Comprehensive Analyses of Mantle Cell Lymphoma with a 17 Gene Panel Reveal an Accumulation of TP53 Mutations in SOX11 Negative Patients

Introduction: Mantle cell lymphoma (MCL) belongs to the mature B-cell neoplasms and is characterized by t(11;14)(q13;q32)/IGH-CCND1 rearrangement resulting in overexpression of CyclinD1 that is encoded by CCND1. CCND1 is a weak oncogene, requiring additional cooperating oncogenic events. MCL show mainly an aggressive course of disease, although a subset of patients have been identified with an indolent clinical course. This has been associated with a mutated IGHV status and the lack of SOX11 expression. However, some additional gene mutations have been identified in recent years, but the underlying biological heterogeneity is still under debate. Also the prognostic impact of SOX11 remains controversially discussed.

Aim: To analyze 1) recently identified molecular mutations in CCND1, UBR5, WHSC1 for their specificity for MCL, 2) in more detail SOX11 negative MCL to get more insights in this group of MCL by sequencing a 17 gene panel.

Patients and Methods: In total 184 patients with mature B-cell neoplasms were investigated. All cases were diagnosed according to WHO classification by cytomorphology, immunophenotyping and cytogenetics. 81 cases were diagnosed as MCL, 78 as chronic lymphocytic leukemia/prolymphocytic (CLL/PL), and 25 as CLL. The cohort comprised 67 females and 117 males. CyclinD1 and SOX11 expression levels were quantified by real time PCR. The 17 gene panel included ATM, BIRC3, BRAF, CCND1, FBWX7, IGHV, KLHL6, KRAS, MYD88, NOTCH1, NRAS, POT1, SF3B1, TP53, UBR5, WHSC1, and XPO1. Next generation sequencing was performed on MiSeq instruments (Illumina, San Diego, CA), except for CCND1, UBR5, WHSC1, and IGHV mutational status, which were analyzed by Sanger sequencing. The latter 4 genes and gene expression levels were analyzed in the total cohort, while the 17 gene panel was applied to 26 MCL patients only (13 SOX11 negative and 13 SOX11 positive cases matched for CCND1 mutations and IGHV mutation status).

Results: 23/184 (13%) patients had CCND1 mutations, while only 3/184 patients (2%) carried a UBR5 mutation, and 4/184 patients (2%) a WHSC1 mutation. Of note, CCND1 and UBR5 mutations occurred exclusively in MCL patients, WHSC1 mutations were found in MCL and CLL/PL. Therefore CCND1 mutation was, as expected, specific for MCL in comparison to the other 2 mature B-cell neoplasms (p<0.001).

Of 81 MCL patients 68 (84%) showed SOX11 overexpression, 23 (28%) had CCND1 mutations and 3 (4%) each had UBR5 and WHSC1 mutations, respectively. The IGHV mutational status was evaluable in 73/81 cases, revealing 32/73 (44%) patients with a mutated IGHV status. A negative correlation of CCND1 mutations and SOX11 overexpression was found: 8/13 (62%) SOX11 negative patients were CCND1 mutated as compared to 15/68 (22%) SOX11 positive patients (p=0.007). Furthermore, CCND1 mutations were more frequent in patients with a mutated IGHV status than in those with unmutated status (15/32 (47%) vs. 8/41 (20%), p=0.021). Accordingly, SOX11 overexpression occurred more often in patients with an unmutated than with a mutated IGHV status (88% vs. 75%; n.s.). Regarding clinical data, more males were SOX11 positive (51/54, 94% vs. 17/27, 63%; p=0.001), and correspondingly more females were CCND1 mutated (12/27, 63% vs. 11/54, 20%; p=0.036).

To get more insight in the SOX11 negative patients (n=13) we addressed all these cases and a SOX11 positive control group, matched for IGHV mutational status and CCND1 mutations (n=13), by comprehensive mutational analyses. Overall, beside CCND1 the most frequently mutated gene was TP53 (8/26, 31%), followed by ATM (6/26, 23%), BIRC3 (2/21, 10%), and KRAS (2/26, 8%). No mutations were detected in any of the other genes analyzed. Addressing differences in gene mutations between SOX11 negative and SOX11 positive cases revealed that TP53 mutations were found more frequently in SOX11 negative cases (6/13, 46% vs. 2/13, 15%; n.s.), while ATM mutations were more frequent in SOX11 positive cases (5/13, 39% vs. 1/13, 8%; n.s.).

Conclusions: 1) CCND1 mutations are specifically found in MCL, correlate with SOX11 negativity and IGHV mutated status, and are more frequent in females. 2) TP53 is frequently mutated in SOX11 negative patients and its prognostic impact has to be further evaluated. 3) Thus, the differences in clinical course between SOX11 positive and negative MCL patients might correlate with a different spectrum of additional molecular alterations.