Lymphoplasmacytic Cells Research Articles

Modeling Lymphoplasmacytic Lymphoma/Waldenström Macroglobulinemia in Transgenic Mice Using Human MYD88L256P Mutated Protein

Lymphoplasmacytic lymphoma (LPL)/Waldenström macroglobulinemia (WM) is a B-cell disorder resulting from the accumulation of cloncal lymphoplasmacytic cells in the bone marrow (BM) along with excessive secretion of immunoglobulin M paraprotein. Despite recent advances in treatment LPL/WM remains incurable. Generating a refined LPL/WM mouse model that would facilitate studying this disease as well as enable stringent in vivo validation of potential therapeutic approaches has been a long-sought goal for the filed. Interestingly, among the most common mutations identified by recent high-throughput studies there was a single gain of function point mutation in the MYD88 gene, with a predicted non-synonymous amino acid (L265P) change, found in over 91% of patients. Therefore, we hypothesized that generation of transgenic mice overexpressing MYD88L256Pcould recapitulate the human setting of LPL/WM.To overcome problems inherently related to embryonic lethality, we generated MYD88WT and MYD88L256P conditional transgenic mice expressing human proteins. To remove a transcriptional stop cassette and activate MYD88 expression in vivo, we mated the conditional mice with mice expressing Cre recombinase under the control of Activation-Induced Cytidine Deaminase (AID) gene promoter to generate AIDCre/-;MYD88WT/- and AIDCre/-;MYD88L256P/- double-mutant mice. Recombinase activity driven by the AID gene promoter caused removal of the stop cassette and expression of MYD88 in germinal center (GC) and post-GC B cells, the appropriate B-cell compartment from which LPL/WM clones are thought to originate in humans. Young (up to 30 weeks of age) AIDCre/-;MYD88WT/- and AIDCre/-;MYD88L256P/- mice where indistinguishable from AIDCre/- mice as assessed by external physical examination. Moreover, obvious histologic and IHC differences in spleens, lymph nodes, and bone marrows between the mutant genotypes and the AIDCre/- control mice were not found. No clonal bands for the IgH gene, nor M-spikes were detected in Southern blots and serum protein electrophoresis, respectively. However, after 30 weeks of age, and at variable times thereafter, some of the AIDCre/-; MYD88L256P/- aging cohort (n=27) mice developed skin rash and excoriation as well as loss of hair in the submandibular areas. A fraction of AIDCre/-; MYD88L256P/- animals that weren't sacrificed because of the severe dermatitis had enlarged lymph nodes and appeared sick. To assess whether MYD88 transgenic mice develop B-cell lymphomas or major pathologic alterations consistent with the diagnosis of LPL/WM, animals were euthanized at different ages and lymphoid organs were rigorously examined. In spleens and lymph nodes of 8 of the AIDCre/-; MYD88L256P/- animals, a nodular lymphoid infiltrate composed predominantly of small atypical cells varyingly infiltrated with CD138+ plasma cells was observed. Lymphoid cells infiltrated other organs including the BM. Enhanced expression of MYD88 and activation of NF-κB activity within atypical lymphoid nodules in diseased transgenic mice was documented by IHC staining for MYD88 and p65. However, no clonal bands of the IgH gene were detected in Southern blots, suggesting that overexpression of MYD88L256P/- mutant protein in activated B-cells gave rise to a polyclonal lymphoproliferative disorder. Consistently, serum protein electrophoresis analysis showed diffuse, polyclonal increase in γ-globulins. Moreover, stains of peripheral blood smears revealed rouleaux formation suggesting increased concentrations of plasma proteins. In addition, one of AIDCre/-; MYD88L256P/- animals developed a more aggressive monoclonal disease resembling human diffuse large B-cell lymphoma. However, large atypical lymphoid cells in this mouse lacked expression of MYD88 and nuclear p65. All AIDCre/- controls and AIDCre/-; MYD88WT/- mice lacked described phenotypes and were histologically unremarkable.Taken together, these results demonstrate that although MYD88L256P/- alone might not be sufficient to induce a malignant transformation in the mature B-cell compartment, it gave rise to a low-grade mature non-clonal B-cell lymphoproliferative disorder with some of the characteristic futures of LPL/WM. Therefore, the MYD88L256P/- mutation might be necessary but not sufficient for LPL/WM development and must be present with concomitant “second hit” genetic alterations for a neoplasm to develop. DisclosuresTreon:Pharmacyclics: Consultancy, Research Funding.

The Bendamustine Plus Rituximab Regimen Is Active and Safe in Previously Untreated Patients with Waldenström Macroglobulinemia, a Study on Behalf of the French Innovative Leukemia Organization (FILO)

