B-cell Lymphoproliferative Disorders Research Articles

Modified Risk-Stratified Sequential Treatment in B-Cell Post-Transplant Lymphoproliferative Disorder (PTLD) after Solid Organ Transplantation (SOT): The Prospective Multicenter Phase II PTLD-2 Trial

Modified Risk-Stratified Sequential Treatment in B-Cell Post-Transplant Lymphoproliferative Disorder (PTLD) after Solid Organ Transplantation (SOT): The Prospective Multicenter Phase II PTLD-2 Trial

Frontline Therapy with Bendamustine-Rituximab (BR) in Patients with Treatment-Naïve Waldenstrom's Macroglobulinemia Confers Similar Long-Term Outcomes to R-CVP in a Real-World Setting: A Population-Based Analysis

Frontline Therapy with Bendamustine-Rituximab (BR) in Patients with Treatment-Naïve Waldenstrom's Macroglobulinemia Confers Similar Long-Term Outcomes to R-CVP in a Real-World Setting: A Population-Based Analysis

Preliminary Clinical Response Data from a Phase 1b Study of Mavorixafor in Combination with Ibrutinib in Patients with Waldenström's Macroglobulinemia with MYD88 and CXCR4 Mutations

Preliminary Clinical Response Data from a Phase 1b Study of Mavorixafor in Combination with Ibrutinib in Patients with Waldenström's Macroglobulinemia with MYD88 and CXCR4 Mutations

P04 Primary mediastinal large B-cell lymphoma in a Caucasian female with anti-SAE-1 positive dermatomyositis

Abstract Case report - Introduction This is a case of a female patient who was diagnosed with dermatomyositis with anti-Small ubiquitin-like modifier Activating Enzyme-1 (SAE-1) and anti-Ro52 autoantibodies. She had extensive skin involvement with mild extracutaneous features and responded well to a combination of prednisolone, hydroxychloroquine and azathioprine. She subsequently developed primary mediastinal large B-cell lymphoma about 3 years after diagnosis of dermatomyositis. Case report - Case description A 59-year-old Caucasian female presented in January 2018 to the dermatologist with a 6-month history of itchy, dry, flaky skin around her eyes with periorbital erythema which later progressed to fixed periorbital oedema. At that point, a diagnostic biopsy was taken from the left lower eyelid which showed a deep inflammatory infiltrate with significant oedema and dilated blood vessels. A few weeks later, she developed a widespread erythematous rash. Other than some cramps and myalgia in the thighs, there was no muscle weakness or dysphagia. There were no red flag symptoms, but some dry cough without dyspnoea. Clinical examination revealed periungual erythema, periorbital erythema and oedema, widespread erythematous eruption on the torso and limbs and papular eruptions on the hand dorsum. Muscle power in upper and lower limbs scored 5 in the MRC scale. Blood tests showed slight lymphopaenia, antinuclear antibodies titre 1:400, dsDNA and ENA negative, raised IgG kappa monoclonal protein at 10g/L with normal kappa/lambda ratio, creatine kinase initially 93 that later rose to 209, slightly raised lactate dehydrogenase 262, otherwise unremarkable biochemistry and inflammatory markers. Extended myositis screen with immunoblot revealed positive SAE-1 and anti-Ro52 autoantibodies. MRI of the femurs showed mild oedema in the adductors. Electromyogram showed some myopathic motor units, not florid but convincingly present. Computed tomography of neck, thorax, abdomen and pelvis did not reveal any underlying malignancy at the time. The diagnosis of SAE-1-positive dermatomyositis was made and she was treated with high-dose prednisolone (1mg/kg), azathioprine and hydroxychloroquine with good response and dermatomyositis went into remission. In December 2020, she developed dyspnoea and was found to have left-sided pleural effusion and anterior mediastinal mass, a biopsy of which confirmed primary mediastinal large B-cell lymphoma. She is currently receiving chemotherapy with rituximab-cyclophosphamide-doxorubicin-vincristine-prednisolone and azathioprine was discontinued. Her dermatomyositis remains in remission. Case report - Discussion The SAE-1 autoantibody was first identified in 2007. Its prevalence ranges from 1% to 8% in different cohorts. The main clinical feature of anti-SAE-1-positive disease tends to be a widespread cutaneous involvement which is typically pruritic, either amyopathic or associated with mild muscle involvement. Other extracutaneous manifestations include arthralgia, dysphagia and interstitial lung disease (ILD) with variable frequency in different cohorts. ILD is usually milder than in other amyopathic dermatomyositis subgroups. Our patient presented with rather extensive cutaneous disease (clinical photography available) requiring high-dose prednisolone and we decided to add azathioprine as a steroid-sparing agent. She had minimal muscle disease and no other extracutaneous manifestations. Interestingly, she developed lymphoma 3 years following the diagnosis of dermatomyositis. The association between dermatomyositis and malignancy is well-established. The generally accepted definition of cancer-associated myositis is malignancy within 3 years of disease onset. Dermatomyositis as opposed to polymyositis, increasing age, male sex, dysphagia, cutaneous ulceration and the presence of anti-transcription intermediary factor-1 gamma antibodies (TIF1γ) were all associated with increased cancer risk in a recent meta-analysis by Oldroyd et al. Anti-SAE-1 is variably reported to be associated with cancers. A recently published study of a North American cohort of anti-SAE-1-positive dermatomyositis patients reported five cases of cancers, mainly internal malignancies and one case of B-cell lymphoproliferative disorder. We are not aware of any more reported cases of lymphoma in patients with anti-SAE-1 autoantibodies. A 2018 study did report two cases of dermatomyositis patients who developed lymphoma without specifying the autoantibody profile in those patients. Our case contributes to the fact that dermatomyositis can be associated with malignancies. It also raises awareness that this rare subgroup of dermatomyositis with SAE-1 autoantibodies can be associated with lymphoma. However, this association has not been widely reported in the literature. Case report - Key learning points

