ALBERT

Description: Splenic marginal zone B-cell lymphoma (SMZL) is a rare clinical and pathological entity recognized by the WHO classification. SMZL usually presents with isolated splenomegaly, bone marrow involvement, and leukemic picture. Nodal disease is generally rare at diagnosis. When lymphocytes with villous projections are found in peripheral blood, the disease is termed splenic lymphoma with villous lymphocytes (SLVL). High HCV-seroprevalence is frequently reported in SMZL. The disease commonly pursues an indolent course; however, about a third of pts follow a more aggressive course. SMZL is also heterogeneous with respect to the preferential usage of IgVH genes, as well as the percentage of mutation load. No previous studies correlated IgVH rearrangement features with the clinical characteristics of SMZL. The aim of the present study was to determine the tumor-related IgVH gene rearrangement and compare the gene usage and the mutation status with clinical features of 59 pts. Diagnosis was made according to the WHO classification and to the diagnostic criteria for SMZL (Matutes et al., Leukemia 2008). Paraffin sections from lesional tissues (14 spleens, and 59 bone marrow trephines) were available for all pts. Histology and blood smears were reviewed. Total RNA was extracted from PB (n=13) or BM (n=46) mononuclear cells. IgVH gene rearrangements were amplified using 6 family-specific VH leader primers coupled with a 3′ heavy chain joining (JH) primer or with a constant region of cμ chain primer. The sequence obtained from direct sequencing of the clonal PCR product was compared with germline in the IMGT database. Sixty VDJ rearrangements were amplified and 54 were functional. VH1 family was used in 19 rearrangements (32%), VH3 family in 33 (55%) and VH4 family in 8 (13%). The most frequent VH genes were VH1-02 (n=13), VH3-23 (n=15), VH3-30 (n=7) and VH4-34 (n=5) (67% of all sequences). VH was unmutated in 25%. DH segments were assignable in 56/60 rearrangements (all of the VH unmutated and 41/45 of VH mutated). The most frequent DH families were DH2 (n=9) and DH3 (n=21); the most frequent DH genes were DH3-03 (n=8), DH3-22 (n=6) and DH2-02 (n=6). Most rearrangements used JH4 family (n=21), JH5 (n=8), and JH6 (n=19), accounting for 80% of all rearrangements. JH4b (n=14), JH6b (n=10), and JH6c (n=8) were the most common JH genes. Median length of HCDR3 region was 17 aa (range 11–32). For antigen selection analysis P value was 〈 0.05 in 41% for CDRs and in 55% for FRs. No correlation was found among VH and DH families (p=0.1), among VH and JH families (p=0.09) and among DH and JH families (p=0.4). Forty-two % of pts were unmutated in VH1 family, 15% in VH3 family and 25% in VH4 family (p=0.09). Unmutated cases were 7% in VH3-23 group, 46% in VH1-02 group and 14% in VH3-30 group (p=0.03). For clinical correlations we considered only the 54 functional rearrangements. Villous lymphocytes 〉10% were detected in 53% of VH1 pts, in 57% of VH4 pts, and in 17% of VH3 pts (p=0.01). Villous lymphocytes 〉10% were detected in 21% of VH3-23 pts, in 50% of VH1-02 pts and in no VH3-30 pt (p=0.05). Liver involvement was present in 9% of VH3-23 pts, in no VH1-02 pt and in 50% of VH3-30 pts (p=0.009). HCV serology was positive in 7% of VH3-23 pts, 17% of VH1-02 pts and 50% of VH3-30 pts (p=0.04). Serum MC was detected in 50% of VH3-23 pts, in 9% of VH1-02 pts and in 17% of VH3-30 pts (p=0.06). BM was involved in 86% of VH3-23 pts, in 100% of VH1-02 pts and in 50% of VH3-30 pts (p=0.02). A sinusal localization was detected in 67% of VH3-23 pts, in 20% of VH1-02 and in 100% of VH3-30 (p=0.03). Unmutated status was significantly related to a higher percentage of BM infiltration (p=0.003). The proportion of intermediate and high risk patients according to the SMZL score (Arcaini et al. Blood 2006) was higher in the unmutated respect to the mutated group (69% vs 32%, p=0.05). After a median F-UP of 3 yrs, 10 pts died (7 of lymphoma, 3 of causes non related to lymphoma). The median OS was 12 yrs and the median PFS was 2 yrs. OS did not differ among VH families (p=0.9). Median PFS was 2 yrs for mutated pts and 1 yr for unmutated pts. We performed clustering of pts by applying a 2-means clustering algorithm, using as parameters nodal disease, villous lymphocytes 〉10%, HCV serology positive, BM involvement: in cluster 1 VH1 family pts accounted for 20%, VH3 family for 71%, VH4 family for 9%; in cluster 2 VH1 family pts accounted for 47%, VH3 family for 29% and VH4 family for 24% (p=0.01). In conclusion, VH rearrangement analysis in SMZL reveals a non-random preference for VH1-02, VH3-23 and VH3-30 genes, whose use differs according to distinctive clinical features at presentation.

