A Quantitative Multiplex PCR Assay, Validated by a High Resolution Array Comparative Genomic Hybridization Analysis, Provides Information Regarding Genomic Imbalances and Prognosis in Diffuse Large B-Cell Lymphomas.
Authors: Jardin Fabrice , Ruminy Philippe , Parmentier Francoise , Kerckaert Jean-pierre , Rainville Vinciane , Picquenot Jean Michel , Buchonnet Gerard , Penther Dominique , Bastard Christian , Tilly Herve 
Published: April 2020

Abstract
Genomic gains and losses may play a crucial role in the development and progression of diffuse large B-cell lymphomas (DLBCL). Some gains or losses are associated with particular morphologic or clinical manifestations and correlate with the “germinal center B-cell like” (GCB)/ non-GCB phenotype, as defined by gene expression profiles. To date, chromosomal-comparative genomic hybridization (CGH) and higher resolution array-CGH provide the more comprehensive view of these genomic imbalances. However, a CGH approach is expensive and not routinely applicable. By contrast, quantitative Multiplex PCR of Short Fluorescent Fragments (QMPSF) is an inexpensive and sensitive method for the detection of genomic deletions or duplications based on the simultaneous amplification of short genomic fragments using dye-labelled primers under quantitative conditions. We designed and evaluated a QMPSF assay to determine simultaneously frequencies of gain/loss of several genes and their clinical relevance in DLBCL. A series of 68 newly diagnosed DLBCL with available conventional cytogenetics and treated with anthracycline-based frontline therapy was studied (median age = 59 years, IPI 0–1: 35%; 2–3: 41%; 4–5: 24%). 15 cases were selected for both CGH (using a high resolution, high performance 60-mer oligonucleotide-based microarray that contains ~ 43,000 probes, with an average spatial resolution of 35 kB) and QMPSF experiments. Considering CGH as the reference method, and using primers located on chromosome 3q27 (BCL6), 6q16 (SIM1), 6q22 (PTPRK), 8q24 (MYC), 9p24 (JAK2), 9p21 (CDNK2A), 12q13 (cdk2), 12q15 (mdm2), 12p13.1 (p27) and 17p13 (p53), the sensibility and specificity of our QMPSF assay to detect abnormal DNA copy numbers ranged between 80–100% and 90–100% respectively. A gain of DNA copy numbers of MYC, p27, and BCL6 was observed in 19, 30 and 19 % respectively. Losses of DNA copies of p53 or CDNK2A were observed in 20 and 34 % of cases. Some genes were almost exclusively amplified (MYC, p27, cdk2) or exclusively deleted (p53, CDNK2A, PTPRK), indicating genomic imbalances selection during oncogenesis. Interestingly, 6q16 (SIM1), 6q22 (PTPRK) and 9p21 (CDNK2A) deletions were predominantly observed in the non-GCB subtype, whereas MYC gains were mainly observed in the GCB subtype and especially in de novo t(14;18) positive DLBCL (p =.002). The prognostic value of a QMPSF assay including 5 genes (p53, MYC, CDNK2A, SIM1 and p27) was tested. With a median follow-up of 81 months, detection of more than 2 gene copy number abnormalities was related to a worse event-free and overall survival (OS = 10 months, n = 13) as compared to patients with 0–2 abnormalities (OS not reached, p = .003, n = 55). The prognosis value of this PCR remains regardless the GCB/non-GCB subtype or the IPI score. To conclude, we developed a reliable and routinely applicable PCR assay, which provided information regarding gain/loss of relevant genes, and prognosis in DLBCL.