CAR-T after Stem Cell Transplantation in B-Cell Lymphoproliferative Disorders: Are They Really Autologous or Allogenic Cell Therapies?

Simple SummaryB-cell malignancies, like leukemias and lymphomas, are neoplasms that emerge from the malignant proliferation of B cells. Hematopoietic stem cell transplant (HSCT) is an effective medical treatment for these malignancies, but unfortunately, relapse of the disease after HSCT remains a challenge and is associated with poor long-term survival. A cell-based immunotherapy, the chimeric antigen receptor (CAR) T-cells, has proven to improve the clinical outcome of relapsed/refractory HSCT B-cell lymphoproliferative disorders patients in clinical trials. Even though results are promising, in this review, we discuss about the importance to determine T cell chimerism in HSCT patients, the origin of the manufactured CAR T-cells (autologous vs. allogenic) and the future perspective of the CAR-T cells in transplanted patients.Allogenic hematopoietic stem cell transplantation (allo-HSCT) is one of the standard treatments for B-cell lymphoproliferative disorders; however, deep relapses are common after an allo-HSCT, and it is associated with poor prognosis. A successful approach to overcome these relapses is to exploit the body’s own immune system with chimeric antigen receptor (CAR) T-cells. These two approaches are potentially combinatorial for treating R/R B-cell lymphoproliferative disorders. Several clinical trials have described different scenarios in which allo-HSCT and CAR-T are successively combined. Further, for all transplanted patients, assessment of chimerism is important to evaluate the engraftment success. Nonetheless, for those patients who previously received an allo-HSCT there is no monitorization of chimerism before manufacturing CAR T-cells. In this review, we focus on allo-HSCT and CAR-T treatments and the different sources of T-cells for manufacturing CAR T-cells.

Clinicopathologic Analysis of Primary Adrenal Diffuse Large B-Cell Lymphoma: A Reappraisal of 23 Japanese Patients Based on EBV Association and PD-L1 Expression in Tumor Cells.

Primary adrenal diffuse large B-cell lymphoma (PA-DLBCL) is rare. We investigate 23 Japanese patients with PA-DLBCL to understand the clinicopathologic features and biological behavior of this disease. The 17 males and 6 females had a median age of 74 years (range: 40 to 86 y). Tumor cells harbored Epstein-Barr virus-encoded small RNA (EBER) in 9 (39%) samples, including samples from the 2 patients with methotrexate-associated B-cell lymphoproliferative disorder. Programmed cell death ligand 1 (PD-L1) expression was detected in tumor cells of 6 (26%) samples, including 1 EBER+ and 5 EBER- samples. Four (17%) patients exhibited an intravascular proliferating pattern, and all 4 patient samples showed positive staining for PD-L1 in tumor cells. Among those patients, 3 showed intravascular proliferating pattern accompanied by a diffuse extravascular proliferation of tumor cells, and 1 patient was diagnosed with intravascular large B-cell lymphoma. We divided the 23 patients into 3 groups: EBER+ (n=9, 39%), EBER-PD-L1+ (n=5, 22%), and EBER-PD-L1- (n=9, 39%). A comparison of the outcomes among the 3 groups showed significant differences in overall survival (P=0.034). The EBER+ group had the worst prognosis, and the EBER-PD-L1- group had the best prognosis. We also compared the outcomes among the 3 groups that received rituximab-containing chemotherapies. Both the overall survival and progression-free survival were significantly different among these groups (P<0.001 and P=0.002, respectively). In conclusion, we evaluated 3 types of PA-DLBCL and found that each had unique clinical, pathologic, and prognostic features. Our results suggested that immune senescence, iatrogenic immunodeficiency, and immune evasion contribute to the development of PA-DLBCL.

