Prognostic Impact of MYD88 L265P Mutation By Droplet Digital PCR in IgM MGUS and Smoldering Waldenström Macroglobulinemia

BACKGROUND: MYD88 L265P mutation is highly prevalent in IgM monoclonal gammopathy of undetermined significance (MGUS), smoldering Waldenström macroglobulinemia (SWM) and symptomatic WM. Allele-specific PCR (AS-PCR) has been used routinely to assess MYD88 mutation; however, with the advent of more precise high-throughput technologies such as droplet digital PCR (ddPCR), absolute quantification can be achieved. There is no data regarding ddPCR applicability in asymptomatic IgM monoclonal gammopathies or as a prognostic biomarker. Here, we aimed to compare MYD88 quantification by ddPCR with clinical and laboratory features and to analyze the prognostic impact in a series of patients (pts) with IgM MGUS and SWM. METHODS: We analyzed bone marrow (BM) and peripheral blood (PB) samples stored from pts diagnosed with IgM MGUS and SWM at our institution from 1980 to 2020. DNA extraction methods followed manufacturer instructions (Qiagen) to obtain genomic DNA from unsorted BM samples and cell-free DNA (cfDNA) from PB. MYD88 L265P mutation was quantified by ddPCR using a Bio-Rad commercial assay (HEX-labeled wild-type allele; FAM-labeled mutant allele). We used OCI-Ly3 DLBCL ABC cell line, homozygous for MYD88 L265P, as a positive control. ddPCR was performed following Bio-Rad technical specifications using the QX200 droplet reader. Data was analyzed using QuantaSoft v.1.0 software (Bio-Rad). Absolute quantification of the mutation was expressed as percentage of fractional abundance. For survival analysis, we used a competing risk analysis to evaluate the prognostic impact of MYD88 mutation on progression to symptomatic WM. RESULTS: A total of 217 unsorted samples were analyzed (187 BM and 30 PB). Genomic DNA from unsorted BM samples was extracted from pts diagnosed with IgM MGUS (46%), SWM (44%), and symptomatic WM (10%). cfDNA was obtained from a subgroup of pts with IgM MGUS (52%) and SWM (48%). Median age at diagnosis was 68 (range 61 to 76). AS-PCR could detect the mutation in 22 (31%) pts with IgM MGUS and 49 (75%) with SWM. ddPCR improved precision detection up to 48 (55%) pts with IgM MGUS and 68 (83%) with SWM. All pts with symptomatic WM harbored the MYD88 mutation, as identified by both techniques. Median absolute quantification from BM was 2.3% and 7% for pts with IgM MGUS and SWM, respectively (p<0.001). Pearson correlation coefficients comparing BM MYD88 mutation quantification by ddPCR with serum M-protein size, IgM concentration, BM lymphoplasmacytic infiltration rate and BM CD19+ cells were 0.3, 0.4, 0.6, and 0.9 (p<0.0004), respectively. Similar coefficients were observed in symptomatic WM regarding BM infiltration rate (0.6; p=0.001) and BM CD19+ cells (0.9; p<0.0001). Spearman correlation coefficients comparing cfDNA MYD88 mutation quantification with BM lymphoplasmacytic infiltration rate and BM CD19+ cells were 0.4 and 0.5 (p<0.008), respectively. Agreement regarding MYD88 mutation detection by ddPCR in BM DNA and cfDNA samples was 82% (Cohen kappa index 0.6). With a median overall survival of 13 years in pts with IgM MGUS and SWM, 13% of them progressed to symptomatic WM while 22% died without progression. Cox univariate analysis using continuous values for MYD88 quantification (p=0.004), serum IgM (p<0.001), BM lymphoplasmacytic infiltration (p<0.001), and serum albumin (p=0.04) were significant. X-tile software was used to find the optimal cutoff point of MYD88 quantification as a biomarker. 4.5% was established for pts with IgM MGUS while 25% for SWM. Using the Fine and Gray regression model in a competing risk analysis taking death without progression as a competing event, higher MYD88 mutation burden negatively impacted the risk of progression of IgM MGUS (SHR 4.6; p=0.003) and SWM (SHR 6; p<0.001) (Figure 1). CONCLUSION: Quantification of the MYD88 L265P mutation by ddPCR has higher precision and sensitivity compared to AS-PCR; thus ddPCR could be used as a potential new and useful biomarker. MYD88 tumor burden correlated with well-known laboratory parameters used for diagnosis and risk stratification, whether using genomic DNA from unsorted BM samples or cfDNA. Risk of progression was higher in patients harboring an increased mutant allele burden. This is the first report showing the prognostic impact of MYD88 quantification in a series of patients with asymptomatic IgM gammopathy and long-term follow up. Figure 1 Figure 1. Disclosures Cibeira: Akcea: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Bladé Creixenti: Janssen, Celgene, Takeda, Amgen and Oncopeptides: Honoraria. Rosinol: Janssen, Celgene, Amgen and Takeda: Honoraria. Fernandez de Larrea: BMS: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Takeda: Honoraria, Research Funding; GSK: Honoraria; Sanofi: Consultancy; Janssen: Consultancy, Honoraria, Research Funding.