BackgroundWaldenström macroglobulinemia (WM) is a rare low-grade B-cell malignancy characterized by the infiltration of IgM-secreting lymphoplasmacytic cells in the bone marrow. There is no standard therapy for WM, but several drugs have been shown to be effective either alone or in combination. Bendamustine (B)-Rituximab (R) has been found to be effective in salvage (Tedeschi , Leukemia Lymphoma 2015) or first setting in MW (Treon, ASH2016).BR- regimen has however reported to be associated with an increase in secondary cancer and the development of secondary myelodysplastic syndrome (MDS) or acute myeloblastic leukemia (AML) in patients with previously treated non-Hodgkin lymphoma. Prolonged neutropenia after R-chemotherapy treatment of lymphoma or CLL has also been associated with an increase of MDS/AML, and a shorter interval between the occurrence of the two malignancies. However, this has not been evaluated in previously untreated WM, nor the possible relationship with secondary cytopenia to WM treatment.MethodsForty-four symptomatic previously untreated patients with WM based on the second WM Workshop recommendations for diagnostic criteria were enrolled in this retrospective multi-center study between January 2012 and January 2017 in ten French centers. MYD88L265P and CXCR4 mutations were searched using q PCR and or next generation sequencing. Del (17) p and/ or TP53 mutation were investigated for 22 patients. All patients received a BR regimen consisting of 375 mg/m2 of rituximab on day 1 and 90 mg/m2 of bendamustine on days 1 and 2, repeated every 4 weeks, with a maximum of 6 cycles. Long-term complications of BR, 6 months or more after treatment discontinuation were analyzed. PFS and OS were plotted using the log rank test and Kaplan-Meier comparison.ResultsThe median age at WM diagnosis was 69 years old (range 45-88) and the median age at initiation of therapy was 70 years old (range 46-89). The MYD88L265Pmutation was present in 24/30 (80%) of the cases. CXCR4 mutations were observed in 2/14 cases, alteration of TP53 in 1/22 cases.The median time from diagnosis to therapy was 2 months (range 0-123 months). The median serum IgM level was 3,870mg/dl (range 650-12000). The criteria for initiating BR treatment were: anemia (54%), B-symptoms (45%), hyper viscosity (25%), adenopathy and/or splenomegaly (18%), thrombocytopenia (16%), neuropathy (11%), and amyloidosis (4%). According to the IPSSMW score, 22% of patients were low risk, 35% intermediate risk, and 43% high risk. Overall 82% of patients completed the planned 6 courses.Using consensus response criteria, the ORR was 95% (CR15%, VGPR 40% PR38% MR 2%) and the major response rate (CR+VGPR+PR) 93%.Toxicities attributed to BR led to dose reduction and/ or incomplete regimen of intended therapy in 18% of patients. Their median age was 78 years old. Toxicities were related to excessive or prolonged myelosuppression in 5 patients and infection in 3.Most of the adverse events were hematological and infectious toxic effects. Grade 3-4 neutropenia occurred in 4 (11%) cases and grade 3-4 thrombocytopenia in one (3%). Minor infections developed in eight cases: three cutaneous infections, two herpes zosters, one upper respiratory tract infection, one sinusitis, and one oral-herpes. Grade 3-4 pneumonia was observed in two cases and sepsis in one.Among 38 evaluable patients for toxicities after BR therapy, 16(42%) developed one or more long-lasting cytopenias: neutropenia (34%), anemia (10%), and thrombocytopenia (8%), all grade I and II.The median duration for prolonged neutropenia, anemia, and thrombocytopenia were 6 months (range: 6-24 months), 7.5 months (range: 6-9 months) and 12 months (6-30 months), respectively.After a median observation time of 27 months (range 5-51 months) after BR-regimen, the PFS and OS were 92% and 100%, respectively. Two patients relapsed at 24 and 45 months. One patient with a history of breast cancer prior to WM treatment relapsed from this solid tumor at 23 months. None of the patients developed aggressive lymphoma or secondary MDS/AML at the last follow-up.ConclusionThe BR regimen is efficient in patients with previously untreated WM. In elderly patients, the dose of bendamustine needs to be lowered. Long-lasting cytopenia occurred in half of patients and resolved in most cases within one year after the end of treatment. Longer follow-up is needed to assess the long-term risks of BR treatment. DisclosuresLaribi:MUNDIPHARMA: Research Funding; NOVARTIS: Honoraria, Research Funding; ROCHE: Research Funding; TEVA: Research Funding; HOSPIRA: Research Funding; AMGEN: Honoraria; TAKEDA: Honoraria, Research Funding. Leblond:SERVIER: Honoraria, Membership on an entity's Board of Directors or advisory committees; GILEAD: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; JANSSEN: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Honoraria; ABBVIE: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; ROCHE: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; NOVARTIS: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Continuous MYD88 Activation Is Associated With Expansion and Then Transformation of IgM Differentiating Plasma Cells.

Activating mutations of MYD88 (MYD88L265P being the far most frequent) are found in most cases of Waldenström macroglobulinemia (WM) as well as in various aggressive B-cell lymphoma entities with features of plasma cell (PC) differentiation, such as activated B-cell type diffuse large B-cell lymphoma (DLBCL). To understand how MYD88 activation exerts its transformation potential, we developed a new mouse model in which the MYD88L252P protein, the murine ortholog of human MYD88L265P, is continuously expressed in CD19 positive B-cells together with the Yellow Fluorescent Protein (Myd88L252P mice). In bone marrow, IgM B and plasma cells were expanded with a CD138 expression continuum from IgMhigh CD138low to IgMlow CD138high cells and the progressive loss of the B220 marker. Serum protein electrophoresis (SPE) longitudinal analysis of 40 Myd88L252P mice (16 to 56 weeks old) demonstrated that ageing was first associated with serum polyclonal hyper gammaglobulinemia (hyper Ig) and followed by a monoclonal immunoglobulin (Ig) peak related to a progressive increase in IgM serum levels. All Myd88L252P mice exhibited spleen enlargement which was directly correlated with the SPE profile and was maximal for monoclonal Ig peaks. Myd88L252P mice exhibited very early increased IgM PC differentiation. Most likely due to an early increase in the Ki67 proliferation index, IgM lymphoplasmacytic (LP) and plasma cells continuously expanded with age being first associated with hyper Ig and then with monoclonal Ig peak. This peak was consistently associated with a spleen LP-like B-cell lymphoma. Clonal expression of both membrane and secreted µ chain isoforms was demonstrated at the mRNA level by high throughput sequencing. The Myd88L252P tumor transcriptomic signature identified both proliferation and canonical NF-κB p65/RelA activation. Comparison with MYD88L265P WM showed that Myd88L252P tumors also shared the typical lymphoplasmacytic transcriptomic signature of WM bone marrow purified tumor B-cells. Altogether these results demonstrate for the first time that continuous MYD88 activation is specifically associated with clonal transformation of differentiating IgM B-cells. Since MYD88L252P targets the IgM PC differentiation continuum, it provides an interesting preclinical model for development of new therapeutic approaches to both WM and aggressive MYD88 associated DLBCLs.

