Splenic Lymphoma Research Articles

Primary splenic diffuse large B-cell lymphoma (PS-DLBCL) presenting in an asymptomatic patient after sports trauma

Primary splenic diffuse large cell lymphoma is unusual and accounts for 2% of all splenic lymphomas. The symptoms included left upper abdominal pain, indigestion, loss of appetite, generalized weakness, fever, anemia, and thrombocytopenia or elevated C Reactive Protein. The current case report, to our knowledge the first of its kind, presents unusual clinicopathologic findings, where the patient remained asymptomatic until the time of diagnosis but showed extensive splenic involvement with stage IV disease. Alternatively, the morphologic findings in this case were like to those seen in other Primary Splenic Diffuse Large B Cell Lymphoma reported in the literature. In conclusion, the present report emphasizes the need for additional case studies and research on the biology of this unusual subset of lymphoma.

Closing the gap between biology and classification in splenic B-cell lymphomas.

The mature splenic B-cell lymphomas are an enigmatic group of lymphoid neoplasms that have long caused significant difficulty for the practicing pathologist due to overlapping diagnostic features among entities and the decreasing availability of splenic tissue for assessment. While some entities have highly characteristic and specific clinicopathological features (e.g. hairy cell leukaemia), others are substantially more difficult to recognise (e.g. splenic diffuse red pulp lymphoma). At the same time, classification systems have been evolving, resulting in multiple changes to the boundaries among these entities and even the existence of some entities in their own right. Moreover, unbiased multi-omic interrogation (whole genome/transcriptome sequencing, methylome) of the splenic B-cell lymphomas over the past decade has given us significant insights into the underling biology of these neoplasms. We present a clinicopathological perspective on the historical, current and future state of the diagnosis and classification of splenic B-cell lymphomas integrating multi-omic data and highlighting areas of focus for the field in order to continue to strive to improve patient outcomes through accurate diagnosis.

A Remarkable Case Report of Splenic Lymphoma with Villous Lymphocytes in a Male Sudanese Patient

A Remarkable Case Report of Splenic Lymphoma with Villous Lymphocytes in a Male Sudanese Patient

Understanding splenic B-cell lymphoma/leukaemia with prominent nucleoli: Diagnosis, underpinnings for disease classification and future directions.

The 5th edition of the WHO classification of haematolymphoid tumours (WHO-HAEM5) introduced a new category, splenic B-cell lymphoma/leukaemia with prominent nucleoli (SBLPN). The diagnostic entity B-cell prolymphocytic leukaemia (B-PLL) has been discontinued and the category of hairy cell leukaemia variant (HCLv) has been conceptually reframed. B-PLL and HCLv diagnoses were uncommon. Overlap existed between B-PLL and other indolent lymphomas like chronic lymphocytic leukaemia/small lymphocytic lymphoma (CLL/SLL). HCLv lacked consistent cytomorphological, immunophenotypic and genetic features. To address these issues, the WHO-HAEM5 classification has introduced SBLPN to serve as a temporary holding ground for entities that do not neatly fit into the existing classification. Cases previously classified as CD5-negative B-PLL and HCLv fall under the SBLPN category. Some splenic marginal zone lymphoma and splenic diffuse red pulp small B-cell lymphoma cases with higher number of medium or large nucleolated B cells would also be classified as SBLPN under the WHO-HAEM5. This review explores the rationale for discontinuing B-PLL and HCLv diagnoses. It then examines the concept of SBLPN, offers practical guidance for diagnosis and discusses future directions in classifying splenic B-cell lymphomas.

Splenic cyst deroofing complicated with B lymphoma

BackgroundSplenic cysts are uncommon and very rarely malignant therefore their treatment isn’t standardized. In case of symptomatic cysts different surgical approaches have been suggested. Primary malignant lymphoma of the spleen comprises less than 1% of non-Hodgkin’s lymphomas. To our knowledge, only 203 cases of splenic large B-cell lymphoma (LBCL) have been reported to date and only 2 of them were fibrin-associated splenic cysts.Case presentation27-year-old model with a 19 × 13 cm splenic cyst without data of malignancy in the preliminary study and therefore treated with laparoscopic deroofing. After histological diagnosis of LBCL with a fibrin/EBV-associated splenic pseudocyst, the patient received 4 cycles of Rituximab and a laparoscopic splenectomy was performed due to resurgence of the pseudocyst. No evidence of malignancy has been found during follow up (EBV viral load every 3 months during the first year, PET-CT every 6 months during the first year and annual afterwards) performed after the splenectomy.Discussion and conclusionsThe value of tumor markers and radiology for diagnosis of splenic cysts is put into question.Only 60 cases of Fibrin-associated LBCL (FA-LBCL) have been described in the literature therefore there are no treatment guidelines for them even though surgery together with systemic treatment has been the prevalent route with good results in most cases.

Diagnostic ability of [18F]FDG PET/CT for distinguishing benign from malignant spleen lesions.