Programmed cell death-ligand 1 (PD-L1)+ tumour cells and low-reacting programmed cell death 1 (PD1)+ tumour-infiltrating lymphocytes predict poor prognosis in Epstein-Barr virus+ diffuse large B-cell lymphoma.

Epstein-Barr virus (EBV)+ diffuse large B-cell lymphoma (DLBCL) has specific tumour cell characteristics, and these patients have worse outcomes than EBV-negative DLBCL patients. We compared 38 EBV+ DLBCL patients with 43 methotrexate-associated EBV+ B-cell lymphoproliferative disorders (MTX+/EBV+ BLPDs) and 30 non-germinal centre (GC) subtype DLBCL. Lymphoma cells of the EBV+ DLBCL group were positive for BCL2 in 17 patients (44.7%), CMYC in 23 patients (60.5%), and p53 in 33 patients (86.8%), which was significantly higher than in the MTX+/EBV+ BLPD group (P < 0.05), and were positive for CD30 in 29 patients (76.3%), compared with two in non-GC subtype DLBCL (6.7%) (P < 0.0001). Significantly more EBV+ DLBCL patients (n = 16, 42.1%) had programmed cell death-ligand 1 (PD-L1)+ tumour cells than patients with non-GC subtype DLBCL (n = 5, 16.7%; P = 0.024), and PD-L1+ tumour cells were more common in advanced stages than in early stages (P = 0.048). Twenty-five EBV+ DLBCL patients (69.4%) had few reactive PD1+ tumour-infiltrating lymphocytes (TILs), compared with 12 patients with MTX+/EBV+ BLPDs (37.5%) (P = 0.008). In the EBV+ DLBCL group, CD30, BCL2, CMYC, and p53 expression was not related to patient prognosis. Poor outcomes were associated with PD-L1+ tumour cells (P = 0.001) and low-reacting PD1+ TILs (P = 0.02), while their combination conferred a worse outcome (P < 0.0001). Immune evasion by PD-L1+ tumour cells and exhaustion of PD1+ TILs may occur in EBV+ DLBCL patients, and PD-L1/PD1 interactions may influence tumour progression and poor prognosis.

Rituximab-Associated Flare of Cryoglobulinemic Vasculitis

BackgroundPatients with cryoglobulinemic vasculitis (CV) can develop disease flare after rituximab administration. The objective of our study was to describe the prevalence, clinical characteristics, predisposing factors, and outcomes of patients with rituximab-associated flare of CV.MethodsWe conducted a retrospective study in a tertiary referral center until March 25, 2020.ResultsAmong 64 patients with CV who received rituximab therapy in our center, 14 (22%) developed disease flare. Median age was 67.5 years. Seven patients (50%) had type II CV and the other half had either type I (n = 6) or type III (n = 1). Twelve patients (86%) had an underlying B-cell lymphoproliferative disorder as the cause of their CV. CV flare occurred after a median time of 5.5 days (range: 2–8 days). The organ systems most involved were the skin (n = 10), kidneys (n = 5), and peripheral nerves (n = 3). Five patients (36%) developed acute kidney injury (AKI), 3 of whom presented with nephritic syndrome secondary to biopsy-proven membranoproliferative glomerulonephritis. Treatment was directed against the underlying disease in addition to supportive care. Patients who developed flare were more likely to have B-cell lymphoproliferative disorder as the underlying etiology of their CV (P = 0.03). Eight patients (57%) died after a median time of 27 months.ConclusionsRituximab-associated flare can occur in all types of CV, tends to arise approximately 2 days and less than 1 week after rituximab administration, and is more likely to happen in patients with an underlying B-cell lymphoproliferative disorder. It does not indicate treatment failure, and rituximab should not be abandoned altogether. AKI is a common manifestation, and mortality rate at 2 years is high.