High Frequency of CNS Involvement in Transformed Waldenström Macroglobulinemia

INTRODUCTION Central nervous system (CNS) relapse is a challenging situation in diffuse large B-cell lymphoma (DLBCL). High CNS-International Prognostic Index (IPI), activated B-cell (ABC) subtype and MYD88 L265P mutation, features often found in transformed Waldenström macroglobulinemia (WM), are associated with a higher risk for developing CNS relapse. This study was aimed to describe CNS involvement in a large cohort of transformed WM. METHODS This international multicenter retrospective study included patients with a diagnosis of WM and a concurrent or sequential histological diagnosis of DLBCL. CNS disease was diagnosed by detection of DLBCL cells in the cerebrospinal fluid and/or by brain biopsy. Patients with CNS involvement by lymphoplasmacytic cells (Bing-Neel syndrome) were excluded. Of 254 identified patients with a diagnosis of histological transformation (HT) between 1988 and 2020, 19 were excluded due to lack of data on extranodal involvement. The first part of the analysis focused on baseline CNS involvement. Clinicobiological characteristics were compared between groups using Chi-square or Fisher's exact tests or Mann Whitney tests as appropriate. We analyzed CNS recurrence in the second part of the study. Forty-eight additional patients were excluded due to baseline CNS involvement (n = 25), absence of treatment at HT (n = 14) and lack of details on follow-up (n = 9). Cumulative incidence of CNS relapse was analyzed using competing-risk models that accounted for other events like systemic relapse or death from any cause, reporting sub-hazard ratio (SHR). RESULTS Baseline CNS involvement was present in 25 patients (11%) with transformed WM, including 10 (4%) with parenchymal disease, 10 with leptomeningeal, 4 (2%) with both, and 1 with unspecified CNS involvement. Characteristics associated with baseline CNS involvement were performance status 2-4 (P=0.03) and ≥2 extranodal sites (P=0.02). Median survival after HT was 1 year [0.7-2.5], comparable to the one of patients without CNS disease (1.8 year [1.2-2.6], P=0.74). We observed no difference in survival based on isolated CNS involvement (n = 10) compared to CNS and systemic involvement (n = 15) (P=0.94). Twenty-three CNS relapses occurred (12%). The 2-year and 3-year rates of CNS relapse were 9% (95% CI, 6-14) and 11% (95% CI, 7-16) (Figure 1). The median time to relapse in the CNS was 11 months (95% CI, 7-25). Thirteen CNS recurrences (57%) occurred during the first year of follow-up. Seventy percent were isolated CNS relapses. The location was leptomeningeal in 43% of cases, parenchymal in 35%, both in 17%, and unspecified in 4%. According to CNS-IPI risk groups (data available for 20 patients), 9 patients (45%) belonged to the high-risk group, 10 (50%) to the intermediate-risk group and 1 (5%) to the low-risk group. Prior to CNS relapse, 87% of patients had received rituximab, and 39% had received CNS prophylaxis (30% intrathecal chemotherapy, 4% high-dose methotrexate (HD-MTX), and 4% both). After CNS recurrence, 96% of patients received salvage treatment: combination of HD-MTX and HD-cytarabine (48%), HD-MTX alone (30%), or HD-cytarabine alone (9%). Four patients underwent consolidative autologous stem cell transplantation. The median survival after CNS relapse was 5.6 months. Factors associated with 3-year cumulative incidence of CNS recurrence in univariate analysis were involvement of kidney/adrenal glands (HR, 4.4; P=0.01) and MYD88 L265P mutation (P=0.01) (Figure 2A and B). Of note, among 74 patients (over 187, 40%) with data available for MYD88 mutation status, 11 CNS relapses occurred in patients with MYD88 L265P mutation (n = 54, 