Incidence and Clinicopathological Relevance of Gene Copy Number Abnormalities Detected by a Single Quantitative PCR Assay in Mantle Cell Lymphomas.
Authors: Jardin Fabrice , Picquenot Jean-Michel , Parmentier Francoise , Cornic Marie , Vaudaux Sandrine , Ruminy Philippe , Penther Dominique , Lanic Helene , Cassuto Ophelie , Rainville Vincianne , Bastard Christian , Tilly Herve 
Published: April 2020

Abstract
The genetic hallmark of mantle cell lymphoma (MCL) is the t(11;14)(q13;q32). Fluorescence in situ hybridisation (FISH) and microarray based comparative genomic hybridisation (array CGH) experiments have demonstrated that additional genetic clonal alterations occur in the majority of MCL and may have prognostic or pathological relevance. We previously developed and validated by CGH or FISH an inexpensive and sensitive genomic PCR assay (Multiplex PCR of Short Fluorescent Fragments, QMPSF) to detect gene copy number abnormalities in diffuse large B-cell lymphoma and chronic lymphocytic leukemia (Haematologica 2008,93:543-Leukemia, 2007,21:1460). In the aim to determine the incidence and clinical relevance of recurrent additional genomic number abnormalities, we specifically designed a single QMPSF assay dedicated for MCL and correlated results with pathological and clinical data. For this purpose, a series of 42 newly diagnosed MCL cases with available frozen-and paraffin-embedded lymph node tissues and clinical features were selected [median age=67y; median MCL international prognostic index (MIPI)=6.1; low risk 21%, intermediate risk 33%, high risk 45%; 3-year overall survival (OS) rate=38%]. The assay was designed according to the most frequent gene copy number abnormalities reported in MCL, allowing simultaneous analysis of 8 relevant genes (CDKN2A, RB1, ATM, CDK2, TP53, MYC, CDKN1B, and MDM2) and 2 reference genes (SEM4F and CECR1). DNA copy number gains of MYC, CDK2, CDKN1B (p27kip1) and MDM2 are observed in an equal frequency (10%). Losses of DNA copies of RB1, CDNK2A, ATM, or TP53 are observed in 38, 31, 26, and 10 % of cases respectively. Some genes are almost exclusively gained (MYC,CDK2), deleted (RB1, ATM, TP53, CDNK2A) or both (CDK2, CDKN1B). Deletions of ATM and RB1 appear strongly associated (21% of cases, p=.001). CDKN2A (p14arf and p16ink4a) homozygous deletions and CDKN1B gains are more frequently observed in blastoid variants (p=.04 and .005 respectively). According to the MIPI score, the number of gene copy abnormalities tends to increase in the high/intermediate risk group (median = 1, range 0–6), as compared to the low risk group (median=0, range 0–3, p=.07). More specifically, MYC gain is exclusively observed in the high risk group (p=.04) and CDKN2A deletions are observed in patients with the highest MIPI. The prognostic relevance of the assay was tested in 42 patients. With a median follow-up of 22 months, CDK2 (3y OS=0%) or MDM2 (3y OS=0%) gains and CDKN2A (3y OS=20%) or TP53 (3y OS=25%) losses correlate to a shorter OS (p &lt;.0001, p=.0007, p=.003 and p=.03 respectively). CDKN2A deletions remain predictive of the outcome in patient with a high MIPI (p =.0005). Furthermore, PCR performed in 5 cases at the time of relapse showed an increase of gene copy number abnormalities compared with initial diagnosis (median = 4 vs.1; p =.02), suggesting that gene losses/gains are involved in a dynamic and selected process. To conclude, we developed a reliable and routinely applicable PCR assay which delineates distinct MCL oncogenic pathways with strong prognostic impact that could be used in combination with the recently defined MIPI.