[18F]Fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT) is a non-invasive imaging modality used in the differential diagnosis of splenic lesions, although ideal parameters and thresholds remain unclear. The present study evaluated the ability of [18F]FDG PET/CT, including its visual and quantitative parameters, to differentiate between benign and malignant splenic lesions. Patients who underwent [18F]FDG PET/CT following the detection of splenic lesions on contrast-enhanced CT were retrospectively analysed. Visual parameters assessed on [18F]FDG PET/CT included whole spleen uptake intensity, lesion multiplicity, and lesion uptake, and quantitative parameters included maximum standardised uptake value (SUVmax), lesion-to-background ratio (LBR), metabolic tumour volume (MTV), total lesion glycolysis (TLG), and lesion size. Parameters differentiating between benign and malignant lesions were evaluated by Pearson's chi-square test, Mann-Whitney U-test, and receiver operating characteristics (ROC) curve analysis. Splenic lesion uptake (p = 0.001) was the only visual parameter significantly distinguishing between benign and malignant lesions. ROC curve analysis demonstrated that SUVmax had the largest area under the ROC, 0.91 (p < 0.001), with an optimal cut-off > 5.3 having a sensitivity of 90.3% and a specificity of 80.6%. Subgroup analysis of malignant lesions showed that SUVmax (p = 0.013), LBR (p = 0.012), and TLG (p = 0.034) were significantly higher in splenic lymphomas than in splenic metastases. Of the [18F]FDG PET/CT parameters investigated, SUVmax had the highest accuracy in diagnosing malignant splenic lesions and was significantly higher in splenic lymphomas than in splenic metastases. Visual determination of [18F]FDG uptake by splenic lesions may be an easily evaluated parameter. SUVmax and visual grade of [18F]FDG PET/CT help to differentiate spleen lesions. [18F]FDG PET/CT is useful for discriminating between benign and malignant spleen lesions. Many splenic lesions are difficult to diagnose on anatomical imaging, with histopathologic analyses are required. SUVmax of PET/CT provided the diagnostic ability to differentiate between benign and malignant splenic lesions. More than normal spleen uptake can be a convenient parameter to diagnose malignant spleen lesions.

Laparoscopic Partial Splenectomy May Be Valuable for the Diagnosis of Malignant Lymphoma: A Case Report.

Diagnosing primary splenic malignant lymphoma (PSML) is challenging due to the non-specific nature of splenomegaly, necessitating splenic biopsy for confirmation. However, performing partial splenic resection for diagnostic purposes is an elective procedure due to the risk of major hemorrhage. Despite the longstanding practice of splenectomy over the past few decades, it remains invasive and may result in severe early or late complications. Hence, we present laparoscopic partial splenectomy (LPS) in a patient suspicious of PSML for diagnostic purposes in this study. An 81-year-old woman presented to our hospital with a one-month history of fever and dry cough. Atypical cells had been detected in her peripheral blood nine months ago. However, at that time, a bone marrow examination did not reveal any atypical cells. The laboratory tests revealed a soluble interleukin receptor-2 levels of 4,667 U/dl and atypical cells were also found in peripheral blood. Abdominal computed tomography showed splenomegaly without any other relevant findings. These findings are suspicious of PSML and LPS without vessel ligation was performed and a small fraction of the spleen from the inferior pole measuring 1.8×1.0 cm was resected. The operation lasted for 63 min with minimal estimated blood loss. Histopathological findings were compatible with the diagnosis of diffuse B-cell lymphoma. The postoperative clinical course was uneventful, and splenomegaly demonstrated improvement six months after the operation. LPS without vessel ligation for biopsy may be valuable for the diagnosis of malignant lymphoma, particularly when there are no swollen lymph nodes, as it offers a less invasive approach.

Gastrosplenic fistula due to splenic lymphoma: two case reports and review of the literature

BackgroundGastrosplenic fistula is a rare and potentially fatal complication of various conditions. Lymphoma is the most common cause. It can occur spontaneously or after chemotherapy. Gastrosplenic fistula diagnosis can be confused with a splenic abscess because of the presence of air into the mass. The computed tomography identification of the fistulous tract is the key to a right diagnosis. Treatment modalities include surgical resection, chemotherapy, or a combination of both.Case presentationHere we report two patients with gastrosplenic fistula due to diffuse large B cell lymphoma. The first patient was a 54-year-old Caucasian woman with an enormous primary splenic diffuse large B cell lymphoma leading to the development of a spontaneous fistula in the stomach. The second patient was a 48-year-old Caucasian male patient with an enormous splenic diffuse large B cell lymphoma complicated by fistula after chemotherapy. Both patients died of septic shock several days after surgery.ConclusionGastrosplenic fistula is a rare complication with a poor-prognosis, for which surgery is currently the preferred treatment.

Hairy cell leukemia 2024: Update on diagnosis, risk-stratification, and treatment-Annual updates in hematological malignancies.