20%) whereas no relapse were observed in MYD88 WT cohort (n = 20). A trend toward higher risk of CNS relapse for ≥2 extranodal sites (HR, 2.3, 95% CI 0.98-5.3; P=0.06) was observed. Cumulative incidence according to CNS-IPI risk groups (0% in the low-risk, 9% in the intermediate-risk and 14% in the high-risk group) was not statistically significant (P=0.47). CONCLUSION CNS involvement occurs frequently in transformed WM. Rate of CNS relapse seems similar to DLBCL patients belonged to the CNS-IPI high-risk group. Special attention should be paid to patients with kidney/adrenal involvement and MYD88 L265P mutation. Figure 1 Figure 1. Disclosures Vos: Sanofi: Membership on an entity's Board of Directors or advisory committees; Celgene: Other: Travel reimbursement. Treon: X4: Research Funding; Janssen: Consultancy, Research Funding; Dana Farber Cancer Institute: Current Employment; BMS: Consultancy, Research Funding; Self: Patents & Royalties: Holder of multiple patents related to testing and treatment of MYD88 and CXCR4 mutated B-cell malignancies; AbbVie: Consultancy, Research Funding; BeiGene: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding. Dimopoulos: Janssen: Honoraria; Beigene: Honoraria; Amgen: Honoraria; BMS: Honoraria; Takeda: Honoraria. Kapoor: Cellectar: Consultancy; Karyopharm: Consultancy; BeiGene: Consultancy; Pharmacyclics: Consultancy; Sanofi: Consultancy; Amgen: Research Funding; Ichnos Sciences: Research Funding; Regeneron Pharmaceuticals: Research Funding; Glaxo SmithKline: Research Funding; Karyopharm: Research Funding; Sanofi: Research Funding; Takeda: Research Funding; AbbVie: Research Funding. Castillo: Abbvie: Consultancy, Research Funding; BeiGene: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Janssen: Consultancy; Roche: Consultancy; TG Therapeutics: Research Funding.

MYD88 L265P mutation in primary central nervous system lymphoma is associated with better survival: A single centre experience

Background The Myeloid differentiation primary response gene (MYD88) mutation in primary central nervous system lymphomas (PCNSL) may be associated with unfavourable prognosis, however current evidence remains limited. We aimed to characterise PCNSLs by integration of clinicopathological, molecular, treatment and survival data. Methods We retrospectively identified and validated 57 consecutive patients with PCNSLs according to the 2017 WHO classification of lymphoid neoplasms over 13 years. Formalin-fixed paraffin-embedded tumour samples underwent polymerase chain reaction assay to detect MYD88 mutation. We used Cox regression for survival analysis including age, treatment and MYD88 as covariates. We searched the literature for studies reporting demographics, treatment, MYD88 and survival of PCNSL patients, and incorporated individual-patient data into our analyses. Results The median age was 66 years and 56% were women. All 57 patients had PCNSL of non-germinal centre cell subtype and the majority (81%) received either single or combined therapies. There were 46 deaths observed over the median follow-up of 10 months. MYD88 mutation status was available in 41 patients of which 36 (88%) were mutated. There was an association between MYD88 mutation and better survival in the multivariable model (hazard ratio [HR] 0.277; 95% confidence interval [CI] 0.09-0.83; p=0.023) but not in a univariable model. After incorporating additional 18 patients from the literature, this association was reproducible (HR 0.245, 95% CI 0.09-0.64, p=0.004). Conclusions Adjusting for confounders, MYD88-mutant PCNSL appears to show improved survival. While further validation is warranted, detection of MYD88 mutation will aid identification of patients who may benefit from novel targeted therapies.

