Clonal Evolution in Relapsed or Refractory Diffuse Large B Cell Lymphoma

Background:

Despite modern chemoimmunotherapy more than a third of patients with diffuse large B cell lymphoma (DLBCL) will experience relapse or refractory disease. Multiple mutations associated with the biology of DLBCL have recently been identified by next-generation sequencing in primary tumor samples, but little is known about their prognostic role. Furthermore, histological comparison of the primary tumor and relapsed disease is often not available in clinical practice due to the lack of centralized assessment and complicated by the difficulties to perform exome-wide sequencing in formalin fixed tissues. Therefore, the role of certain mutations and their mechanisms in clonal evolution during relapse is unknown and the rising of chemo-resistant DLBCL subclones has not been described in the literature so far.

Methods:

We identified all patients with available histologically confirmed relapsed or refractory DLBCL in our single center cohort of 346 patients with aggressive lymphoma treated at our tertiary cancer center in Salzburg, Austria. Primary formalin fixed paraffin embedded tumor sample, sample of refractory or relapsed disease and matched germ line were available for targeted next generation sequencing in 27 patients. A targeted exon capture and next-generation sequencing of all coding exons of 104 selected genes known to be frequently mutated in lymphoma were performed on a HiSeq 2500.

Results:

Sequencing was successful in 96.8% of all samples resulting in 25 patients with sequencing of the primary tumor and 24 patients with available pairs of primary lymphoma and histologically confirmed relapse. In these 24 patients two relapse samples were available in 10 patients and three relapse samples in one patient. Non-synonymous mutations were present in 74 of the 104 genes tested. Individual tumor samples showed between 0 and 29 non-synonymous mutations (median: 10). Less than six non-synonymous mutations in the primary tumor were associated with a better median OS than more mutations (28 versus 15 months p=0.031).

We also compared the frequency of mutated genes in our cohort consisting of high risk patients defined by actual relapse with the literature containing patients with extensive sequencing but only little clinical data and clinical follow-up. Common mutated genes such as CARD11, CD58, CD79B, CREBBP, EZH2, BTG1 or B2M showed no difference in frequency to our patient cohort indicating no or only small driver function in resistant or refractory disease. Nevertheless, mutations previously reported to be at low frequency in DLBCL were significantly more often observed in our primary samples (NOTCH1, MYC, RB1, FAT2, ATM, SMARCA4, BCL7A) and relapsed samples (TP53, MCL1, ATM, FAT2, MYC, RB1, SMARCA4) of high risk patients when compared to the literature.

We also observed the gain and the loss of several mutations between first diagnosis and histologically confirmed relapse. Overall, we observed an increase of the amount of non-synonymous mutations at first relapse in 12, no change in 6 and a decrease in 6 paired cases. A completely stable pattern of non-synonymous mutations was detected in 4 cases, but in the majority of cases relevant dynamic was observed: e.g.: gain of non-synonymous mutations in the p53 gene was seen in 3 patients (5 mutations), in the TNFRSF-14 gene in 2 patients (3 mutations), in the RB1 gene in 1 patient (1 mutation), in the NOTCH2 gene in 3 patients (4 mutations) and in the MYD88 gene in 1 patient (1 mutation) or loss of non-synonymous mutations in the CREBBP gene in 3 patients (3 mutations) or in the CRAD11 gene in 2 patients (2 mutations). Monitoring of subclones during disease was also possible using the allelic fraction over time e.g.: showing an increase of the NOTCH1 mutation burden in 2 biopsies after first diagnosis.

Discussion:

To the best of our knowledge clonal evolution detected by next generation sequencing has not been reported in DLBCL so far. We demonstrate the feasibility of such an approach from fixed tissue samples and using a curated set of target genes. In analogy to other lymphoid malignancies we can show the increase of allelic burden of certain mutations over time and the loss or gain of several others. While this approach is limited by the bias introduced by the selection of genes in the gene set, we feel that deep sequencing of selected mutations will provide further insights into subclone dynamics, which may be responsible for clonal evolution.