Primary testicular lymphoma (PTL) and primary diffuse large B-cell lymphoma of the central nervous system (PCNSL) are considered disease entities that arise in extranodal immune-privileged sites, share common molecular genetic features with an ABC phenotype, and show frequent mutations of MYD88L265P and CD79B.1 These tumors usually display inferior responses to conventional chemoimmunotherapy.2,3 At relapse, PTLs often involve extranodal sites including the central nervous system (CNS), skin, pleura, and contralateral testis.2 Extracerebral involvement by PCNSL is, however, rarely found in clinical practice, accounting for ∼12% of relapses of PCNSL in large retrospective series.4,5 Testicular relapse of PCNSL has been occasionally described.5 Here we describe a case of primary CNS large B-cell lymphoma treated with methotrexate (MTX), high-dose chemotherapy, and auto-stem cell transplantation that relapsed 18 months after the initial diagnosis. Location at relapse included testicular and bilateral adrenal glands, without lymph node involvement detectable by positron emission tomography/computed tomography. Both tumors showed equivalent morphology, phenotype, cytogenetic features, and clonal relationship. Somatic mutation analysis by next generation sequencing (NGS) with a targeted exonic approach demonstrated MYD88L265P mutation in both tumors and de novo CD79B Y196S mutation exclusive to the relapse. The pattern of mutations suggest that the 2 tumors might have evolved from a common progenitor clone with MYD88L265P being the founder mutation.
A 60-year-old man presented with dysphasia and hearing loss of 3 months duration. Cerebral magnetic resonance imaging disclosed an expansive lesion in the left temporal lobe. Surgical biopsy was diagnosed as diffuse large B-cell lymphoma (DLBCL), non-Germinal Center B cell type, according to WHO classification.1 After clinical evaluation with whole-body computed tomography scan, no evidence of involvement outside the CNS was found, and the case was considered to be PCNSL. The patient was treated with PCNSL protocol including MTX and high-dose intensification, before autologous stem cell transplantation, which was performed without complications. Eighteen months after the initial diagnosis, a suspicious mass in the left testis was found by ultrasound scan. Left orchiectomy was diagnosed as DLBCL. The tumor showed identical morphology and phenotype when compared with the CNS lesion. Translocations involving MYC, BCL2, and BCL6 were ruled out by fluorescent in situ hybridization (FISH), and clonality testing confirmed the distant relapse at the molecular level. Clonality analysis found clonal peaks of identical size in FR3. Furthermore, direct sequencing of IgH showed nearly identical CDR3 sequences in both samples, confirming involvement of the 2 sites by a single tumor clone (Fig. 1). After orchiectomy, the patient was treated with bilateral testicular radiotherapy (30 Gy), bilateral adrenal radiotherapy (40 Gy), and Nivolumab. The patient achieved remission of adrenal gland lesions but developed CNS relapse, as detected by magnetic resonance imaging findings. Holocraneal radiotherapy was initiated (36 Gy).
Targeted NGS was performed, after generation of a custom library for the detection of mutations in exonic regions of 36 genes of interest, using DNA extracted from formalin fixed paraffin embedded material from both tissue samples and germline DNA from peripheral blood granulocytes of the patient. All 3 samples were sequenced on a Miseq (Illumina) machine. Miseq Reporter software and IgV were used for variant calling. Only missense, frameshift, and nonsense somatic mutations with variant frequency>20% were considered. Sixty-seven somatic mutations (60 missense, 5 nonsense, 2 frameshift deletions) in 22 genes were found (Supplementary Table 1, Supplemental Digital Content 1, http://links.lww.com/AIMM/A207). Only 1 mutation (MYD88 p.L265P) was shared by both tissue samples with similar variant allele frequency. Most mutations were found only in the first PCNSL sample, whereas CD79B p.Y196S appeared de novo in the testicular lymphoma sample (Supplementary Table 1, Supplemental Digital Content 1, http://links.lww.com/AIMM/A207 and Fig. 2).
