Clinical Cancer Imaging, Diagnosis, Prognosis Research CDKN2A/B Alterations Impair Prognosis in Adult BCR-ABL1– Positive Acute Lymphoblastic Leukemia Patients Ilaria Iacobucci1, Anna Ferrari1, Annalisa Lonetti1, Cristina Papayannidis1, Francesca Paoloni2, Stefania Trino1, Clelia Tiziana Storlazzi4, Emanuela Ottaviani1, Federica Cattina5, Luciana Impera4, Maria Chiara Abbenante1, Marco Vignetti2, Antonella Vitale3, Leonardo Potenza6, Stefania Paolini1, Simona Soverini1, Fabrizio Pane7, Mario Luppi6, Robin Foa3, Michele Baccarani1, and Giovanni Martinelli1 Abstract Purpose: The 9p21 locus, encoding three important tumor suppressors (p16/CDKN2A, p14/ARF, and p15/CDKN2B), is a major target of inactivation in the pathogenesis of many human tumors. Patients and Methods: To explore, at high resolution, the frequency and size of alterations affecting this locus in adult BCR-ABL1–positive acute lymphoblastic leukemia (ALL) and to investigate their prognostic value, 112 patients (101 de novo and 11 relapsed cases) were analyzed by genome-wide single-nucleotide polymorphism arrays and gene candidate deep exon sequencing. Paired diagnosis–relapse samples were further available and analyzed for 19 (19%) cases. Results: CDKN2A/ARF and CDKN2B genomic alterations were identified in 29% and 25% of newly diagnosed patients, respectively. Deletions were monoallelic in 72% of cases, and in 43% of them, the minimal overlapping region of the lost area spanned only the CDKN2A/B gene locus. An analysis conducted at relapse showed an increase in the detection rate of CDKN2A/ARF loss (47%) compared with the time of diagnosis (P ¼ 0.06). Point mutations within the 9p21 locus were found at very low levels, with only a nonsynonymous substitution in the exon 2 of CDKN2A. Of note, deletions of CDKN2A/B were significantly associated with poor outcomes in terms of overall survival (P ¼ 0.0206), disease free-survival (P ¼ 0.0010), and cumulative incidence of relapse (P ¼ 0.0014). Conclusions: Inactivation of the 9p21 locus by genomic deletion is a frequent event in BCR-ABL1– positive ALL. Deletions are frequently acquired during leukemia progression and are a poor prognostic marker of long-term outcomes. Clin Cancer Res; 17(23); 7413–23. Ó2011 AACR. Introduction signaling and defective responses to antimitogenic stimuli that contribute to unscheduled proliferation and genomic Disruption of tumor suppressor genes and/or activation instability in tumor cells. Taken together, these alterations of oncogenic pathways result in constitutive mitogenic result not only in proliferative advantages but also in increased susceptibility to the accumulation of additional genetic alterations that contribute to tumor progression and 1 Authors' Affiliations: Department of Hematology and Oncological acquisition of more aggressive phenotypes. In almost all Sciences "L. and A. Seragnoli" S.Orsola Malpighi Hospital, University of Bologna, Bologna; 2Gruppo Italiano Malattie EMatologiche dell'Adulto tumors (1–5), these cell-cycle defects are mediated by the (GIMEMA) Data Center, GIMEMA Foundation; 3Department of Cellular Bio- inactivation of a region located in humans at chromosome 4 technologies and Hematology, "Sapienza" University, Rome; Department of arm 9p21. Biology, University of Bari, Bari; 5Chair of Hematology and BMT unit, Uni- versity of Brescia, Brescia; 6Hematology, Department of Oncology, Hema- This region has a complex and unique genomic organi- tology and Respiratory Diseases University of Modena and Reggio Emilia, zation containing 2 candidate tumor suppressor genes, Modena; and 7CEINGE Biotecnologie Avanzate and Department of Biochem- CDKN2A istry and Medical Biotechnology, University of Naples Federico II, Naples, Italy [cyclin-dependent kinase (CDK) inhibitor 2A] and CDKN2B, which encode 3 critical factors for the reg- Note: Supplementary data for this article are available at Clinical Cancer INK4A Research Online (http://clincancerres.aacrjournals.org/). ulation of cell cycle and/or apoptosis (6): p16/ (INhibitor of CDK4) and p14/ARF (alternative reading Current address for S. Trino: Department of Onco-Hematology, Centro di frame) encoded by CDKN2A and p15/INK4B encoded by Riferimento Oncologico della Basilicata (CROB), IRCCS, Rionero in Vulture, CDKN2B Italy. Moreover, this region includes a recently discov- ered noncoding RNA, designed ANRIL (antisense noncod- Corresponding Author: Giovanni Martinelli, Molecular Biology Unit, CDKN2BAS Department of Hematology/Oncology "Seragnoli", University of Bologna, ing RNA in the INK4 locus) or , with a first exon Via Massarenti, 9–40138 Bologna, Italy. Phone: 0039-051-6363829; Fax: located in the promoter of the ARF gene and overlapping the 0039-051-6364037; E-mail: [email protected] 2 exons of CDKN2B. Its