POS-629 PERITONEAL MEMBRANE HISTOPATHOLOGIC CHANGES CAUSED BY PERITONITIS IN CONTINUOUS AMBULATORY PERITONEAL DIALYSIS PATIENTS: TWO CASE REPORTS

Long-term Peritoneal Dialysis (PD) causes morphologic and functional changes in the peritoneal membrane (PM) which is defined as peritoneal fibrosis, a known cause of loss of peritoneal ultrafiltration capacity. Inflammation, angiogenesis , and bioincompatible of peritoneal dialysis solution are a key event during the pathogenesis. Peritonitis remains a major problem in Peritoneal Dialysis. Excessive peritoneal inflammatory responses lead to mesothelial cell exfoliation and thickening of the submesothelial, resulting in peritoneal fibrosis and sclerosis. We report two cases of the patient in Continuous Ambulatory Peritoneal Dialysis (CAPD), focus on PM histopathologic changes. Case 1. A male, 47 years old, suffered from repeated peritonitis. After fourth episode of infection, the catheter was malfunction and removed. He was done on CAPD for 7 years with glucose base solution. Histopathologically, peritoneal membrane showed diffuse fibrosis and hyalinization, without mesothelial cell lining nor inflammatory cell infiltrate. He was transferred to hemodialysis. Case 2. A 28 years old male, came with chief complaint of fever, abdominal pain, and cloudy effluent in CAPD fluid. On third episode of peritonitis, Empirical treatment with cefotaxime intraperitoneal was done , however CAPD catheter was malfunctioned and removed. He was done on CAPD for 6 years with glucose base solution. The peritoneal membrane histopathology showed diffuse fibrosis and hyalinization, involving vessels, foci of lymphoplasmacytic cells infiltration, without mesothelial cell. He was transferred to hemodialysis. The success of PD was dependent on the structural and functional integrity of the PM. The mesothelium lines the PM and is the first line of defense against chemical / bacterial insult. Peritonitis remains a major complication of PD and cause of ultrafiltration/technique failure, morbidity and mortality. The mortality risk of PD-related peritonitis was 16-18%. Bioincompatible PD solution is also associated with production of proinflammatory and profibrotic cytokines. High peritoneal glucose exposure is associated with increased incidence of relapsing/recurrent peritonitis and conduct proinflammatory and profibrotic reactions in the peritoneal cavity leading to fibrosis and malfunction after 6-7 years. Peritoneal fibrosis was detected in 50% and 80% of PD patients within one and two years on PD. Pathological alterations in the vasculature of peritoneal include hyalinization of the blood vessels, vasculopathy, and submesothelial thickening.Figure 2Case 2.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Peritonitis episode altered peritoneal structure and functional. Neglected infection and repeated infection led to PD catheter removal. Glucose based solution may a contributing factor for membrane failure due to inflammation and fibrosis after 6-7 years.

Glomerulonephritis with severe nephrotic syndrome induced by immune complexes composed of galactose-deficient IgA1 in primary Sj\xf6gren\u2019s syndrome: a case report

BackgroundPrimary Sjögren’s syndrome (pSS) is an auto-immune disease characterized by sialadenitis and dacryoadenitis with lymphoplasmacytic cell infiltration. In pSS, not only sicca symptoms, but also extra-glandular involvement induced by immune abnormalities based on pSS occurs. Renal involvement is one such important life-threatening extra-glandular involvement. Although the aberrant glycosylated IgA in pSS as a product of over-activated B cells is a risk factor of renal involvement, its association has not been clarified. Here we report a case of glomerulonephritis (GN) induced by immune complexes (IC) composed of galactose-deficient IgA1 (Gd-IgA1) in a patient with pSS.Case presentationA 48-year-old Japanese woman with pSS was admitted to our hospital because of a two-month history of nephrotic syndrome. Seven years before she had been diagnosed with pSS from keratoconjunctivitis sicca, elevation of serum anti-Ro/SSA antibody titer and lymphoplasmacytic cell infiltration around salivary ducts of the small salivary glands. Renal biopsy revealed diffuse bubbling appearance in glomerular basement membrane (GBM) with scarce mesangial proliferation. Immunofluorescence showed granular IgA, C3 and Gd-IgA1 staining of GBM. Light chain staining showed no monoclonality. Electron microscopy showed electron dense deposits mainly in the intra-membranous and paramesangial areas and slightly in the subepithelial area. Additional serum analysis confirmed elevation of Gd-IgA1 (13.5 μg/mL), which was comparable with that seen in IgA nephropathy, and qualitative enzyme-linked immunosorbent assay of IgA-containing circulating immune complex (IgA-CIC) was positive. Thus, we diagnosed GN induced by IC composed of Gd-IgA1. Furthermore, retrospectively performed immunofluorescence of the small salivary gland evaluated at the diagnosis of pSS showed positive Gd-IgA1 staining of infiltrating lymphoplasmacytic cells. Therefore, we concluded that Gd-IgA1 produced by over-activated B cells in pSS formed circulating IC and thereby induced GN. After induction therapy with high dose prednisolone and mycophenolate mofetil, the nephrotic syndrome remitted within 3 weeks, the serum Gd-IgA1 level decreased to the normal range (3.8 μg/mL), and serum IgA-CIC disappeared in the 6th month after induction therapy.ConclusionsOur findings clearly demonstrate an association between aberrant glycosylated IgA and the renal involvement seen in pSS, thereby helping to clarify the renal significance of aberrant glycosylated IgA in pSS.

The type of interstitial inflammatory cell infiltrate and the severity of glomerular injury; an underestimated morphologic correlation

Introduction: The interrelation between the type of interstitial inflammatory cells and the severity of glomerulonephritis was not considered in most of the relevant medical literature. Objectives: To investigate the relationship between the type of interstitial cell infiltrate and the morphological severity of glomerular injury in different types of proliferative glomerulonephritides. Patients and Methods: We retrospectively reviewed 138 native kidney biopsies and assessed the relationship between the type of interstitial inflammatory cell infiltrate and the severity of glomerular injury in the form of cellular crescents and fibrinoid necrosis. Results: The predominant type of interstitial inflammatory cell infiltrate was lymphocytic, noted in more than half of the cases. Lymphoplasmacytic inflammatory cell infiltrate was the second most common type which observed. Fifty-five of patients had inflammation in areas of fibrosis. Cellular/fibrocellular crescents were observed in 44% of cases, and fibrinoid necrosis in 30% of cases. As compared to the ‘lymphocytic’ group, patients in the ‘lymphoplasmacytic’ group had ~3 times higher probability of presenting with crescents and fibrinoid necrosis. Conclusion: Our study highlights the significance of morphological correlations that may predict the severity of glomerular injury. Such findings would be helpful in limited or inadequate renal biopsy samples where the pathologist can alert the clinician, in the appropriate clinical context, to the possibility of having crescents and/or necrotizing lesions in the unsampled glomeruli.