Hairy cell leukemia (HCL) and HCL-like disorders, including HCL variant (HCL-V) and splenic diffuse red pulp lymphoma (SDRPL), are a very heterogenous group of mature lymphoid B-cell disorders characterized by the identification of hairy cells, a specific genetic profile, a different clinical course and the need for appropriate treatment. Diagnosis of HCL is based on morphological evidence of hairy cells, an HCL immunologic score of 3 or 4 based on the CD11c, CD103, CD123, and CD25 expression, the trephine biopsy which makes it possible to specify the degree of tumoral bone marrow infiltration and the presence of BRAFV600E somatic mutation. Progression of patients with HCL is based on a large splenomegaly, leukocytosis, a high number of hairy cells in the peripheral blood, and the immunoglobulin heavy chain variable region gene mutational status. VH4-34 positive HCL cases are associated with a poor prognosis, as well as HCL with TP53 mutations and HCL-V. Patients should be treated only if HCL is symptomatic. Chemotherapy with risk-adapted therapy purine analogs (PNAs) are indicated in first-line HCL patients. The use of chemo-immunotherapy combining cladribine (CDA) and rituximab (R) represents an increasingly used therapeutic approach. Management of relapsed/refractory disease is based on the use of BRAF inhibitors (BRAFi) plus R, MEK inhibitors (MEKi), recombinant immunoconjugates targeting CD22, Bruton tyrosine kinase inhibitors (BTKi), and Bcl-2 inhibitors (Bcl-2i). However, the optimal sequence of the different treatments remains to be determined.

Gastrosplenic fistula due to diffuse large B-cell lymphoma: case report

BackgroundGastrosplenic fistula (GSF) is a rare complication of various benign and malignant etiologies. Splenic lymphoma is the most common cause of GSF. Most cases are due to diffuse large B-cell lymphoma (DLBCL).Case presentationA 39-year-old male patient presented at the emergency department with left upper quadrant abdominal pain. CT scan showed necrotic splenic mass with fistulous tract between the spleen and the gastric cavity. The patient underwent splenectomy and partial gastrectomy. The histopathological diagnosis was DLBCL.ConclusionsGastrosplenic fistula is a rare complication of diffuse large B-cell lymphoma. Radiologists should be aware of it and consider it in the diagnosis.

Splenic Lymphoma: A Series of Seven Cases

Splenic Lymphoma (SL) is a lymphoproliferative disorder characterised by involvement of the spleen, with or without bone marrow involvement. However, advancements in imaging modalities have led to more frequent diagnosis of isolated splenic lesions. There is always a dilemma in diagnosis as to whether the pathology truly originates from the spleen or if it is part of systemic involvement in Non-Hodgkin Lymphoma (NHL). Clinical manifestations vary widely, and treatment outcomes for SL depend on accurate diagnosis. However, prognosis still remains poor in these cases compared to systemic NHL. The present case series provide a comprehensive overview of patients diagnosed with SL, focusing on their clinicopathological characteristics, therapeutic details, prognosis and outcomes. Previously isolated case reports have been published, and there is still a lack of consensus on the management of this entity. The present case series included seven cases over a two-year period, from January 2022 to December 2023. In the current case series, authors have highlighted the heterogeneity and variability in the presentation of SL and how the clinical behaviour ranges from indolent to highly aggressive. Nevertheless, the presenting clinical, laboratory and pathological features of these diseases often display significant overlaps and pose confusion in arriving at an exact diagnosis. The authors emphasise how flow cytometry helps facilitate the diagnosis and systemic therapy is the modality of choice for treatment decided based on the histology type; however, outcomes still remain dismal, highlighting the unmet need to improve accurate diagnosis and explore newer treatment options. The novelty in this series lies in the detailed analysis and comparison of different subtypes of SL, a rare group of lymphomas primarily originating in the spleen. The present case series discusses the latest World Health Organisation (WHO) classification, which redefines the Hairy Cell Leukaemia (HCL) variant as Splenic B-cell Leukaemia/ Lymphoma with Prominent Nucleoli (SBLPN). Detailed molecular and genetic characteristics of various SLs are highlighted, such as the presence of the v-raf murine sarcoma viral oncogene homolog B1 (BRAF) p.V600E mutation in HCL, Mitogen-activated Protein Kinase 1 (MAP2K1) and Immunoglobulin Heavy Variable 4-34 (IGHV4-34) mutations in the HCL-variant, and 7q distortion in Splenic Marginal Zone Lymphoma (SMZL). Emphasis is placed on the individualised treatment approach based on specific patient profiles, genetic markers and disease characteristics. The study underscores the need for further research and clinical trials to validate emerging therapies, such as the use of Rituximab plus Doxorubicin, Bleomycin, Vinblastine and Dacarbazine (R-ABVD) in Hodgkin lymphoma and combinations of novel agents in aggressive SLs like HCL-variant and CD20-negative B-cell NHL.

An Unusual Case of a Central Pontine Myelinolysis Associated with Primary Splenic Diffuse Large B-cell Lymphoma.