Description: Findings of recent studies indicate that flow cytometry (FCM) may be valuable in the diagnosis and prognostication of myelodysplastic syndromes (MDS). This approach appears particularly promising in patients with low-risk MDS without ringed sideroblasts and excess of blasts (i.e., with refractory anemia tout court) who have normal karyotype. These patients lack in fact any specific morphological or cytogenetic marker. However, the analytical methods reported so far require considerable technical skill, and therefore FCM has not yet become a routine procedure in the work-up of MDS patients. In this work, we developed a simple, reproducible FCM protocol for MDS and tested its validity prospectively. This study has been approved by the Ethics Committee, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy and by the Institutional Review Board of Nippon Medical School. The cytological diagnosis of MDS was made according to the WHO criteria by two independent cytologists who were blinded to clinical data. Three-color FCM was conducted at two laboratories (Tokyo and Pavia), which had received the details of the analytical method beforehand. The FCM protocol was developed in Tokyo and a part of which was reported previously (Leuk Res, 2008 32(5):699–707). The mandatory FCM parameters were CD34+ myeloblasts (% in all nucleated cells), CD34+ B-cell progenitors (% in all CD34+ cells), CD45 expression of CD34+ myeloblasts, and side scatter of mature myeloid cells. The optional parameters were CD11b, CD15, and CD56 expressions on CD34+ myeloblasts. These seven parameters were quantitatively analyzed and their reference ranges (RR) were determined using data from the cohort reported previously (Blood. 2006; 108(3): 1037–44). Bone marrow samples from 80 MDS patients with refractory anemia and normal karyotype, and from 82 controls were analyzed. Controls are patients who underwent routine diagnostic procedures for cytopenia and were eventually found to have conditions other than MDS and other clonal diseases. Abnormal data (outside the RR) in 2 or more parameters were common in MDS and were observed in 7 of 24 (29%) Japanese patients and 37 of 56 (66%) Italian patients when the four mandatory parameters alone were analyzed, and in 16 of 24 (67%) Japanese patients and 40 of 46 (87%) Italian patients when all seven parameters were analyzed (56 of 70 [80%] in total). A decreased CD34+ B-cell progenitor was the most common abnormality. By contrast, the occurrence of abnormalities in 2 or more FCM parameters was rare in control patients and was observed in 5 of 82 (6%) patients when all seven parameters were analyzed (56/70 versus 5/82, P 〈 .0001). Therefore, when bone marrow samples lacking ringed sideroblasts and blast excess, and having normal karyotype show 2 or more abnormal FCM parameters, the likelihood ratio of MDS is 13.1 (95% confidence interval [CI], 6.4 to 29.3): the diagnostic sensitivity and specificity were 80% (95% CI, 74 to 84%) and 94% (95% CI, 89 to 97%), respectively. In conclusion, the findings of this study strongly indicate that the adopted FCM protocol is feasible and useful for diagnosing MDS in patients who lack specific morphological or cytogenetic markers.

Description: Impaired immune responses have been hypothesised to be a possible trigger of unfavourable outcomes in coronavirus disease 2019 (COVID-19). We aimed to characterise IgM memory B cells in patients with COVID-19 admitted to an internal medicine ward in Northern Italy. Overall, 66 COVID-19 patients (mean age 74 ± 16.6 years; 29 females) were enrolled. Three patients (4.5%; 1 female) had been splenectomised and were excluded from further analyses. Fifty-five patients (87.3%) had IgM memory B cell depletion, and 18 (28.6%) died during hospitalisation (cumulative incidence rate 9.26/100 person-week; 5.8–14.7 95% CI). All patients who died had IgM memory B cell depletion. A superimposed infection was found in 6 patients (9.5%), all of them having IgM memory B cell depletion (cumulative incidence rate 3.08/100 person-week; 1.3–6.8 95% CI). At bivariable analyses, older age, sex, number of comorbidities, and peripheral blood lymphocyte count

Description: Background. The World Health Organization defines Waldenström Macroglobulinemia (WM) as a lymphoplasmacytic lymphoma associated with a serum immunoglobulin M (IgM) monoclonal protein. Since bone marrow (BM) infiltration is always present, while extramedullary involvement is infrequent, trephine BM biopsy is mandatory based on currently used diagnostic criteria, while multiparameter flow cytometry (MFC) studies are recommended to support the diagnosis (Owen et al, 2003). Next generation flow cytometry using panels with 8 to 17 monoclonal antibodies (MoAb), high number of cell acquisitions and appropriate software have shown an increased sensitivity, being able to detect one tumor cell among 10.000 normal cells (10-4) or even more. Aims of the study. Here we report an extensive phenotypic characterization by eight color MFC of a large prospective cohort of newly diagnosed WM patients, enrolled in the multicenter, observational trial