Mast cells (MC) excess in a bone marrow (BM) associated with lymphoplasmacytic cells (LPC) in a 55-Y-old patient with Waldenström's macroglobulinemia (WM).

Mast cells (MC) excess in a bone marrow (BM) associated with lymphoplasmacytic cells (LPC) in a 55-Y-old patient with Waldenström's macroglobulinemia (WM).

Waldenström macroglobulinemia: 2021 update on diagnosis, risk stratification, and management.

Waldenström macroglobulinemia (WM) is a lymphoplasmacytic lymphoma with immunoglobulin M (IgM) monoclonal protein. Clinical features include anemia, thrombocytopenia, hepatosplenomegaly, lymphadenopathy, and rarely hyperviscosity. Presence of IgM monoclonal protein associated with ≥10% clonal lymphoplasmacytic cells in bone marrow confirms the diagnosis. The L265P mutation in MYD88 is detectable in more than 90% of patients and is found in the majority of IgM MGUS patients. Age, hemoglobin level, platelet count, β2 microglobulin, LDH and monoclonal IgM concentrations are characteristics that are predictive of outcomes. Not all patients who fulfill WM criteria require therapy; these patients can be observed until symptoms develop. Rituximab-monotherapy is inferior to regimens that combine it with bendamustine, an alkylating agent, a proteosome inhibitor, or ibrutinib. Purine nucleoside analogues are active but usage is declining in favor of less toxic alternatives. The preferred Mayo Clinic induction is rituximab and bendamustine. Bortezomib, fludarabine, thalidomide, everolimus, Bruton Tyrosine Kinase inhibitors, carfilzomib, lenalidomide, and bendamustine have all been shown to have activity in relapsed WM. Given WM's natural history, reduction of therapy toxicity is an important part of treatment selection.

Epidemiology and Outcomes of Hospitalizations Due to Waldenstrom Macroglobulinemia: A National Perspective

Introduction:Waldenstorm's macroglobulinemia (WM) is a type of indolent B cell lymphoma characterized by immunoglobulin M monoclonal gammopathy and bone marrow infiltration by lymphoplasmacytic cells. Being an uncommon hematologic malignancy, studies on epidemiology of this disease are limited. Median survival is 5-11 years and depends on disease progression, treatment complication and transformation to high-grade lymphoma. In the nationally representative database, we aimed to find out the trends and outcomes of hospitalizations primarily due to WM. Methods:We used National Inpatient Sample (NIS) for the years 2007-2017 by Healthcare Cost and Utilization Project. We extracted a study cohort of adult hospitalizations due to WM using International Classification of Diseases (9th/10th editions) Clinical Modification diagnosis codes. Our primary and secondary objectives were to estimate the trends of hospitalization and outcomes as well as identify predictors of poor outcomes. Poor outcomes were defined as in-hospital mortality and discharge to facility. We utilized Cochran Armitage trend test and multivariable survey regression modeling to analyze trends and predictors of poor outcomes using SAS software, version 9.4 (SAS Institute, North Carolina, USA). Results:We studied a total of 7,379 patients who were hospitalized with WM during 2007-2017. Overall from 2007 to 2017 the burden of hospitalizations has decreased from 804 to 645 . The cohort consisted of elderly patients with a median age of 69-years (IQR:61-78), 60.2% were males, 78% Caucasians, and 10.5% African American. Mean length of stay was 7-days which remained stable over the period. A decline in the proportion of WM patients being discharged to facility (20.1% in 2007 to 13.9% in 2017; pTrend<0.001) and in-hospital mortality (4.7% in 2007 to 2.3% in 2017; pTrend<0.001) was observed. Furthermore, in multivariable logistic regression analysis, rural hospitals (OR 4.3; 95%CI 1.5-12.4; p<0.001), lower median household income (OR 4.1; 95%CI 1.4-12.2; p<0.05) and concurrent conditions like septicemia (OR 10.7; 95%CI 4.2-26.7; p<0.001), and pulmonary circulatory disease (OR 3.6; 95%CI 1.4-9.1; p< 0.001) were associated with higher odds of in-hospital mortality. Conclusion:In this nationally representative study, we observed that the hospitalizations due to WM had mildly declined and outcomes were improved during the study period which might be an indication of improvements in treatment modalities. WM in benign state is rarely a grave prognosis and does not require frequent hospitalization but mortality is usually due to other comorbidities and hyperviscosity. We were able to identify several risk predictors that were associated with poor outcomes which require further studies to better risk stratification and develop preventive measures. Disclosures No relevant conflicts of interest to declare.