A 54-year-old woman with persistent fatigue and a high fever presented with central pontine myelinolysis (CPM) on brain imaging, despite the absence of neurological symptoms. The patient had a slightly low serum sodium concentration. Further investigation led to a diagnosis of primary splenic diffuse large B-cell lymphoma (PS-DLBCL). Brainstem auditory-evoked potentials (BAEP) indicated minor abnormalities. CPM was successfully resolved after splenectomy and immunochemotherapy. This is the first reported case of CPM in a patient with PS-DLBCL confirmed by a pathological diagnosis following splenectomy and BAEP-detected abnormalities that improved with immunochemotherapy.

Primary Splenic Hodgkin Lymphoma and its Prognosis: A Report of Two Cases

Primary Splenic Lymphomas (PSL) constitute an extremely uncommon variety of splenic neoplasm and can present with grave complications such as splenic rupture. Hodgkin Lymphomas (HL), a type of hematopoietic neoplasm, are typically diagnosed between the ages of 20 and 30 years and present with supra-diaphragmatic lymphadenopathy, often accompanied by systemic B symptoms. A histopathological diagnosis involving Reed-Sternberg (RS) cells in an inflammatory background is crucial. However, other differentials such as reactive hyperplasia, infectious mononucleosis, anaplastic large cell lymphoma, or various other lymphomas may mimic Hodgkin disease both clinically and histologically. Therefore, accurate diagnostic evaluation of Hodgkin lymphoma is crucial, especially as it is highly curable with combination chemotherapy, even in higher stage disease. In this report, the authors present two cases of primary splenic HL diagnosed through histopathological and immunohistochemical examination of splenectomy specimens from January 2020 to December 2021 at the Department of Pathology, IPGME&amp;R, Kolkata, India. While reporting the histopathological sections, the authors also considered other differentials such as reactive changes, tuberculosis, and other non-Hodgkin lymphomas. Both patients were male, aged 10 years and 18 years, and presented with abdominal distension and fever, without any palpable peripheral lymph nodes, for the last six months to one year. After chemotherapy, the patients were regularly monitored to identify any signs of recurrence or relapse. Therefore, the initial recognition and proper diagnosis of Hodgkin lymphoma presenting in the spleen may vary in clinical presentation and morphology, but early accurate diagnosis carries a good prognosis, as survival is best determined by its histopathological type.

Diagnosis of chronic B-cell lymphoproliferative disease in peripheral blood=how machine learning may help to the interpretation of flow cytometry data.

Flow cytometry (FCM) has become a method of choice for immunologic characterization of chronic lymphoproliferative disease (CLPD). To reduce the potential subjectivities of FCM data interpretation, we developed a machine learning random forest algorithm (RF) allowing unsupervised analysis. This assay relies on 16 parameters obtained from our FCM screening panel, routinely used in the exploration of peripheral blood (PB) samples (mean fluorescence intensity values (MFI) of CD19, CD45, CD5, CD20, CD200, CD23, HLA-DR, CD10 in CD19-gated B cells, ratio of kappa/Lambda, and different ratios of MFI B-cells/T-cells [CD20, CD200, CD23]). The RF algorithm was trained and validated on a large cohort of more than 300 annotated different CLPD cases (chronic B-cell leukemia, mantle cell lymphoma, marginal zone lymphoma, follicular lymphoma, splenic red pulp lymphoma, hairy cell leukemia) and non-tumoral selected from PB samples. The RF algorithm was able to differentiate tumoral from non-tumoral B-cells in all cases and to propose a correct CLPD classification in more than 90% of cases. In conclusion the RF algorithm could be proposed as an interesting help to FCM data interpretation allowing a first B-cells CLPD diagnostic hypothesis and/or to guide the management of complementary analysis (additional immunologic markers and genetic).

Untangling hairy cell leukaemia (HCL) variant and other HCL-like disorders: Diagnosis and treatment.

The variant form of hairy cell leukaemia (HCL-V) is a rare disease very different from hairy cell leukaemia (HCL), which is a very well-defined entity. The 5th WHO edition (Leukemia, 36, 2022 and 1720) classification (WHO-HAEM5) introduced splenic lymphomas/leukaemias including four different entities: (1) HCL, (2) splenic marginal zone lymphoma (SMZL) with circulating villous cells in the peripheral blood, (3) splenic lymphoma with prominent nucleolus (SLPN), which replaced HCL-V and CD5 negative B-prolymphocytic leukaemia (B-PLL), and (4) splenic diffuse red pulp lymphoma (SDRPL). All these entities have to be distinguished because of a different clinical course and the need for a different treatment. The diagnosis can be challenging because of complex cases and overlap and/or grey zones between all the entities and needs integrating clinical, histologic, immunophenotypic, cytogenetic and molecular data. We review the diagnostic criteria including clinical, immunophenotypic and molecular characteristics of patients with HCL-V and other HCL-like disorders including HCL, SDRPL, SMZL, B-PLL and the Japanese form of HCL. We also discuss the different criteria allowing us to separate these different entities and we will update the recent therapeutic options that have emerged, in particular the advances with chemoimmunotherapy and/or targeted therapies.

