maintenance therapy in reducing MRD and the role of monitoring the response to predict clinical relapse. Patients and Methods newly diagnosed MM patients enrolled in the RV-MM-EMN-441 trial (NCT01091831) and achieving at least a very good partial response (VGPR) after consolidation were included in the study. Patients received 4 Lenalidomide-Dexamethasone (RD) courses as induction, Cyclophosphamide to mobilize bone marrow stem cells (BMSC) and then were randomized to receive 6 cycles of Cyclophosphamide-Lenalidomide-Dexamethasone (CRD) or Autologous Stem Cell Transplantation (ASCT) with Melphalan 200 mg/m2. All patients received maintenance therapy with Lenalidomide (R) or Lenalidomide-Dexamethasone (RD) until relapse. MRD analysis was performed in a single laboratory (University of Turin, Italy) using flow cytometry according to European Myeloma Network guideline (Rawstron AC, Haematologica 2008). Samples of bone marrow (BM) were collected at diagnosis, after consolidation, after 3 and 6 courses of maintenance and then every 6 months until clinical relapse. The samples were considered MRD +ve if ≥ 0.01% of PC were detected. Immunophenotypic (IF) relapse was defined as an increase of ≥ 25% in the amount of malignant plasma cells in BM compared to the previous determination. Results Fifty patients (27 female/23 male) with a median age of 57 yrs (40-65) entered the study. According to ISS, 27 patients were stage I, 15 stage II and 8 stage III. Fish risk profile was standard in 31 patients, high in 11 and not available in 8. Twenty-five patients received CRD as consolidation and 25 underwent ASCT. The median follow-up was 28.6 months. After consolidation 16 (32%) patients achieve a complete response (CR) and 34 (68%) a VGPR. MRD was negative in 19/48 (40%) patients, of which 12 received ASCT (out of 23, 52%) and 7 received CRD (out of 25, 28%). Patients receiving ASCT showed a lower value of residual cells (median 0.08%, range 0 – 1.00) compared to patients receiving CRD (median 0.5%, range 0 – 2.9%, p=0.0134). The lower MRD value was achieved after consolidation in 31 patients (62%), after 3 courses of maintenance in 6 patients (12%) and after 6 or more courses of maintenance in 13 patients (26%). The increase in quality of response was observed primarily in patients receiving CRD: the average amount of residual plasma cells in bone marrow was 71/uL after induction, lowering to 51/uL after 6 and 12 courses of maintenance therapy. Nine patients clinically relapsed after an average time of 25.6 months from the beginning of the therapy and in all patients this was anticipated by immunophenotypic relapse. Conclusion 1) consolidation therapy with ASCT determines a deeper response compared to CRD; 2) maintenance therapy can improve the quality of response, in particular in patients not receiving ASCT; 3) Immunophenotypic relapse anticipate the clinical relapse. These results suggest the possible role of MRD monitoring to better assess the response to therapy also during maintenance and as marker of early relapse. Disclosures: Ladetto: Celgene: Research Funding, Speakers Bureau; Janssen Cilag: Research Funding, Speakers Bureau; Mundipharma: Research Funding, Speakers Bureau; Roche: Research Funding, Speakers Bureau; Amgen: Research Funding, Speakers Bureau. Boccadoro:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Palumbo:Amgen: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Millenium: Consultancy, Honoraria; Onyx: Consultancy, Honoraria.