The Cytogenetic Aberrations of Lymphoplasmacytic Lymphoma/ Waldenström’s Macroglobulinemia in China

Object:Lymphoplasmacytic lymphoma/ Waldenström's macroglobulinemia (LPL/WM) is a rare B-cell proliferative malignancy characterized by immunoglobulin M monoclonal gammopathy and /or bone marrow infiltration by lymphoplasmacytic cells. Recently, there are no large-scale studies to detect the clinical features, genetic background in patients with LPL/WM in China even Asia. The aim of this study is to explore the clinical and genetic differences, observe the impact of cytogenetic abnormalities on survival and prognosis in Chinese patients. Methods:305 patients with LPL/WM admitted in our hospital from June 1998 to December 2018 were retrospectively analyzed. Baseline characteristics, laboratory detection and therapy were compared using Chi-square test and Kaplan-Meier methodology was undertaken for survival analysis. Results:The median age of 305 patients in the study was 62 years (range 32-87) and male to female ratio was 2.45:1(216/89). The most common symptoms at diagnosis were fatigue (64.9%) and bleeding (14.1%).The patients in low, intermediate and high risk group of the international prognostic scoring system for WM (ISSWM) was 17.6%, 36.5% and 45.9%,respectively.The median hemoglobin was 8.5g/dl(range 2.4-18.7). About 41.3% patients had IgM higher than 4000mg/dl. The cytogenetic abnormalities were uncommon in LPL/WM patients. The chromosomal karyotypes were successful in 194 cases, of which 20 patients showed clonal abnormalities. 12 patients had two or more chromosome abnormalities. The most common chromosome abnormalities were deletion 6/6q(25%) and Y chromosome loss (20%). The median progression-free survival (PFS) and overall survival (OS) in patients with normal and abnormal chromosome karyotypes were similar. 194 patients had FISH detection, of which 33 patients (17%) had FISH abnormalities, 9 (4.6%) patients had two kind of abnormalities. The patients with more than one cytogenetic abnormality had lower survival than just had one abnormality or normal patients. The most common aberrations were 6q deletion and trisomy 12. 17p deletion was detected in 11 patients (11/184,6.0%) and it was a poor prognostic marker of PFS and OS. Single 13q14 deletion had not impact on PFS or OS, but when companied with other cytogenetic aberration, it could worsen the PFS and OS . Besides related to survival, cytogenetic abnormal was also related to the clinical features. Conclusions:Despite the low incidence and abnormal cytogenetic detection rate of LPL/WM, the clinical and genetic features are unique in Chinese patients. The cytogenetic aberrations play an important role in predicting prognosis. Disclosures No relevant conflicts of interest to declare.

High-Dose Methotrexate Based Therapy for the Treatment of Bing Neel Syndrome

BACKGROUND Bing-Neel Syndrome (BNS), characterised by the direct infiltration of central nervous system (CNS) with clonal lymphoplasmacytic lymphoma cells, is a rare cause of neurological complications in patients with Waldenström Macroglobulinaemia (WM). The pathophysiology/neurotropism of BNS is not well understood. Various treatments that appear to cross the blood-brain barrier are used, including chemoimmunotherapy combinations and Ibrutinib. High-dose methotrexate (HDM)-based regimens, including MATRix (methotrexate [MTX], cytarabine, thiotepa, rituximab)1and R-IDARAM (rituximab, MTX [intrathecal and intravenous], cytarabine, dexamethasone) are also used, based on the experience in high-grade CNS lymphoma; however, there are concerns regarding the appropriateness of these regimens in the indolent setting, largely due to their toxicity. This study aimed to determine the efficacy and tolerability of HDM-based, frontline treatment in a large cohort of BNS patients. METHODS A single-centre retrospective review of consecutive patients with BNS treated with HDM based regimens was undertaken. Comprehensive laboratory and CNS imaging results were collated (including whole-brain/spine MRI, bone marrow biopsy, and CSF biochemistry, cytology and MYD88 L265P mutation). Best responses achieved, according to IWWM response assessment criteria, were determined. RESULTS Between 2011 and 2020, 27 patients were treated with HDM-based chemoimmunotherapy: 14 (51.9%) with MATRix, 5 (18.5%) with MTX/cytarabine/rituximab (MAR), 2 (7.4%) with HDM only, and 6 (22.2%) with R-IDARAM. Median time from WM diagnosis to BNS diagnosis was 60 months (0-572 months), including 10 patients (37.0%) who were diagnosed with WM at the time of BNS presentation/diagnosis. Median age at diagnosis with BNS was 66 years (43-75 years), and 15/27 (55.6%) were male. CR was achieved in 6 patients (22.2%), PR in 7 (25.9%), and non-response in 8 (29.6%); PD on therapy occurred in 4 cases (14.8%) and 2 patients died before response could be assessed. Of the 14 patients who received MATRix, 5 (31.2%) achieved PR, 6 (42.9%) had stable disease and 3 (21.4%) progressed while on treatment (Figure 1A). Of the 7 who received MAR or HDM only, 4 (57.1%) achieved CR, with 1 PR and 1 SD; 1 patient died before response could be assessed. Response was not affected by prior chemotherapy (for WM), and no biological or radiological feature predicted response in this highly heterogenous population. Predicted overall survival plateaued at 71.5% 1 year after start of treatment (Figure 1B). At a median follow up of 19.4 months, 7 patients (25.9%) had died: 3 (11.1%) from progressive BNS (1 patient had received each of MATRix, R-IDARAM and HDM only), and 4 (14.8%) from treatment-related toxicity (including infection). Of the latter 4 patients (all male), 2 had received MATRix and 1 had received each of MAR and R-IDARAM. No objective response was seen in 6 of the 7 deceased patients; the remaining 1 patient achieved PR following full-dose MATRix, but subsequently relapsed and died of BNS 7 months later. Regarding dose intensity, 5/15 MATRix recipients (33.3%) received full-dose regimens (i.e. 3.5mg/m2 of MTX per cycle for 3-4 cycles); a further 5 patients received a dose reduction of MTX (to 50-75%) and 9 patients (60%) received <3 cycles. Treatment was discontinued early in 6 of these patients primarily due to inadequate response (SD or PD), in 2 patients due to toxicity, and in 1 patient after desirable response had been achieved. CONCLUSION In the setting of indolent CNS lymphoma, the rationale for chemoimmunotherapy is unclear. Although HDM-based therapy is used in BNS, its administration is limited by toxicity, and dose reductions may undermine dose intensity in the CSF. We observed higher rates of treatment-related mortality and only modest response rates with MATRix vs other HDM-based regimens; this primarily related to treatment related toxicity necessitating dose reduction requirements. These data suggest MATRix offers no response benefit over MAR or HDM only; indeed, the omission of thiotepa appears to allow dose optimisation of MTX and cytarabine, and does not negatively impact on outcome. If patients survive the first year of their disease/treatment, survival outcome is excellent. 1. Ferreri, A.J.M., et al.The Lancet Haematology3, e217-e227 (2016). Figure 1 Disclosures Carr: Lupin: Honoraria; Grifols: Other: Travel support; CSL: Honoraria. Wechalekar:Janssen, Takeda, Caelum, Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. D'Sa:Janssen: Honoraria, Research Funding; BeiGene: Honoraria, Research Funding; Sanofi: Honoraria.