MM-191: Association between IL17A Polymorphism and Clinicopathological Parameters in Egyptian Multiple Myeloma Patients

<h3>Context</h3> Multiple myeloma (MM) is a B-cell lymphoproliferative disorder in which the bone marrow (BM) microenvironment plays a key role in pathogenesis. T-helper 17 cells present within the BM microenvironment and secrete the pro-inflammatory cytokine interleukin-17 (IL-17). Single nucleotide polymorphisms (SNPs) in IL-17 (<i>IL17A</i>) are found to be involved in the pathogenesis of many cancers and autoimmune diseases. <h3>Objective</h3> To investigate the role of <i>IL17A</i> SNPs in MM pathogenesis and their impact on clinical features of MM. <h3>Design</h3> A prospective study conducted on 77 MM patients and healthy controls of matched age and gender. <h3>Setting</h3> The study was carried out in the Oncology Center, Mansoura University. <h3>Patients</h3> Seventy-seven patients with MM (female; 46.8%, male; 53.2%) with mean age of 54.6 years. <h3>Interventions</h3> We obtained genomic DNA from 77 patients with MM and healthy controls and detected <i>IL17A-197</i> G/A (rs2275913) genotypes using the polymerase chain reaction-restriction fragment length polymorphism method. <h3>Main Outcome Measures</h3> The significance of <i>IL17A</i> polymorphism in relation to clinical data, laboratory data, and survival outcome. <h3>Results</h3> The <i>IL17A</i> polymorphism was not involved in susceptibility to MM in the Egyptian population, and there was no statistically significant association with gender (p=0.14), hypercalcemia (p=0.28), hypoalbuminemia (p=0.49), renal impairment (p=0.13), elevated LDH (p =0.62), osteolytic bone lesions (p=0.26), pathological fracture (p=0.96), plasmacytoma (p=0.42), and ISS stage III (p=0.26). <i>IL17A</i> polymorphism showed no statistically significant differences in OS (p=0.83) of MM patients, while there was a statistically significant worse OS among patients with thrombocytopenia (p=0.006), renal impairment (p=0.047), and ISS stage III (p=0.02). <h3>Conclusion</h3> In this study, <i>IL17A</i> polymorphism was not associated with susceptibility to MM and did not influence its severity. In addition, OS survival in MM patients was not affected by <i>IL17A</i> polymorphism.

IBCL-114: Clinical and Biological Prognostic Factors in Follicular Lymphoma Patients During Treatment

Introduction Follicular lymphoma (FL) is an indolent yet heterogeneous B-cell lymphoproliferative disorder. Most people respond to treatment well. However, a particular group of patients has a poor prognosis, and these patients are difficult to define. Patients and Methods We retrospectively analyzed 143 FL patients treated at the University of Debrecen since 2009. We investigated the prognostic factors that may influence the survival of FL patients. Results We found a standardized uptake value (SUV)max cut-off of 9.85 at the staging PET/CT to significantly separate FL patients’ progression-free survival (PFS) (p=0.0001, HR: 0.2535, 95%CI: 0.1118-0.4878). Lymphocyte/monocyte (Ly/Mo) ratio of 3.41 drawn at diagnosis also significantly predicted PFS (p=0.0027, HR: 2.997, 95% CI: 1.463-6.142). Combining patients with staging SUVmax >9.85 and Ly/Mo 9.85 and interim SUVmax >3.15, a high-risk group of FL patients can be identified (p Discussion Biological prognostic factors, such as the Ly/Mo ratio, may improve the prognostic assessment of staging PET/CT. Nevertheless, PFS difference is translated into OS when using a combination of staging and interim SUVmax. We consider investigating additional biological prognostic factors while currently highlighting PET/CT’s role in FL.