Splenic B Cell Lymphoma Innate Immune Cells: Detection of Red Versus White Pulp Lymphoma Differences in NK Cell Populations By High-Parameter Flow Cytometry

Introduction Splenic B-cell lymphomas, (SLs, total incidence ~1:100,000/year) comprise four distinct indolent B-cell non-Hodgkin lymphomas. Three predominantly involve the red pulp of the spleen (classical hairy cell leukemia (HCL), hairy cell leukemia variant (HCLv) and splenic diffuse red pulp small B-cell lymphoma (SDRPL)) and the fourth predominantly involves the white pulp (splenic marginal zone lymphoma (SMZL)). HCL is usually diagnosed by detection of an activation mutation in BRAF and responds well to treatment. We focused our study on the remaining three diseases, which have overlapping clinical presentations with only subtle differences in their morphology and phenotypes, but have considerable differences in prognosis, response to treatment and outcome. The white pulp lymphoma SMZL generally has good treatment responses and longer survival, whereas the red pulp SLs, HCLv and SDRPL are less responsive to treatments with shorter survival. Cells of the tumor microenvironment (TME) may have distinct characteristics between these three diseases. We hypothesized that TME innate immune cells from red pulp SLs (HCLv, and SDRPL) would be similar to each other and distinct from the white pulp SL, SMZL.In this study, we report that natural killer (NK) cells, which are important innate immune cells critical for immune surveillance, vary in proportions among red and white pulp SLs. Methods We studied splenectomy specimens from patients with SMZL (n=8), HCLv (n=8), SDRPL (n=7), and controls (healthy, post-trauma, n=8). Cryo-preserved bulk splenic suspension cell isolates from fresh (&amp;lt;4 hour) surgical specimens and intact formalin-fixed paraffin embedded (FFPE) tissue sections were used. Human specimen collection and usage was conducted with written informed consent after approval of the Institutional Research Subjects Review Board. FFPE tissue sections were studied by immunohistochemical (IHC) staining and splenic suspension cells were analyzed by high-parameter fluorescence flow cytometry using a Cytek Aurora spectral flow cytometer with a 25-color myeloid and a 28-color NK/B-cell panel. Results IHC characterization showed an extensive network of innate immune cells as reflected by CD163 + macrophages. Using high-parameter flow cytometry, we focused on innate immune cells in the non-B- non-T-cell (CD3 -CD19 -) population. The average proportions of this population were: NK cells 25-36%, monocyte/macrophages 20-34%, dendritic cells 5-11%, and granulocytes 2%. Among the patient groups, the median percentage of NK cells was greater for controls (34%) and SMZL (35%) as compared to red pulp lymphomas (HCLv (28%), SDRPL (24%)). Next, we examined NK subtypes: 1) cytotoxic NK cells (CD56 +CD16 +), 2) cytokine-secreting weakly cytotoxic NK cells (CD56 +CD16 -), and 3) undefined NK cells (CD56 -CD16 +) as a median percentage of total NK cells. The major subtype for control and SMZL was CD56 +CD16 - with values of 58 and 44%, respectively. This subtype comprises only 38%-40% of red pulp SLs. Proportionally the CD56 +CD16 + subtype was the largest for red pulp SLs at 52-57% as compared to 30% for control and 38% for SMZL. The remaining CD56 -CD16 + NK cell subtype was generally higher among red pulp SLs (HCLv (9%), SDRPL (12%)) as compared to control (6%) and SMZL (4%) patients. The increase in cytotoxic CD56 +CD16 + subtype among red pulp SLs was confirmed by measuring the percentage of granzymeB +perforin + cells among mature cytotoxic NK cells (CD56 +CD16 +/ CD57 +NKG2a -). SLs had higher proportions of this NK phenotype (78-86% compared to control 69%) and red pulp SLs had the most granzymeB +perforin + proportions with 82 and 86% for HCLv and SDRPL, respectively. Discussion In summary, spleens from patients with SMZL had similar percentages of total NK cells to controls, with lower percentages in red pulp SLs. All SLs had a higher percentage of cytotoxic NK cells than control, with the red pulp SLs having the most. These data suggest that monoclonal antibody treatments targeting SL antigen, such as anti-CD20, may be effective by activating cytotoxic NK cell to target lymphoma cells by antibody-dependent cellular cytotoxicity. Thus, uncovering the characteristics of the TME may aid the development of non-invasive diagnostic procedures and/or precision therapies.