18F-FDG PET/CT in Hyalinized Cholecystitis.

Hyalinizing cholecystitis is a rare type of chronic cholecystitis. Moderate patchy transmural especially perivascular lymphoplasmacytic inflammatory cell infiltration is observed in the gallbladder wall. We present the 18F-FDG PET/CT and MRI findings of this rare subtype of chronic cholecystitis. Hyalinizing cholecystitis should be kept in mind in the differential diagnosis of gallbladder wall thickening with intense 18F-FDG uptake.

Abstract IA13: Treatment of COVID-19 pulmonary failure by targeting BTK

Abstract Pulmonary failure is the main cause of mortality related to COVID-19 infection. Up to 80% of patients hospitalized for COVID-19 infection require supplemental oxygenation, of whom 30-40% may require mechanical ventilation. SARS-CoV-2 binds via the ACE2-receptor that is highly expressed on alveolar type II (ATII) cells in the lung. ATII cells constitute 5-15% of the lung epithelium. While alveolar type I cells are highly adapted for gas exchange, alveolar type II cells have a specialized role in innate immune response. ATII cells express Toll receptors (TLRs) and can trigger inflammatory cytokines and chemoattractants in response to pathogens that recruit and activate other immune cells, including macrophages and neutrophils. We and others previously showed that BTK, and its upstream activator HCK, were involved in TLR-mediated signaling. Both BTK and HCK are triggered by MYD88, a TLR-adaptor protein that signals for all Toll receptors except TLR3 in response to viral and bacterial pathogens, including coronaviruses. ATII cells express TLRs, as do alveolar macrophages that coordinate inflammatory responses with ATII cells. As components of TLR/MYD88 signaling, BTK and HCK can drive inflammatory cytokine production through ERK1/2. The potential for BTK inhibitors to abrogate lung injury and death was demonstrated in an experimental model wherein mice challenged with a lethal intranasal inoculum of a mouse-adapted strain of H1N1 influenza virus were protected against lung injury. Control mice developed respiratory failure, along with histologic and CT findings consistent with lung injury in sharp contrast to the mice that received ibrutinib. Control mice also lost weight and died, whereas those treated with ibrutinib recovered their weight after a brief loss and all survived. Mice treated with ibrutinib also showed decreased inflammatory cell infiltration as well as proinflammatory cytokines in lung tissues that included proinflammatory and chemoattractant cytokines such as IL-1β, IL-6, KC/CXCL1, TNFα, and MCP-1 observed in SARS-Cov-1 and SARS-CoV-2 patients. In a series of Waldenstrom’s macroglobulinemia (WM) patients who were on full-dose ibrutinib (420 mg/day) and contracted COVID-19, none experienced dyspnea. In this series, one WM patient who contracted COVID-19 on reduced-dose ibrutinib (140 mg/day) experienced dyspnea and became hypoxic when ibrutinib was stopped and required mechanical ventilation. He was started on full-dose ibrutinib and showed rapid improvement, was extubated the next day, and did not require any oxygen supplementation two days later. Consistent with his rapid course of improvement on ibrutinib, his C-reactive protein level showed a marked decrease. In a subsequent and larger experience, Roschewski and colleagues administered acalabrutinib to 19 COVID-19 patients, 11 on supplemental oxygenation and 8 on mechanical ventilation. Eight of 11 (73%) and 2/8 (25%) on supplemental oxygenation and mechanical ventilation were discharged on room air, respectively. Ex vivo analysis of peripheral blood samples showed attenuation of activated monocyte BTK expression and IL-6 production following acalabrutinib. Serially collected blood samples from patients with chronic lymphocytic leukemia (CLL), WM, and chronic graft-versus-host disease (cGVHD) on ibrutinib monotherapy also showed marked reductions in proinflammatory and chemoattractant cytokines that greatly overlapped with those reported elevated in the plasma of SARS-Cov-1 and SARS-COV-2 patients, and in ACE2+ cells from lung tissue of SARS-CoV-1 patients. In the iLLUMINATE randomized study, CLL subjects treated with ibrutinib immediately prior to infusion with obinutuzumab also showed significantly decreased levels of inflammatory cytokines associated with infusion related reactions (a cytokine release syndrome). These findings are consistent with a shift from an M1 to M2 polarized macrophage response following ibrutinib and are supported by preclinical and clinical studies showing dependence of macrophage lineage commitment on BTK function. BTK-inhibitors may therefore provide protection against lung injury and even improve pulmonary function in hypoxic patients with COVID-19. Randomized clinical trials to examine the benefit of BTK-inhibitors in COVID-19 patients in pulmonary distress and/or accompanying corollary studies to identify response biomarkers have been initiated: NCT04439006, NCT04382586, NCT04346199, NCT04380688. Selected

Majocchi\u2019s granuloma on the forearm caused by Trichophyton tonsurans in an immunocompetent patient

Majocchi's granuloma is an uncommon fungal infection of the dermis and subcutaneous tissue. The most frequently identified cause of Majocchi’s granuloma is anthropophilic Trichophyton rubrum, and it is most commonly located on the anterior aspect of the lower limbs in women. Here, we report a case of Majocchi’s granuloma on the forearm, a site that is rarely involved, in a 62-year-old woman who had been bitten by a dog. Histological examination revealed a dense dermal infiltrate composed of lymphoplasmacytic cells and neutrophils, with hyphae in the dermis. The presence of the fungus, Trichophyton tonsurans, was confirmed by mycological examination and molecular methods. Therefore, histological and mycological examination confirmed the diagnosis of Majocchi’s granuloma. The patient was treated with local moxibustion and itraconazole, 200 mg/day, for 60 days, which facilitated a complete resolution of the lesions.