IBCL-034: Hairy Cell Leukemia: Single-Center Experience

Context Hairy cell leukemia (HCL) is an uncommon, chronic B-cell lymphoproliferative disorder that represents approximately 2% of all leukemias. The lack of large national and international registries on HCL has limited the ability to identify clear clinico-epidemiological characteristics in this rare disease. Objective To evaluate the HCL patients in the Egyptian population with regard to epidemiological, pathological, immunophenotypic, and clinical data, as well as treatment modalities and outcomes. Patients This is a retrospective observational study in which 92 patients diagnosed with HCL were reviewed through electronic health records between January 1, 2008, and December 31, 2019. Results Median age of patients was 50 years. The prevalence of hepatitis C virus (HCV) infection was triple that of the general population and appeared to have no effect on overall survival (OS). Most of the patients had varying degrees of cytopenia, varying from pancytopenia (50%), bicytopenia (43.5%), thrombocytopenia (69.3%), leukopenia (53%), and anemia only (6.5%). Hairy cells in peripheral blood samples were observed in 18.5% of cases. Sixty-one patients received cladribine, five patients received interferon (INF-a), six patients had palliative splenectomies, five patients were subjected to watchful waiting, and 15 patients received supportive therapy. The presence of comorbidities, age > 60 years, fever, pancytopenia, hemoglobin level Conclusions HCL in Egypt is a rare NHL that has different clinicopathological features, especially in association with hepatitis C infection, which necessitates participation in international registries for HCL, e.g., Hairy Cell Leukemia Foundation, to investigate the most optimal treatment strategies for these neoplasms in the real-world population.

EBV-positive B-cell lymphomas and lymphoproliferative disorders: Review from the perspective of immune escape and immunodeficiency.

BackgroundEpstein‐Barr virus (EBV) is detected in a variety of B‐cell lymphomas (BCLs) and B‐cell lymphoproliferative disorders (B‐LPDs). Immunodeficiency has been considered to play a key role in the pathogenesis of these diseases. In addition, immune escape of tumor cells may also contribute to the development of EBV+ BCLs and B‐LPDs. The PD‐1/PD‐L1 pathway is particularly important for immune escape of tumor cells that contribute to development of lymphoma through suppression of cytotoxic T‐cell function. We now consider PD‐L1 immunohistochemistry (IHC) a very useful method for predicting whether tumor cells of lymphoid malignancies are characterized by the immune escape mechanism.MethodsWe reviewed articles of EBV+ BCLs and B‐LPDs from the perspective of immune escape and immunodeficiency, particularly focusing on PD‐L1 IHC.ResultsBased on PD‐L1 IHC, we consider that EBV+ BCL and B‐LPD can be classified into three types: “immunodeficiency”, “immune escape”, and “immunodeficiency + immune escape” type. The immunodeficiency type includes EBV+ diffuse large BCL (DLBCL) of the elderly, EBV+ sporadic Burkitt lymphoma, EBV+ mucocutaneous ulcer, and methotrexate (MTX)‐associated B‐LPD. The immune escape type includes EBV+ classic Hodgkin lymphoma (CHL) and EBV+ DLBCL of the young. The immunodeficiency + immune escape type includes CHL type MTX‐associated LPD and a minor subset of EBV+ DLBCL of the elderly.ConclusionsRecently, good results have been reported for immune check‐point inhibitors in treating lymphoma. Lymphomas and LPDs characterized by immune escape are regarded as good candidates for PD1/PD‐L1 blockade therapy. Therefore, from both the clinical and pathological perspective, we suggest that lymphoma diagnosis should be made considering immune escape and immunodeficiency.

Epstein-Barr Virus Positive B-Cell Lymphoproliferative Disorder of the Gastrointestinal Tract.

Simple SummaryEpstein-Barr virus (EBV) contributes to the reactive and neoplastic lymphoid proliferation of B-, T-, and NK-cell lineages, which represent a vast clinicopathological spectrum ranging from indolent, self-limited disease to aggressive lymphomas. EBV-positive B-cell lymphoproliferative disorder (EBV+ B-LPD) is the most common of these diseases, accounting for 3% to 15% of diffuse large B-cell lymphomas. The spectrum of EBV+ B-LPD is expanding with advances in understanding immunosenescence and iatrogenic immunodeficiency in the era of immune-oncology. We review EBV+ B-LPD affecting the gastrointestinal tract with a focus on the PD-L1 expression in tumor and non-malignant immune cells to better understand this peculiar disease.Epstein-Barr virus positive B-cell lymphoproliferative disorder (EBV+ B-LPD) encompasses a broad clinicopathological spectrum and distinct clinical behavior that relatively favors the gastrointestinal (GI) tract. In this review, we provide an update on the clinicopathological features and biological behavior of EBV-positive mucocutaneous ulcer (EBVMCU) and primary EBV+ diffuse large B-cell lymphoma (DLBCL) of the GI tract. EBVMCU is a newly recognized entity but well known as an indolent and self-limited EBV+ B-LPD occurring in various immunodeficiencies. In contrast, EBV+ DLBCL constitutes the largest group of EBV+ B-LPDs and is regarded as an aggressive neoplasm. These two distinct diseases have historically been distinguished in the reappraisal of age-related EBV-associated B-LPDs but are challenging in routine practice regarding their differential diagnostic and therapeutic approaches. An increasing number of reports indicate that they are epidemiologically prevalent beyond western and eastern countries, but their comprehensive analysis is still limited. We also describe the PD-L1 positivity of tumorous large cells and non-malignant immune cells, which is relevant for the prognostic delineation among patients with primary DLBCL of the GI tract with and without EBV on tumor cells.