- Published
- 2013
28. Improved Igh-Based MRD Detection By Using Droplet Digital PCR: a Comparison With Real Time Quantitative PCR In MCL and MM
- Author
-
Daniela Drandi, Lenka Kubiczkovà, Nadia Dani, Simone Ferrero, Luigia Monitillo, Barbara Mantoan, Elisa Genuardi, Daniela Barbero, Manuela Gambella, Davide Barberio, Paola Ghione, Guglielmo Guarona, Elona Saraci, Paola Omedè, Roberto Passera, Roman Hajek, Sergio Cortelazzo, Antonio Palumbo, Mario Boccadoro, Giorgio Inghirami, and Marco Ladetto
- Subjects
Pathology ,medicine.medical_specialty ,Thermal cycler ,Serial dilution ,business.industry ,Immunology ,Context (language use) ,Cell Biology ,Hematology ,Gene rearrangement ,030204 cardiovascular system & hematology ,Biochemistry ,Minimal residual disease ,Molecular biology ,3. Good health ,Standard curve ,03 medical and health sciences ,0302 clinical medicine ,Real-time polymerase chain reaction ,Medicine ,Digital polymerase chain reaction ,business ,030215 immunology - Abstract
Background In mature lymphoid disorders, minimal residual disease (MRD) detection based on real time quantitative PCR (RQ-PCR) of immunoglobulin heavy chain gene rearrangement (IgH) has a well-established role in prognostic assessment, particularly in Mantle cell Lymphoma (MCL) and Multiple Myeloma (MM). RQ-PCR has excellent sensitivity and specificity but has a major limitation in its relative quantification nature, as it requires a reference standard curve usually built with dilutions of diagnostic tumor DNA or on plasmids containing the target rearrangement. Droplet Digital PCR (DD-PCR), applying the principle of limiting dilution of DNA and single molecule detection allows a reliable absolute quantification of target. In this study we compared IgH-based MRD detection by RQ-PCR and DD-PCR, to assess whether DD-PCR could achieve the same performances of RQ-PCR in the absence of the limitation mentioned above. Methods Bone marrow (BM) and peripheral blood (PB) samples were collected from patients affected by MCL and MM in which RQ-PCR based MRD analysis was already performed in the context of prospective clinical trials. In all trials patients gave the informed consent for MRD determination. IgH-based MRD detection by RQ-PCR was carried out as previously described [Ladetto et al. BBMT 2000] and results were interpreted according to the Euro-MRD guidelines [van der Velden et al. Leukemia 2007]. DD-PCR was performed by the QX100 Droplet Digital PCR system (Bio-RAD Inc.) on 500 ng of genomic DNA combined with the same Allele Specific Oligonucleotides (ASO)-primers and TaqMan-probes used in the RQ-PCR. Droplets were generated by QX100 droplet generator. End-point PCR (40 cycles) was performed on a T100 Thermal cycler (Bio-RAD Inc). The PCR product was loaded in the QX100 droplet reader and analyzed by QuantaSoft 1.2 (Bio-Rad Inc). For data interpretation RQ-PCR and DD-PCR results were expressed as amount of target copies per 1E+05 cells. Comparability of MRD results by DD-PCR and RQ-PCR was assessed by means of bivariate correlations between methods analysis (R2.15.1 package irr). Discordances were classified as follows: a positive/negative discordance was defined as major when the positive result was >1E-04 and minor when ≤1E-04; a quantitative discordance was defined as the presence of two positive results with a quantitative discrepancy >1 log. Results Overall, 161 samples belonging to 35 patients (18 MCL and 17 MM), 66 MCL and 95 MM were analyzed. 35 samples were taken at diagnosis and 126 at follow-up. 118 were BM while 43 were PB. A significant correlation was found between DD-PCR and RQ-PCR (R2=0.89, p Conclusions Here we report for the first time the use of DD-PCR in the context of IgH-based MRD evaluation in lymphoproliferative disorders. DD-PCR is a feasible tool for IGH-based MRD monitoring in MCL and MM, reaching similar sensitivities compared to standardized RQ-PCR. Moreover DD-PCR allows bypassing the need of building a standard curve thus considerably reducing the complexity of IgH-based RQ-PCR (need of purified diagnostic tissue or Flow Cytometry-based quantification of tumor load or diagnosis, or building of a plasmid-derived standard curve). Finally DD-PCR might potentially overcome the problem of positive non-quantifiable samples. These features make DD-PCR a feasible and attractive alternative method for IgH-based MRD assessment. Disclosures: Kubiczkovà: GAP304/10/1395 : Research Funding; MUNI/11/InGA17/2012: Research Funding.