IBCL-019: Waldenström Macroglobulinemia (WM) in a Public Health Service in Sao Paulo, Brazil

Context: WM is a rare indolent non-Hodgkin lymphoma that mainly affects people over 60 years old. It is characterized by lymphoplasmacytic cells bone marrow infiltration and IgM gammopathy. Patients have asthenia, weight loss, mucosal bleeding, lymph node enlargement, splenomegaly and neurological changes resulting from hyperviscosity and/or cytopenias. Treatment goal is to relieve symptoms. Recently, targeted drugs have been used, which may increase its median overall survival in the next years. Notwithstanding these drugs are not equally available around the world. Objective: To recognize clinical, epidemiological profile and treatment evaluation of WM patients at Santa Casa de Sao Paulo, Brazil. Design: WM retrospective analysis cohort diagnosed between January 1, 2000 and December 31, 2016. Patients or other participants: We analyzed patients with WM diagnosis. HIV, HBV, and HCV positive serology patients were excluded. Results: There were 18 patients, 10 men/8 women, with 61 years old median age. Fifteen patients presented with splenomegaly; 14 had B symptoms at diagnosis. Median serum IgM was 3019 mg/dL; 8 patients were submitted to plasmapheresis because of symptomatic hyperviscosity syndrome. 70% were classified as High/Very-high risk and all patients were treated upfront. As 1st line treatment we used alkylant + prednisone in 12 patients; alkylant + prednisone + anthracycline in 3 patients; fludarabine in 2 patients and to 1 patient only prednisone. Less than 25% got CR/VGPR; 5.5% got PR and 72% were refractory. There were 2 deaths before 2nd line treatment for which we used fludarabine-based therapy in 9, alkylant + prednisone + anthracycline in 2 and alkylant + prednisone in 1 patient. Ten patients were dead before 3rd line therapy. Mean OS was 55 months. Hb under 9.5 g/dl and platelets under 120×103mm3 had statistically significant worse median OS. Conclusions: Our Waldenstrom macroglobulinemia cohort showed poor response to 1st line chemotherapy regimens. IPSS-WM showed that they arrive in severe clinical conditions making it a challenging treatment. The absence of new available target drugs might have resulted in bad 1st line response and a high relapse rate.

A Case Report of Hashimotos Thyroiditis

Background: Hashimoto?s thyroiditis is a common lifelong autoimmune disease of the thyroid gland. Case Presentation: A 63-year-old woman was clinically diagnosed as non toxic goiter. Thyroidectomy was done and specimen was sent for histopathological examination. A brownish and soft mass was received with size 6x5x4cm. Microscopic examination showed that proliferation of lymphoid follicles with germinal center of lymphoplasmacytic cells between acini. Some of acini were destroyed and atrophic, while some of them formed microfollicular structures. Acini were lined by simple cuboid epithelium with round oval nuclei, fine chromatin, and eosinophilic cytoplasm. Most of acini contained colloid inside lumen. Lying in between acini were found oncocytic cells with round nuclei, some with prominent nucleoli, fine chromatin, abundant and granular cytoplasm. Stroma consisted of fibrous connective tissue. Conclusion: Histopathological examination yielded the diagnosis of Hashimoto?s thyroiditis.

Waldenström Macroglobulinemia: A Clinicopathological Profile and Review of Six Cases

AbstractWaldenström macroglobulinemia (WM) is an indolent B-cell neoplasm in which lymphoplasmacytic cells are accumulated in the bone marrow. This rare disease is incurable with the available treatments and its management is generally based on risk-adapted methods. We present here six cases of WM with their clinicopathological profile and response to therapy. This is a retrospective observational study conducted in a tertiary referral center in Eastern India from October 2014 to December 2016. The clinical manifestations, diagnostic challenges, and response to therapy for WM were analyzed. A total of six cases of WM were analyzed, of which five were male; the mean age was 65 years. The most common presentation was transfusion-dependent anemia, followed by hyperviscosity. All the patients had bone marrow infiltration by lymphoplasmacytoid cells, and serum protein electrophoresis and immunofixation showed immunoglobulin M kappa monoclonal protein. Bortezomib, dexamethasone, and rituximab (BDR) was the most commonly used regimen. Complete and very good partial responses were seen in one patient (16.7%) each and partial response in 3 (50%) patients. There was disease progression to diffuse large B-cell lymphoma in one patient (16.7%). After a 26-month follow-up, four of six patients (66.67%) remained free of disease progression. A very low incidence of WM was reported, and patients had a varied clinicopathological spectrum. A combination of BDR is a promising frontline therapy in the patients of WM.

The BLIMP1-EZH2 nexus in a non-Hodgkin lymphoma.

Waldenström's macroglobulinemia (WM) is a non-Hodgkin lymphoma, resulting in antibody-secreting lymphoplasmacytic cells in the bone marrow and pathologies resulting from high levels of monoclonal immunoglobulin M (IgM) in the blood. Despite the key role for BLIMP1 in plasma cell maturation and antibody secretion, its potential effect on WM cell biology has not yet been explored. Here we provide evidence of a crucial role for BLIMP1 in the survival of cells from WM cell line models and further demonstrate that BLIMP1 is necessary for the expression of the histone methyltransferase EZH2 in both WM and multiple myeloma cell lines. The effect of BLIMP1 on EZH2 levels is post-translational, at least partially through the regulation of proteasomal targeting of EZH2. Chromatin immunoprecipitation analysis and transcriptome profiling suggest that the two factors co-operate in regulating genes involved in cancer cell immune evasion. Co-cultures of natural killer cells and cells from a WM cell line further suggest that both factors participate in immune evasion by promoting escape from natural killer cell-mediated cytotoxicity. Together, the interplay of BLIMP1 and EZH2 plays a vital role in promoting the survival of WM cell lines, suggesting a role for the two factors in Waldenström's macroglobulinaemia.