Immunophenotypic Profile of Chronic Lymphoproliferative Diseases in the State of Rio Grande Do Norte, Northeast, Brazil

INTRODUCTION: Chronic lymphoproliferative diseases (CLPD) are considered as a heterogeneous group of diseases characterized by monoclonal expansion and accumulation of apparently mature lymphocytes, which have a proliferative and/or survival advantage over normal lymphocytes in different organs such as bone marrow, peripheral blood and lymph nodes. This translates into the progressive accumulation of clonal cells and their products, causing peripheral blood and bone marrow lymphocytosis, in addition to lymphadenopathy, splenomegaly or other organomegaly. According to the World Health Organization, which reflects the international consensus and is based on pathological, genetic and clinical factors, lymphoproliferative neoplasms are classified according to the stage of maturation and the lineage where the neoplastic transformation occurs through flow cytometry (FC). OBJECTIVES: The aim of this study was to demonstrate the immunophenotypic diagnostic profiles of patients with CLPD in Rio Grande do Norte, Brazil by FC. METHODOLOGY: Were investigated 884 patients with CLPD of both sexes through CF with a panel of monoclonal antibodies directed to CLPD. In addition, other patient information, such as age, gender, and clinical and hematological data were also collected. RESULTS: The results showed that 89.7% of the cases were B Cell CLPD (B-CLPD) and 10.3% T and natural killer cells (T/NK-CLPD). The distribution of B-CLPD cases were as follows: 487 Chronic lymphocytic leukemia; 18 B-Cell Pro-lymphocytic leukemia; 25 Hairy cell leukemia; 6 variant Hairy cell leukemia; 6 Follicular lymphoma leukemized; 4 Splenic lymphoma with villous lymphocytes; 33 Mantle cell lymphoma; 4 Waldenstrom's macroglobulinemia; 69 Multiple myeloma; 14 Plasma cell leukemia; 11 Burkitt's lymphoma and 117 Leukemic Non-Hodgkin lymphoma (Table 1). The distribution of CLPD-T/NK was as follows: 12 Large granular lymphocytic leukemia; 14 T-Cell prolymphocytic leukemia; 18 Adult T-cell leukemia; 20 Sézary syndrome; and 24 Peripheral T-Cell lymphoma and 2 DLPC-NK (Table 1). DISCUSSION: The B-CLPD are the most commonly observed corresponding to more than 85% of all cases and are characterized by the presence of clonal B lymphocytes with expression of a single type of immunoglobulin light chain (kappa or lambda) in a proportion greater than 1/10, associated the strong positivity of Pam-B antigens such as CD19, CD22 and CD20 and the absence or low expression of Pam-T antigens. However, other antigens such as CD200, CD5, FMC7, CD79b and CD103, among others, may be present or absent, specifically characterizing these entities as Chronic lymphocytic leukemia (weak CD22+; CD200+, CD5+, FMC7-, CD79b-), Hairy cells leukemia (strong CD22+; FMC7+; CD79b+; CD5-; CD200-; CD103+; CD123+; CD11c+ and Follicular lymphoma (strongCD22+/CD20+, CD10+, CD200-, CD79B+, FMC7+). T/NK-CLPD are less frequent and are characterized by the expression of Pan-T antigens CD2, CD3, CD5, CD7, CD28 and TCL-1, with clonality evidenced by strong positivity of TCRα/β in most cases and increase of TCD4+ or TCD8+ lymphocytes in a ratio above 1:10 in most cases. In this group, large granular lymphocyte leukemia (LGLG) is a rare type of T/NK cell CLPD characterized by peripheral and medullary infiltration of large hypergranular lymphocytic cells, comprising about 2-5% of all cases, the most commonly seen being CD8+ T cells. CONCLUSION: The results presented demonstrate that immunophenotyping is a defining technique for clarifying the diagnosis of patients with CPLD, clonality was also successfully characterized in the investigated cases of CPLP of B ,T and NK lineages.

Lymphoma Cell Sorting to Enrich Low Degree Infiltration Clonal B-Cells for Diagnostic Delineation of Molecular Genetic Background By NGS