- Published
- 2013
29. miRNA in Serum and Bone Marrow Plasma Cells From Multiple Myeloma Patients
- Author
-
Andrew Stiff, Alberto Rocci, Craig C. Hofmeister, Paola Omedè, Susan Geyer, Sara Bringhen, Luciano Cascione, Anissa Bingman, Manuela Gambella, Federica Cavallo, Luciana De Luca, Jingwen Guan, Alessandra Larocca, Jacqueline Corry, Francesca Gay, Yvonne A Efebera, Uccello G, Don M. Benson, Tiffany Talabere, Kevin Murnan, Magarotto Valeria, Mario Boccadoro, Carlo M. Croce, Antonio Palumbo, and Flavia Pichiorri
- Subjects
Pathology ,medicine.medical_specialty ,Creatinine ,biology ,Beta-2 microglobulin ,Myeloma protein ,Immunology ,C-reactive protein ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,Pathogenesis ,chemistry.chemical_compound ,medicine.anatomical_structure ,chemistry ,medicine ,biology.protein ,Bone marrow ,Precordial catch syndrome ,Multiple myeloma - Abstract
Abstract 2921 Background: Multiple myeloma (MM) is a clonal B-cell malignancy characterized by the aberrant expansion of clonal plasma cells (PCs) within the bone marrow. Malignant PCs produce intact or partial monoclonal immunoglobulin (M protein) and cause organ damage. More than 20,000 new cases of multiple myeloma (MM) are diagnosed every year in the US with approximately 10,700 deaths occurring. The pathogenesis of MM is still largely unclear, but several reports suggest that interaction of tumor cells with the bone marrow microenvironment and microRNAs (miRNAs) deregulation may play a role in the etiology and progression of MM. miRNAs are small non-coding RNAs capable of regulating protein expression by binding to mRNA, and have been implicated in the development of MM. First identified inside cells, miRNAs can also be detected in body fluids, including serum and plasma, and may be a valid biomarker. Few studies have investigated the agreement between circulating miRNAs and intracellular myeloma PC miRNAs at diagnosis. Methods: Using Nano-String nCounter technology we first performed a screening analysis on serum samples obtained from MM patients and healthy controls. We identified a candidate set of miRNAs differentially expressed in the serum of MM patients. The levels of these miRNA markers were validated by RT-PCR in both serum and bone marrow PCs from the same cohort. Agreement of the quantitative miRNA marker levels between sample types was evaluated using intraclass correlation coefficients (ICC) (both for normalized and log2 measures). Results: Thirty-nine MM patients (21 male, 18 female) with a median age of 72 years (range: 65 – 83) were included in the analysis. Most were ISS stage I or II (59% vs. 41% ISS stage III) and 39% were high risk according to FISH abnormalities – 21% of patients carried del17p, 24% t(4;14) and 5% t(14;16). Medians and ranges for lab markers were as follows: hemoglobin 10.0 g/dl (7.2 to 15.1), beta2-microglobulin 5.18 ug/ml (1.38 – 12.1), creatinine 0.94 mg/dl (0.65 – 2.49), CRP = 1.6 mg/dl (0.02 – 116.0). Nine age-matched healthy controls were also used for the analysis. After the screening analysis, the following miRNAs were differentially expressed between healthy subjects and MM patients in serum samples: miR-92a, miR-451, miR-19b, miR-21, miR-16, miR-25, miR-30a, and miR-126. There was no significant agreement or correlation between serum and myeloma cell samples using either untransformed as well as log2measures (all p>0.40) (Table 1). Conclusion: Our preliminary results suggest a difference between circulating miRNAs in myeloma patients from controls. This indicates that future studies are needed to better define the role of miRNAs in the peripheral blood as a prognostic and even diagnostic biomarker in myeloma. From our preliminary data it also appears that circulating miRNAs are not simply secreted into the peripheral blood by myeloma PCs as it seems that circulating miRNAs do not reflect those of myeloma PCs. Differential expression could be determined by other cells that can release and or modify their miRNA expression in response to MM. Ongoing studies are examining the origin and function of miRNAs in the peripheral blood. Disclosures: No relevant conflicts of interest to declare.
- Published
- 2012
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.