Issue Highlights-July 2021.

Issue Highlights-July 2021.

CROCIN PROMOTES APOPTOSIS IN HUMAN EBV-TRANSFORMED B-LYMPHOCYTE VIA INTRINSIC PATHWAY

BackgroundAs a major carotenoid in saffron, crocin demonstrates potent anti-cancer impacts. However, its anti-lymphoma effects remain vague, especially in the human EBV-associated B-cell lymphoproliferative disorders. This study examined crocin’s apoptogenic potential and its underlying mechanism in CO 88BV59-1 cell line vs. normal human peripheral blood B cells.MethodsCO 88BV59-1 cells were treated with crocin alone or in combination with vincristine for up to 72 h. The cell viability was examined using a resazurin assay. Flow cytometry using annexin V and propidium iodide labeling was performed to detect apoptotic cells. Also, the expression levels of genes and proteins involved in apoptosis (CASP3, CASP8, CASP9, P53, Bax, and Bcl-2) were respectively determined via real-time PCR and Western blot analysis.ResultsCrocin concentration-dependently reduced cell viability in CO 88BV59-1 cells with no significant toxicity toward normal B cells. Similar to vincristine, crocin significantly increased apoptosis in these cells during 72 h of incubation. Furthermore, the combination of crocin (80 μM) and vincristine (1 μM) enhanced apoptosis in CO 88BV59-1 cells. Therefore, this synergistic effect was detected in human EBV-transformed B-lymphocyte. CASP3, CASP9, P53, and Bax/Bcl-2 ratio expressions were significantly raised in CO 88BV59-1 cells, whereas CASP8 was unaltered. It was proposed that crocin promoted apoptosis in CO 88BV59-1 cells in a time- and concentration-dependent manner via the induction of the intrinsic pathway.ConclusionThe results suggest that crocin may serve as a good alternative/coadjuvant to vincristine in EBV-associated B-cell lymphoproliferative disorders.

Somatic Variants Associated with MDS/AML Are Present in MYD88 Mutated Waldenstrom's Macroglobulinemia

Background: The Rapid Heme Panel (RHP) is a targeted amplicon-based custom next-generation sequencing used as a clinical assay to identify mutations in 95 genes related to hematological malignancies that include acute leukemias (AML, ALL), myelodyplastic syndromes (MDS), and B-cell lymphoproliferative disorders. Clonal hematopoiesis of indeterminate potential (CHIP) has recently been shown to be an age-related precursor to MDS when blast or dysplasia criteria are not met for a formal MDS diagnosis. The RHP is used to identify the top somatic mutated genes closely associated with CHIP or MDS, and includes MYD88 and CXCR4 variant testing for Waldenstrom's Macroglobulinemia (WM). Materials and Methods: Sample processing and sequencing of bone marrow (BM) samples from WM patients was used for isolation of genomic DNA for RHP. Amplicons were designed to cover 250bp and 95 genes. Analyses were performed by identifying and separating somatic variants from those seen as germline. Variants with read percentage depth of 50% were presumed to be of germline origin. Variants with read percentages greater than the mutated MYD88 read percentage depth were identified as potential CHIP or MDS clonal variants. These variants were validated using Sanger sequencing using CD19-selected and CD19-depleted BM mononuclear cells. Mutant variants were annotated against Ensembl Variant Effect Predictor (VEP) database. Results: Three-hundred thirty-six WM patients were included in this analysis. The median age for these patients was 66.7 (range 31-93 years). The median BM disease involvement for these patients was 35%. RHP identified the MYD88 L265P mutation in 267 (80%) patients. The remaining patients were either unmutated (n=34), or lacked MYD88 reads (n=35). Therefore the percentage of patients with MYD88 L265P among those with MYD88 reads was 88.7%. CXCR4 C-terminal domain mutations were also observed in 55/267 (20.6%) WM patients with MYD88 L265P mutations. In addition to MYD88 and CXCR4, variants in 66 other genes contained within the RHP profile were identified that included variants in genes associated with MDS/AML in 9 (3.3%) patients. The median age for these patients was 64 (range 44-80 years) and included variants at: EP300S1400N, a histone acetyltransferase regulating transcription (n=1); TET2T1270fs, a methylcytosine dioxygenase that converts methylcytosine to 5-hydroxymethylcytosine (n=1); and DNMT3AV338fs, DNMT3AY584fs, DNMT3AG587fs, and DNMT3AL754R, a DNA methyltransferase that represses transcription (n=4). Variants in DNMT3BR97C, DNMT3BE39Q, and DNMT3BA384T (n=3). Sanger sequencing of BM mononuclear cells confirmed the somatic, non-CD19 origin for DNMT3A and TET2. Four of 9 patients were previously untreated, and two received alkylator based therapy previously. None exhibited BM morphological evidence of MDS or AML. Conclusions: Infrequently variants related to MDS/AML are present in BM of patients with WM, including previously untreated patients. These variants occur in the absence of BM morphological evidence of MDS, and may be consistent with CHIP. DisclosuresTreon:Pharmacyclics: Consultancy, Research Funding.