Introduction: According to WHO 2022 classificationthe vast majority of B-cell lymphoma entities continue to be defined histologically and by their immunophenotype. However, genetics help to define these entities and provide prognostic information. NGS targeted panel sequencing with short turn-around time has emerged as powerful diagnostic tool to uncover relevant genes frequently mutated in B-cell lymphoma. Yet its sensitivity limits its application to lymphoma cases with clone sizes &amp;gt;5%. In case of clinical symptoms and need for therapy small B-cell clones (&amp;lt;5%) can be diagnostically challenging if tissue for histopathology is difficult to obtain and immunophenotype is not characteristic for a specific lymphoma entitiy such as splenic B-cell lymphoma. Aim: To enrich small B-cell clones (&amp;lt;5%) by cell sorting to &amp;gt;15% to enable lymphoma NGS panel sequencing for potential genetic characterization and risk stratification. Methods: Peripheral blood or bone marrow samples (n=43) that containedsmall B-cells clones (&amp;lt;5%) as identified by flow cytometric routine diagnostics were stained for cell sorting by a combination of monoclonal antibodies against 9 B-cell antigens, including CD5, CD19 and CD45. The gating strategy excludes debris and doublets and defines B- and T-cells out of CD45-positive events. By application of cell sorting (SRT Cytoflex, Beckman Coulter) we aimed at &amp;gt;500,000 cells consisting of B- and T-cells, containing &amp;gt;15% clonal B-cells to ensure sufficient NGS sensitivity but minimize sort time. DNA was extracted from sorted samples and lymphoma NGS panel sequencing (66 genes ~ 1,500x coverage, min. 400x) was performed (NovaSeq 6000, Illumina). For technical and biological validation, native sample material was sequenced in parallel for a subset of patient samples (n=30). Results: In total43samples with &amp;lt;5% clonal B-cells were applied for cell sorting and clonal B-cells were enriched to a median of 17% (range, 3-97%) in a median of 800,000 cells (range, 127,400-3,780,000). In a pilot study to optimize the sorting procedure 8 of 13 sorted samples (62%) revealed quantitatively sufficient and good quality DNA and could be successfully analyzed by NGS. In 4 of these 8 samples (50%) mutations were identified. Optimized processing regarding cell sorting and DNA extraction was applied to further 30 samples all of which could be successfully sequenced. Overall, in 50% of all samples with NGS analysis after cell sorting (19/38) one or more mutations (total number of mutations: n=30) associated with B-cell lymphoma were detected. Mutations comprised genes frequently affected in B-cell lymphoma: MYD88 (30% of detected mutations, 9/30), CXCR4 (20%, 6/30), NOTCH1 (7%, 2/30), NOTCH2 (3%, 1/30), KMT2D (7%, 2/30), TP53 (13%, 4/30), BRAF (7%, 2/30), KLF2 (10%, 3/30) and DNMT3A (3%, 1/30). All of the cases negative for NGS after sorting (n=15) were negative also by NGS on matched native sample material without prior enrichment of clonal B-cells. Out of the 15 cases with positive NGS results after sorting NGS on native sample material revealed mutations in only 4 (27%) cases (only 1 case with detection of all mutations) while in 11 (73%) cases no mutations were detected. In the 15 cases positive after sorting median variant allele frequency (VAF) was 8.8% (range 3.4-38.3%). As anticipated, in the 4 cases with mutations detected also without sorting VAF was lower in native sample material (median 3.1%, range 1.98-4.9%) as compared to sorted sample material (median 8.9%, range 6.8-23.2%, p=0.0412). Conclusion: Cell sorting can overcome the diagnostic challenge of low degree infiltration B-cell lymphoma that require molecular genetics for further classification and risk prediction prior to therapy. Analysis of 30 samples and comparison with matched native material revealed that enrichment targeting 15% of clonal B-cells in a total of &amp;gt;500.000 cells are desirable for successful and sensitive NGS panel sequencing. In 50% of such cases mutations are detectable after sorting. Cell sorting to enrich clonal B-cells thus is a powerful tool to enable NGS panel sequencing for low percentage mature B-cell lymphoma and should be considered for routine diagnostics of B-cell lymphoma.

Epidemiology and Survival of Patients with Marginal Zone Lymphoma Treated at the National Cancer Institute of Peru. a Retrospective Analysis of 187 Cases

Background Marginal zone lymphoma (MZL) represents a heterogeneous subgroup, classified as extranodal, splenic and nodal lymphoma. Extranodal cases, also termed mucosa-associated lymphoid tissue (MALT), account for 70% of MZLs. Splenic marginal zone B-cell lymphoma (SMZL) accounts for &amp;lt;2% of all NHL. There is no data about marginal zone lymphoma in Latin American patients. Methods We evaluated retrospectively all patients diagnosed with marginal zone lymphoma at the National Cancer Institute from 2010 to 2019. Baseline characteristics, treatments and survival were retrieved from medical records. Kaplan Meier method was used to analyze the overall survival. The estimate of the survival curves was performed by the Kaplan-Meier method, and the differences were computed by the log-rank test. The Cox proportional hazard models were used to identify predictors of survival of MZL. A level of p &amp;lt;0.05 was considered for a statistical significance. Results During the study period 2010-2019, 4437 patients with B-cell non-Hodgkin lymphoma were evaluated at the INEN, 187 of them were marginal zone lymphomas, representing 4.2% of all cases. The most frequent marginal zone lymphoma was MALT, followed by nodal and splenic lymphoma with 89.8%, 5.3% and 4.8% of cases, respectively. The median age for the entire cohort was 59 years (range 17-89). The median age for the MALT, nodal and splenic MZL cohort were 58.5 years (range 17-89), 75 years (range 52-88), 68 years (range 43-87), respectively. 37.4% were male for the entire cohort, and 37%, 40%, 44% for the MALT, nodal and splenic MZL group. Elevated LDH was seen in 13% of the entire cohort and in 11% and 24% of the MALT and splenic MZL group. Transformed lymphoma was reported in 6.4% of the entire cohort, but in the 20% of nodal MZL and no one case of the splenic MZL. More data you can see in the table 1. The most frequent primary site locations for MALT lymphoma were gastric, ocular adnexal, parotid gland, cutaneous, intestinal and CNS with 36%, 30%, 7%, 5%, 5% and 5%, respectively. The median overall survival was 114 months (IQR 76-133) for the entire cohort and 133 months, 59 and 59 months for the MALT, nodal MZL, and splenic MZL, respectively (p=0.0011), see the figure 1. The 5-year OS was 81.4% (95% CI, 71.6-88.1) for the entire cohort and 85.7% (95% CI, 76.5-91.5) for the MALT lymphoma group. No one patients were alive at 5 years in both nodal and splenic lymphoma groups. Conclusion Marginal zone lymphoma is a relatively rare type of B-cell lymphoma. The most frequent type is MALT lymphoma, followed by nodal MZL. The median age for the entire group was 59 years, however patients with nodal MZL were older with a median age of 75 years. Transformed lymphoma was present in 6% of the entire cohort and in the 20% of the nodal MZL. Gastric, ocular adnexal and parotid gland were the most common primary site of MALT lymphoma. MALT lymphoma had long survival, but nodal and splenic MZL had shorter survival with no one surviving 5 years from diagnosis.

