Page 337 - 2021_02-Haematologica-web

Case Reports
A deep molecular response of splenic marginal zone
lymphoma to front-line checkpoint blockade
Splenic marginal zone lymphoma (SMZL) is an uncom- mon form of B-cell non-Hodgkin lymphoma (B-NHL) that frequently presents with prominent splenomegaly, often with circulating malignant lymphocytes but with modest lymphadenopathy. As SMZL is generally consi- dered an incurable disease, treatment is often deferred until the manifestation of cytopenias, symptomatic splenomegaly, or constitutional symptoms warrant inter- vention. The current approach to frontline treatment typically involves rituximab given with or without chemotherapy, treatment of associated conditions such as hepatitis C infection, and less commonly splenectomy. Immune checkpoint blockade with agents such as anti- PD-1 antibodies have been effective in certain subtypes of B-NHL, but the use of these agents in patients with previously untreated SMZL has not yet been explored.
A 77 year-old man with a history of stage IIc melanoma treated with wide local excision with curative intent presented to the hematology clinic in June 2016 for evaluation of thrombocytopenia (Figure 1). An initial workup revealed what was thought to be an incidental IgA l and IgG κ monoclonal gammopathy of undeter- mined significance (MGUS), and the decision was made to proceed with observation. However, after developing anemia and progressive thrombocytopenia, further workup was initiated, including imaging that revealed mild mediastinal and bilateral axillary adenopathy and prominent splenomegaly. A bone marrow biopsy was performed in June 2017 that showed an atypical lym- phoid population positive for B-lymphoid markers CD19 and CD20, negative for CD5 and CD23, and l light chain restricted. Next-generation sequencing of the bone mar- row aspirate using an in-house gene panel revealed a TP53 I195S mutation with a variant allele frequency of 0.06 and loss of PTEN.1 Though uncommon, adenopathy can occur in SMZL, and while splenic B-cell lymphoma unclassifiable was also considered,2,3 the combination of an aberrant immunophenotype, cytopenias, and signifi- cant splenomegaly made SMZL the most likely diagnosis. However, given the absence of symptoms, therapy was deferred at that time.
Approximately 3 months later, surveillance imaging for his melanoma suggested metastatic disease, which was subsequently confirmed by biopsy of a right lung nodule. Pembrolizumab monotherapy every 3 weeks was recom- mended to treat his melanoma. At the time, he had also developed worsening and symptomatic splenomegaly (22.9 cm), intermittent drenching night sweats, fatigue, and ongoing cytopenias, suggesting the need for SMZL therapy. However, given that the metastatic melanoma was thought to be the more immediately threatening malignancy, treatment of the SMZL was deferred in order to initiate melanoma therapy. On the day he initiated treatment with pembrolizumab in September 2017 his lab- orarory results were notable for a white blood cell count (WBC) of 26 K/uL, of which 69% were lymphocytes, hemoglobin of 11.1 g/dL, and a platelet count of 60 K/uL.
Within 3 months of starting pembrolizumab, the melanoma metastases had significantly decreased in size. Interestingly, a substantial response was also observed in his SMZL: spleen size was reduced to 14.0 cm, WBC was 5.9 K/uL, absolute lymphocyte count was 0.84 K/uL, hemoglobin was 13.8 g/dL, and the B-symptoms had largely disappeared, although his platelets remained at 60. His pembrolizumab treatment course was complicat-
ed by type I diabetes mellitus, which was considered an immune-related adverse event requiring an inpatient admission for diabetic ketoacidosis that was effectively managed. Later in his course he was admitted for sepsis, which was thought to be unrelated to treatment. At the last documented follow-up, he had received 35 cycles of pembrolizumab, the melanoma was in complete remis- sion, the spleen size was normal, and his blood counts had remained stable, with the only abnormality being persistent thrombocytopenia, which had improved, but remained in the 90-105 range. To date he has not received any therapy specifically directed to the SMZL.
After obtaining Instituational Review Board approval, genomic DNA was isolated from peripheral blood mononuclear cells (DNEasy Kit, Qiagen, Hilden, Germany). Hybrid capture was performed on the geno- mic DNA samples followed by library preparation with unique molecular identifiers using a custom bait set from Twist Bioscience (San Francisco, CA, USA). Sequencing was performed on the Illumina platform (Illumina, San Diego, CA, USA) and after deduplication and consensus sequence calling, mutations were identified using Varscan 2.2.3, and annotated using Annovar. Mutations were scored based on allele frequency, strand bias differ- ential, local noise and mapping quality, and frequency in known single nucleotide polymorphism (SNP) databases. These variants were visually inspected in Integrated Genome Viewer (Broad Institute, Cambridge, MA, USA). Bone marrow staining was performed using antibodies to PD-L1 (Clone E1L3N, Cell Signaling Technology, Danvers, MA, USA) and BSAP (Clone PAX5, BD Biosciences, San Jose, CA, USA).
We had banked a pre-treatment peripheral blood mononuclear cell (PBMC) sample just prior to pem- brolizumab initiation, and after observing this dramatic response of therapy-naive SMZL to PD-1 blockade, we obtained another PBMC sample approximately 6 months later. Genomic DNA was extracted from the samples and error-corrected deep sequencing using unique molecular identifiers was performed on a panel of genes that are recurrently mutated in hematologic malignancies. With this technology we are able to detect the presence of mutations to a variant allele frequency (VAF) as low as 0.003.4 Prior to pembrolizumab initiation, the same TP53 I195S mutation identified in the diagnostic bone marrow biopsy was again observed, this time with an elevated VAF of 0.78, consistent with the predominance of lym- phocytes at the time of sample acquisition and the enrichment of lymphoid DNA from PBMC.5 Remarkably, after 6 months of pembrolizumab treatment, the muta- tion was undetectable in the blood, suggesting a deep molecular response of the SMZL. The raw mutation calls visualized in Integrated Genome Viewer are shown in Figure 2A.
We also examined the clinical sequencing that was per- formed on the melanoma biopsy specimen using another custom panel.6 The sequencing data reported two sepa- rate TP53 mutations: I195S (VAF 0.29) and G105V (VAF 0.13). The TP53 I195S mutation was therefore blood- derived, whereas the G105V was tumor derived. This conclusion is also consistent with the finding that the average VAF among all mutations identified in the tumor was 0.11, close to the VAF of the G105V mutation but less than half of that of the I195S mutation (Figure 2B). The “contamination” of solid tumor sequencing by somatic mutations present in blood cells is an increasing- ly recognized phenomenon, can complicate interpreta- tion of these data, and may have important prognostic and therapeutic implications.7-10
haematologica | 2021; 106(2)
651