BIO-WM (NCT03521596). In this study we evaluated the potential role of MFC on BM and peripheral blood (PB) samples as an alternative to BM biopsy in the diagnostic work-up of WM, with the final goal to assess the feasibility of a non-invasive or at least less invasive diagnostics. Methods. The diagnosis of WM was made using the criteria of the Second International Workshop on WM. Patients without bone marrow biopsy were excluded from the analysis. Whole BM and PB samples were processed following the bulk lysis protocol and stained using 8-color combination of the following fluorochrome-conjugated monoclonal antibodies: cIgM, CD56, CD5, CD19, CyK, CyL, CD38, CD45 (screening panel). CD5- lymphoproliferative disorders with two populations in different maturation stage showing identical cytoplasmatic light chain restriction underwent further characterization with six 8 color-panels (characterization panel). A minimum of 1x106/L cells per tube was acquired. The full list of monoclonal antibodies is reported in Table 1. Results. We analyzed 173 prospective patients with a histologic diagnosis of WM, either asymptomatic (n=46, 27%) or symptomatic (n=127, 73%). Their baseline characteristics are reported in Table 2. MFC results on BM samples collected at diagnosis were available in 165 patients. The median white blood cell (WBC) count was 13.5 x 109/L (IQR: 4.9-94) and the median number of cell acquisitions was 1.16 x 106 (IQR: 0.91-1.58). Clonal CD19+ B-cells were detected by MFC in 155/165 cases (94%) and showed k light chain restriction in 76% of cases. The median percentage of clonal CD19+ B-cells out of total BM WBC was 5.9% (IQR: 2.9-12.4). Twelve cases out of 165 (7%) were CD5+. The complete phenotype of clonal B CD19+ lymphocytes is shown in Figure 1. Clonal CD38+ plasma cells were detected in BM samples in 86% of cases and were CD20+ in 51% of cases. The median percentage of clonal CD38+ plasma cells out of total BM WBC was 0.18% (IQR: 0.05-0.47). The complete antigenic expression of clonal plasma cells is shown in Figure 2. The MYD88 (L265P) mutation was found with droplet digital polymerase chain reaction (dd-PCR) in 95% of patients with detectable clonal CD19+ B-cells in BM samples, without significant difference between CD5- and CD5+ cases (95% versus 93%, P=0.698). Paired BM and PB samples were available in 157 patients. Clonal CD19+ B-cells were detected in BM samples in 149/157 cases (95%) and in PB samples in 98/157 cases (58%). Overall, there was a slight agreement between BM and PB MFC results according to Landis and Koch scale (63%, k = 0.138). Conclusions. The comparison of MFC results on BM samples with BM histology in this large prospective cohort of WM patients suggests that eight-color MFC is highly sensitive and is able to identify the majority of patients with WM. Conversely, the comparison of MFC results on paired BM and PB samples indicates that MFC on PB samples has a low sensitivity, missing approximately 40% of cases. The integration of MFC results on BM samples with MYD88 mutation status assessed with a highly sensitive method such as dd-PCR may be considered as an alternative, less invasive method for the diagnosis of WM. Further analyses within this trial will assess the role of MFC combined with molecular profile of patients for the differential diagnosis between WM and other mature B-cell lymphoproliferative disorders as well as for the discrimination between WM and IgM monoclonal gammopathy of undetermined significance. Figure 1 Disclosures Varettoni: Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Other: Travel/accommodations/expenses; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel/accommodations/expenses. Ferrero:Servier: Speakers Bureau; Gilead: Research Funding, Speakers Bureau; EUSA Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Puig:JANSSEN: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES, Research Funding; TAKEDA: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES, Research Funding; AMGEN: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES, Research Funding; THE BINDING SITE: Consultancy, Honoraria; BRISTOL-MYERS SQUIBB: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES, Research Funding, Speakers Bureau; CELGENE: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES, Research Funding, Speakers Bureau. Ferrari:Abbvie: Honoraria. Laurenti:Janssen: Honoraria; Gilead: Honoraria; AbbVie: Honoraria; Roche: Honoraria. Marchetti:Takeda: Other: Sponsored meetings; Pfeizer: Other: Sponsored meetings; AbbVie: Other: Sponsored meetings; Gilead: Consultancy; Novartis: Speakers Bureau; Amgen: Speakers Bureau. Re:BerGenBio ASA: Research Funding. Boccadoro:GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria, Research Funding; Mundipharma: Research Funding; AbbVie: Honoraria; Celgene: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding. Garcia-Sanz:Novartis: Honoraria; Janssen: Honoraria, Research Funding; Incyte: Research Funding; Gilead: Honoraria, Research Funding; BMS: Honoraria; Amgen: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Honoraria; Takeda: Consultancy, Research Funding.