PCNSL is an uncommon large B-cell lymphoma entity. It represents 3.1% of all primary brain tumors and <1% of non-Hodgkin’s lymphomas.1,6 After first-line therapy including high-dose MTX, relapse occurs in the majority of patients (26% to 56% in larger cohorts of patients).4,5,7 While local relapse within the CNS is the rule (≈80% of patients), relapse outside the CNS has been found in a fraction of cases (14% to 16%), involving the testis, breast, lymph nodes, bone marrow, musculoskeletal tissues, renal and adrenal glands, and liver.4,7
The morphology of PCNSL is usually a DLBCL with a postgerminal center B-cell phenotype.1 Mutational profile of PCNSL is characterized by somatic mutations in PRDM1,8 frequent MYD88L265P and CD79B ITAM mutations, biallelic CDKN2A loss, NFKBIZ amplification, MGMT methylation, and frequent 9p24.1/programmed death ligand 1/PD-L2 CNAs.9,10 In particular, the MYD88L265P mutation has been found to be a specific marker for the ABC molecular subtype of DLBCL in a recently published meta-analysis.11 Interestingly, MYD88L265P mutation is found more frequently in DLBCL cases arising in extranodal immune-privileged sites.
We have performed a meta-analysis of 11 recently published cohorts of patients with PCNSL and PTL.9,10,12–20 The MYD88L265P mutation is found in 66% (237/355) of PCNSL cases and in 74% (77/103) of PTL cases. Furthermore these cases may carry the CD79B ITAM hot spot mutations, with 50% (157/314) and 35% (29/82) in PCNSL and PTL cases, respectively. Interestingly, cases with CD79B mutations and concurrent MYD88L265 mutations occur in 71% (118/165) and 93% (27/29) of PCNSL and PTL cases, respectively (Fig. 3). These figures are higher than expected for DLBCL NOS with ABC phenotype (≈30% for MYD88L265P mutation and 10% for both MYD88 and CD79B mutations)21,22 and underscore the role of B cell receptor and nuclear factor kB activation by somatic mutations in these DLBCLs that colonize immune-privileged sites.
Here we report the mutational landscape of a case of PCNSL and its testicular relapse using a targeted NGS approach. Interestingly, our results after the analysis of the primary and relapsed sample reveal significant genetic heterogeneity despite stable phenotypic and cytogenetic features. Three main conclusions are derived from this mutational screening. First, the mutational portrait of the first PCNSL sample is highly heterogenous and complex, with few dominant and potentially driver mutations (i.e., MYD88L265P, EZH2, FAT2, ATM, SMARCA4) and multiple cooccurring subclonal mutations in a variety of genes, including tumor suppressor genes (PRDM1, TP53) and genes involved in the NOTCH pathway (NOTCH1, NOTCH2, and SGK1). This highly heterogenous mutational landscape of PCNSL is consistent with previously published data in larger cohorts of cases with exome and targeted sequencing.15,19 Higher genetic complexity based on targeted sequencing identification of number of nonsynonymous mutations has been associated with a lower overall survival in DLBCL.23
Second, a clonal selection process occurred, with a reduction in the number of mutations found in the testicular relapse. Only MYD88L265P and a novel CD79B Y196S mutation appeared in the second sample from the patient. Our deep sequencing approach demonstrates that CD79B Y196S mutation was not present in a significant fraction of the tumor DNA in the first sample, suggesting a pattern of subclone selection with CD79B Y186S mutation appearing as a second genetic hit in a B-cell population that already carried the MYD88L265P mutation. This implies that the 2 tumors evolved from a common progenitor B-cell clone with MYD88L265P being the founder mutation (Fig. 2). Whether this evolution emerges in relation to therapy as a tumor evasion mechanism or reflects the natural evolution of a B-cell lymphoma with concurrent MYD88 and CD79B mutations relapsing in an immune-privileged environment (i.e., testis) remains unresolved.
Third, based on recently published clinical data, it seems that the presence of the combination of MYD88L265P and CD79B ITAM mutations in the relapse sample might have therapeutic implications.24,25 So far, 1 phase I/II clinical trial has demonstrated benefit for systemic DLBCL of the ABC phenotype with the use of BTK inhibitors, particularly for those tumors with concurrent MYD88L265P and CD79B mutations.25 Recently, 1 phase Ib clinical trial demonstrated clinical benefit with the use of Ibrutinib alone and in combination with chemotherapy in 18 patients with PCNSL. Unfortunately, the mutational profile of the tumors in the PCNSL trial were not available for the majority of patients.24
In summary, targeted NGS for the detection of hot spot somatic mutations in relapsed DLBCL is useful to confirm the ABC phenotype and discovers relevant information that might guide therapeutic decision.
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