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.

Short Course of Bortezomib in Anemic Patients with Refractory or Relapsed Cold Agglutinin Disease. a Phase II Prospective Study By the Gimema Group

Background: Cold agglutinin disease (CAD) is a chronic hemolytic disorder caused by the presence of an IgM autoantibody with specificity towards red cell antigens. The autoantibody is produced by a B-cell clone, which can often be detectable with standard diagnostic work-up. Some patients with CAD require transfusions to compensate for the chronic red cell destruction. There are few therapeutic modalities of proven efficacy which include alkylating agents, rituximab alone or in combination with fludarabine or bendamustine. Bortezomib is a proteasome inhibitor which has been successfully used in Ig-producing diseases, like amyloidosis and Waldenstrom macroglobulinemia (WM). Protein metabolism is a tightly regulated process, and inhibition of its turnover using proteasome inhibitors may lead to apoptosis in protein secreting B-cell malignancies, including multiple myeloma (MM) and WM. Aim: We have conducted a prospective multicenter, phase II, open label study (ClinicalTrials.gov NCT01696474) to evaluate the potential efficacy of a short course of bortezomib in anemic patients with CAD requiring transfusion or with a hemoglobin concentration below 100 g/L determined at least monthly during the two months before entering the trial. Patients and Methods: Patients had to be refractory to at least one previous treatment. The primary endpoint was the achievement of transfusion independence or a significant rise (>20 g/L) of hemoglobin concentration. A single course of bortezomib was given at the dose of 1,3 mg/sqm iv on days 1, 4, 8, 11. Safety evaluation, duration of transfusion independence, and the effects of treatment on the underlying clonal B-cell disorder were secondary objectives of the trial. Patients with a concomitant lymphoproliferative disorder requiring specific treatment for reasons other than CAD or with preexisting peripheral neuropathy were excluded. Results: Between 12-04-2012 and 03-14-2016, 21 patients have been enrolled by 6 Gimema italian centers. Their characteristics were as follows: median age 70 years (range 53 to 85); M/F ratio: 1/2; 6 patients had concomitant or previous clonal B-cell disorder (3 CLL, 3 B-cell lymphoma) not requiring specific therapy. Previous treatments included corticosteroids, rituximab, cyclophosphamide, azathioprine or combinations of them. Median Hb concentration at study entry was 87 g/dL (range 78-102). Ten patients were transfusion-dependent. Treatment response was evaluated at three months. Two patients stopped treatment early, one at day 4 because of pulmonary embolism and the other at day 8 because of headache, respectively, and were excluded from efficacy analysis. One of them actually achieved transfusion independence in spite of treatment stop. Of 19 evaluable patients, 3 achieved complete remission, defined as normalization of hemoglobin levels and absence of symptoms or signs of hemolysis and 3 partial remission, defined as the achievement of transfusion independence or of a stable increase of > 20g/L of hemoglobin concentration in untransfused patients, for an overall response rate of 31,6%. Four of the 6 responding patients achieved transfusion independence. The small number of patients studied precluded an analysis of factors associated with response. Five grade 3 adverse events, including headache, diarrhea, increased bilirubin levels, anemia and upper respiratory tract infection (URTI) in one patient each, and one grade 4 pulmonary embolism. They were considered not related to study drug, except for URTI. Four of 6 responding patients maintained the response after 12 months from study entry (66,7%; 95% CI: 37.9-100). All patients were alive at last follow up except for one who died of septic shock during off treatment follow up, 10 months after study entry. The effects of bortezomib on underlying B-cell lymphoproliferative disorder are being evaluated. Conclusions: The data demonstrate that a short course of bortezomib may be effective in a subset of patients with symptomatic CAD failing previous treatments, with acceptable toxicity. Responses seem to be long-lasting in the majority of responding patients. These data provide a rational for further investigating the potential benefits of bortezomib either by using a more prolonged treatment schedule or in combination with other active agents. The study has been supported by JANSSEN-CILAG Spa which provided bortezomib and a grant for study monitoring. DisclosuresRossi:Gilead: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Teva: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees. Zaja:Celgene: Honoraria, Research Funding; Takeda: Honoraria; Novartis: Honoraria, Research Funding; Janssem: Honoraria; Abbvie: Honoraria; Roche: Honoraria, Research Funding; Gilead: Honoraria. Mauro:Janssen: Honoraria; Abbvie: Honoraria; Roche: Honoraria; Gilead: Honoraria. Barcellini:Alexion: Honoraria; Agios: Honoraria, Research Funding; Novartis: Honoraria.