Hairy Cell Leukemia with Severe Leukocytosis

Introduction Hairy cell leukemia (HCL) is a rare indolent mature B-cells neoplasm that typically manifests with pancytopenia and splenomegaly. Nevertheless, significant splenomegaly, high hairy cells count in the blood, elevated β 2 microglobulin, and increased LDH can predict resistance to purine analogs. The absence of BRAF V600E mutation in HCL is rare and is associated with poorer outcomes. Case Presentation A 68-year-old female patient presented with symptomatic massive splenomegaly, leukocytosis (89,000 /microliter), monocytopenia, and thrombocytopenia. β 2microgobulin level was 4.4 mcg/L. Lactate dehydrogenase was 521 U/L. Peripheral blood and bone marrow examinations revealed typical hairy cells morphology, and the cells were positive for CD19, CD20, CD103, CD25, CD11c, CD200 (dim), and surface-Kappa. CD38 was negative. BRAF V600E result was wild-type. IGHV was mutated (IGHV3-49). She received first-line therapy with a 5-day course of Cladribine (0.15 mg/kg/day) with initial resolution of leukocytosis, normalization of platelets, and a 50% reduction in spleen size. This was followed by consolidation with an 8-weekly course of Rituximab (375 mg/m 2). Soon after the 2 nd dose of Rituximab, leukocytosis recurred, and she was initiated on second-line therapy with Bendamustine (90 mg/m 2 on days 1 and 2) along with Rituximab (BR). She has not achieved any durable response following two cycles of BR. Unfortunately, she suffered a septic shock requiring admission to our intensive care unit. Upon routine mental status evaluation, brain MRI revealed innumerable supratentorial and infratentorial bilateral acute/subacute and old microhemorrhages (Figure 1). This prompted further evaluation of unexplained recurrent episodes of pulmonary hemorrhages while on mechanical ventilation that were found to be secondary to pulmonary end-arterial cavitating lesions suggestive of hairy cells infiltration (Figure 2 arrowheads). As she was clinically unstable, we attempted to control leukocytosis with corticosteroids, leukapheresis aggressively and started the patient on Ibrutinib, but she succumbed to her disease. Discussion Recently, the 5 th edition of the World Health Organization classification of hematolymphoid tumors highlighted the splenic B-cell lymphoma and leukemia family that encompasses hairy cell leukemia (HCL), splenic B-cell lymphoma/leukemia with prominent nucleoli (SBLPN), formerly known as hairy-cell leukemia variant, splenic diffuse red pulp small B-cell lymphoma (SDRPL) and splenic marginal zone lymphoma (SMZL). HCL is typically an indolent mature B-cell neoplasm with characteristic pathologic features that preferentially insinuate the bone marrow and the spleen causing impaired hematopoiesis and splenomegaly. Unlike other disorders within this group, HCL commonly harbors BRAF V600E somatic mutation in almost all cases. Rarely, patients with HCL can have extramedullary involvement by the malignant cells. Leukocytosis in HCL appears to increase the risk of developing extramedullary lesions that can present unique clinical challenges necessitating early and effective therapy. According to the literature, brain lesions in HCL were described in three other cases, and leukocytosis was a common denominator. Our patient with significant leukocytosis had brainand pulmonary lesions, and the latter was the cause of her demise due tounusually aggressive and refractory disease. Therefore, we hypothesize that leukocytosis in HCL can be “the tip of the iceberg”. The higher the leukocytosis, the higher the burden of extramedullary disease, as in our case, who had simultaneous lesions in the brain and lungs. Patients with HCL and leukocytosis may warrant initial diagnostic evaluation to ensure the absence of potentially sinister lesions, which can inform subsequent monitoring and management plans. Additionally, more novel therapies could be considered sooner in the treatment course to control the disease and these lesions. Finally, our patient did not harbor BRAF V600E mutation, which likely resulted in further worse outcomes and limited the usage of targeted therapies like BRAF inhibitors.