transcription occurs in the opposite doi: 10.1158/1078-0432.CCR-11-1227 strand to the CDKN2A/B locus and coclusters mainly with Ó2011 American Association for Cancer Research. p14/ARF in both physiologic and pathologic conditions (7). www.aacrjournals.org 7413 Downloaded from clincancerres.aacrjournals.org on October 6, 2021. © 2011 American Association for Cancer Research. Iacobucci et al. tivation and at investigating the influence of 9p21 inacti- Translational Relevance vation on prognosis. CDKN2A/ARF CDKN2B and encode 3 tumor sup- Materials and Methods pressors involved in the regulation of cell cycle and/or apoptosis and often lost in several cancers. Here, we Patients reported a genetic loss in 29% (CDKN2A/ARF)and All patients gave informed consent to blood collection 25% (CDKN2B) of newly diagnosed Philadelphia- and biological analyses, in agreement with the Declaration positive (Phþ) ALL (acute lymphoblastic leukemia) of Helsinki. The study was approved by the Department of patients and in 47% (CDKN2A/ARF) and 40% Hematology and Oncological Sciences "L. and A. Serag- (CDKN2B) of relapsed cases. The novelty of this article noli", University of Bologna, Bologna, Italy. Overall 112 is represented by the correlation, for the first time, adult BCR-ABL1–positive ALL patients, enrolled from 1996 found in adult Phþ ALL patients, among the genomic to 2008 in different clinical trials of the GIMEMA (Gruppo status of CDKN2A/B and clinical outcome. Our results Italiano Malattie EMatologiche dell’Adulto) Acute Leuke- showed that deletions of CDKN2A/B are significantly mia Working Party or in Institutional protocols, were ana- associated by univariate (P ¼ 0.0206 for overall sur- lyzed. Clinical trials included: LAL0201-B enrolling elderly vival, P ¼ 0.0010 for disease free-survival, and P ¼ (>60 years) Philadelphia-positive (Phþ) ALL patients who 0.0014 for cumulative incidence of relapse) and mul- received imatinib, 800 mg daily, associated to steroids as tivariate analysis (P ¼ 0.0051) with poor outcome. frontline treatment; LAL2000 enrolling adult (>18 years) Our findings strongly suggest that there are genetically ALL patients, including Phþ cases, who received induction distinct Phþ ALL patients with a different risk of and consolidation chemotherapy followed by maintenance leukemia relapse and that testing for CDKN2A/B therapy with imatinib; LAL1205 enrolling adult Phþ ALL alterations at diagnosis may aid risk stratification. patients who received dasatinib 70 mg twice a day for 84 consecutive days, as induction therapy, initially associated to steroids without further chemotherapy as frontline treatment. INK4A and ARF share common second and third exons, but A total of 101 patients (90%) were de novo ALL cases a different first exon (exon 1a for INK4 and exon 1b for ARF) analyzed at the time of diagnosis, whereas 11 (10%) were and therefore are translated in alternate reading frames, relapse cases analyzed only at the time of treatment failure. exhibiting no protein sequence similarity. Functionally, In 19 of 101 (19%) cases, both diagnosis and relapse INK4A is a CDK inhibitor, whereas ARF (p19 Arf in mice) samples were collected and, thereafter, analyzed. Demo- regulates p53 tumor suppressor function through its inter- graphics and main clinical characteristics of the patients are action with MDM2 (8). CDKN2B lies adjacent to INK4/ARF summarized in Supplementary Table S1. and it encodes p15/INK4B, a CDK inhibitor, which forms a complex with CDK4 or CDK6 and prevents the activation of Single-nucleotide polymorphism microarray analysis the CDK kinases; thus, the encoded protein functions as a Genomic DNA was extracted using the DNA Blood Mini cell growth regulator that controls cell-cycle G1 progression. Kit (Qiagen) from mononuclear cells isolated from periph- Losses of ARF and INK4, eliminating the critical tumor eral blood or bone marrow aspirate samples by Ficoll surveillance mechanism, are hypothetically harmful gradient centrifugation. DNA was quantified using the because they facilitate proliferation and growth of incipient Nanodrop Spectrophotometer and quality was assessed cancer cells with oncogenic mutations. Supporting this using the Nanodrop and by agarose gel electrophoresis. A hypothesis, a genomic loss of ARF has been shown to total of 83 samples (63 diagnosis, 20 relapse) were geno- enhance oncogenicity and limit imatinib response in mouse typed with GeneChip Human Mapping 250K NspI and 48 models of BCR-ABL1–induced acute lymphoblastic leuke- samples (38 diagnosis, 10 relapse) with Genome-Wide mia (ALL; ref. 9). Human SNP 6.0 array microarrays (Affymetrix Inc.) follow- Deletions of p16/INK4A, p14/ARF, and p15/INK4B have ing the manufacturer’s instructions and as previously been shown to frequently