Ascites in A Patient with Hairy Cell Leukaemia and Carcinoma of Breast: A Diagnostic Challenge

Hairy cell leukaemia (HCL) is a rare indolent B-cell lymphoproliferative disorder. We report a diagnostic challenge in detecting the cause of ascites, which is a rare, unique manifestation of HCL. A 72-year-old lady presented with 1-month-history of pain in left upper abdomen and loss of weight. There was hepatomegaly, splenomegaly, bilateral inguinal lymphadenopathy, anaemia, and lymphocytosis. She was diagnosed as HCL, based on morphology, immunophenotyping of peripheral blood and bone marrow biopsy examination. In 2009, she was diagnosed as carcinoma of breast when she presented with a mass in left breast; and she received treatment. For HCL, she received intermittent chemotherapy (Chlorambucil+Prednisolone). Her HCL was stable until 2018 when she presented with recurrent ascites which needed frequent, regular peritoneal paracentesis. Since she had HCL and carcinoma of breast, determining the aetiology of ascites was challenging. Possible causes of her ascites included metastatic carcinoma of breast, HCL, cirrhosis of liver with portal hypertension and peritoneal tuberculosis. Cytology of peritoneal fluid showed mature-looking lymphocytes but no malignant cells. Interestingly, flow cytometry analysis of peritoneal fluid showed the presence of clonal B cell population with lambda light chain restriction. Therefore, it was concluded that her ascites was a manifestation of HCL. A few months later, she succumbed to septicaemia. Impact of ascites on disease course of HCL included rapid disease progression, poor prognosis and shortened survival. We highlight the important role of immunophenotyping in addition to cytomorphology to guide us in confirming the aetiology of ascites in a patient with haematological and solid organ malignancies.

Development of anti-human IgM nanobodies as universal reagents for general immunodiagnostics

Nanobodies are the smallest antibody fragments which bind to antigens with high affinity and specificity. Due to their outstanding physicochemical stability, simplicity and cost-effective production, nanobodies have become powerful agents in therapeutic and diagnostic applications. In this work, the advantages of nanobodies were exploited to develop generic and standardized anti-human IgM reagents for serology and IgM+ B-cell analysis. Selection of anti-IgM nanobodies was carried out by evaluating their yields, stability, binding kinetics and cross-reactivity with other Ig isotypes. High affinity nanobodies were selected with dissociation constants (KDs) in the nM range and high sensitivities for detection of total IgM by ELISA. The nanobodies also proved to be useful for capturing IgM in the serodiagnosis of an acute infection as demonstrated by detection of specific IgM in sera of dengue virus patients. Finally, due to the lack of an Fc region, the selected nanobodies do not require Fc receptor blocking steps, facilitating the immunophenotyping of IgM+ cells by flow cytometry, an important means of diagnosis of immunodeficiencies and B-cell lymphoproliferative disorders. This work describes versatile anti-IgM nanobodies that, due to their recombinant nature and ease of reproduction at low cost, may represent an advantageous alternative to conventional anti-IgM antibodies in research and diagnosis.