High-affinity T-cell receptor specific for MyD88 L265P mutation for adoptive T-cell therapy of B-cell malignancies

BackgroundAdoptive transfer of engineered T cells has shown remarkable success in B-cell malignancies. However, the most common strategy of targeting lineage-specific antigens can lead to undesirable side effects. Also, a substantial fraction of patients have refractory disease. Novel treatment approaches with more precise targeting may be an appealing alternative. Oncogenic somatic mutations represent ideal targets because of tumor specificity. Mutation-derived neoantigens can be recognized by T-cell receptors (TCRs) in the context of MHC–peptide presentation.MethodsHere we have generated T-cell lines from healthy donors by autologous in vitro priming, targeting a missense mutation on the adaptor protein MyD88, changing leucine at position 265 to proline (MyD88 L265P), which is one of the most common driver mutations found in B-cell lymphomas.ResultsGenerated T-cell lines were selectively reactive against the mutant HLA-B*07:02-restricted epitope but not against the corresponding wild-type peptide. Cloned TCRs from these cell lines led to mutation-specific and HLA-restricted reactivity with varying functional avidity. T cells engineered with a mutation-specific TCR (TCR-T cells) recognized and killed B-cell lymphoma cell lines characterized by intrinsic MyD88 L265P mutation. Furthermore, TCR-T cells showed promising therapeutic efficacy in xenograft mouse models. In addition, initial safety screening did not indicate any sign of off-target reactivity.ConclusionTaken together, our data suggest that mutation-specific TCRs can be used to target the MyD88 L265P mutation, and hold promise for precision therapy in a significant subgroup of B-cell malignancies, possibly achieving the goal of absolute tumor specificity, a long sought-after dream of immunotherapy.

Frostbite and Cold Agglutinin Disease: Coexistence of Two Entities Leading to Poor Clinical Outcomes

An 83-year-old woman was admitted to the emergency department for a 7-day history of fatigue and progressive cyanosis in the feet and hands after cold exposure despite physical protective measures. Upon arrival, the patient presented with necrotic cutaneous lesions in both hands and distal lower extremities. Upon admission, hemoglobin was 7.6 g/dL and laboratory tests were consistent with cold agglutinin disease (CAD), the presence of monoclonal IgM, and flow cytometry consistent with lymphoplasmacytic lymphoma, but MYD88 L265P mutation was negative. The patient required blood transfusion, resulting in stabilized hemoglobin and a decrease in markers of hemolysis. Treatment with aspirin 250 mg daily and intravenous iloprost 0.5 mL/h was initiated with a poor clinical response at day 4. Amputation was required. Plasma exchange was performed and chemotherapy with rituximab and bendamustine was initiated. The clinical course was marked by further necrosis, prompting discussions regarding an additional amputation that was not performed considering the high surgical risk and refusal by the patient. Supportive treatment was initiated, and the patient expired one month after hospital admission.

Chylothorax as a complication of Waldenström macroglobulinaemia with a patient’s perspective

Chylothorax has rarely been reported as a pleuropulmonary complication of Waldenström macroglobulinaemia (WM). In general, when a unilateral effusion is discovered particularly in patients with a history of cancer or active malignancy, a broad differential including chylothorax needs to be considered. We present the case of a 50-year-old woman found to have chylothorax secondary to progression of WM as confirmed by cytology and presence of MYD88 L265P mutation in the pleural fluid specimen and subsequent resolution with chemotherapy. This review centres particularly on non-traumatic causes of chylothorax with a focus on WM and includes a unique patient perspective.