P1832IGG4 RELATED DISEASE: NEPHROPATHY AND BONE MARROW FAILURE IN A 2 YEAR-OLD CHILD

Abstract Background and Aims IgG4 related disease (IgG4 RD) is a recently recognized systemic immune-mediated disorder. Pathophysiology of the disease is still unclear. It is characterized by fibro-inflammatory damage of the tissues, IgG-4 positive plasma-cells and often by elevated serum IgG4. This systemic disease can potentially affect every organ. Renal involvement can include tubulo-interstitial nephritis (TIN), membranous glomerulopathy (MGP) and retroperitoneal fibrosis. The epidemiology is still poorly described, but the disease seems more frequent in men over 50 years of age. Only few cases of IgG4 RD are reported in pediatric patients. Method We describe the first case of IgG4 related kidney disease (IgG4 RKD) in a child with concomitant bone marrow failure. Results M.C., a 2 year-old child, was diagnosed in 2017 with a trilinear bone marrow failure (HB 7.2 g/dL; PLT 6.000/mmc; PMC 923/mmc). Bone marrow biopsy showed poor and dyshomogeneous cellularity (20%) and lymphoplasmacytic infiltrate organized in two follicular structures. All investigations performed to rule out inherited bone marrow failure were negative (DEB test, telomere lenght measurement, c-Mpl mutation analysis, mithocondrial DNA analysis) IgG subclass analysis showed elevated serum levels of IgG4 subclass (353 mg/dL- normal level < 120 mg/dL). During the hospitalization, kidney failure with tubular acidosis was also found (creatinine 1.3 mg/dL, eGFR 28.5 mL/min/1.73 mq, bicarbonate 8.3 mEq/L). Urine analysis showed microematuria, proteinuria (1.07 g/L) and granular casts. Renal ultrasound did not demonstrate abnormal findings. A renal biopsy was performed and a kidney sample with up to 40 glomeruli was obtained. Light microscopy (H&E, JSM, PAS and Masson’s trichrome stains) showed tubule-interstitial inflammatory infiltrate (mainly composed of lymphoplasmacytic cells), irregular thickening of the glomerular basement membranes. IHC stains for C4D showed subepithelial deposits (++). IF was negative for IgA, IgM, C4 and C1q while showed subepithelial glomerular membrane IgG deposits (+++) with granular pattern and tubular wall deposits; glomerular deposits (+) and focal tubular deposits (+++) of C3. A diagnosis of MGP associated with TIN was made. IHC staining for IgG4 was also performed, which demonstrated plasmacells with overlapping positivity for IgG and IgG4. After the diagnosis of IgG4 RKD, an immune-suppressive therapy with steroids (1 mg/kg/die)– targeting both the haematological disorder and glomerulopathy - was started, without clinical response. Thereafter the patient underwent bone marrow transplant from HLA-identical family donor. Conclusion GMP indeed is a very uncommon disease in childhood. Moreover, the association of GMP with TIN is reported in few cases in the literature. Only in the last decade some case of TIN and GMP have been recognized as IgG4 RD and few cases have been reported in adults of IgG4 RKD with simultaneous TIN and GMP. To exclude primary GMP we will perform also the research of antiPLA2r1 antibodies in kidney biopsy. However, the specificity of antiPLA2r in children is lower than in adults and the association of primary GMP with IgG4 TIN is unlikely. IgG4 RD is an emerging systemic disease and it should be taken into account in differential diagnosis in systemic auto-immune diseases, also in the pediatric age. IgG4 RKD and bone marrow failure IgG4 RD are very rare in children but its real incidence among pediatric patients could be still underestimated.

Lymphoepithelial Carcinoma of Salivary Gland EBV-association in Endemic versus Non-Endemic Patients: A Report of 16 Cases.

Lymphoepithelial carcinoma of salivary glands (LECSG) are rare neoplasms, reported in endemic populations (southeastern Chinese) with a strong Epstein-Barr virus (EBV) association. A retrospective series comparing EBV status within an ethnically diverse population (endemic vs. non-endemic patients) has not been reported. Sixteen LECSG were equally distributed between males (n = 8) and females (n = 8) with a median age of 54years (range 18 to 85years) at initial diagnosis. Ten patients were white, 4 Asian, and 2 black. The patients typically presented with swelling or mass for an average of 11.6months. Tumors affected only major salivary glands: parotid (n = 13); submandibular (n = 3). Tumors were an average of 2.9cm (range 1.5 to 5.8cm). Nine of 16 (56%) patients had cervical lymph node metastases at presentation. No patients had nasopharyngeal or oropharyngeal tumors. Microscopically, the tumors were widely infiltrative, characterized by large polygonal to spindled cells arranged in a syncytial, lattice-like network in a background of lymphoplasmacytic cells. The neoplastic cells showed an open-vesicular nuclear chromatin to a more basaloid-morphology, the latter showing hyperchromatic nuclei and less cytoplasm, whilenearly all of the cases hadassociated lymphoepithelial lesions/sialadenitis. By in situ hybridization, 8 of 16 cases had a strong, diffuse EBER expression (4 of 4 Asians; 4 of 12 non-Asians), while with immunohistochemistry all cases tested were pan-cytokeratin, CK5/6 and p63 reactive; none of the cases tested were p16 reactive. All patients were managed with wide or radical excision, 4 with concurrent chemoradiation, and 6 with radiation alone. Distant metastasis (lung, brain, and bone) developed in 2 patients. Overall follow-up (mean 3.8years) revealed 12 patients alive and 2 dead, none with evidence of disease (mean 4.3years); one white male alive with disease at 1.9years, and one Asian female dead of disease at 4.2years; both of theselatter patients had Group IV stage disease. High stage (GroupIV) patientshad a shorter mean survival than lower stage patients: 3.1 versus 4.8years, respectively. In conclusion,LECSG are uncommon primary neoplasms. Concurrent lymphoepithelial lesions may help suggest a primary tumor. The tumors, irrespective of race or ethnicity, may express EBER. There is an overall good survival, perhaps better for EBV-negative patients and for those with lower stage disease.