Small Lymphocytes Research Articles

THE UNFORTUNATE PERFECT STORM - SEPTIC SHOCK DUE TO CHRONIC IMMUNOSUPPRESIVE MEDICAL THERAPY AND MEDICATION-INDUCED LYMPHOMA

Abstract INTRODUCTION Pharmacological treatment of Crohn's Disease (CD) involves immunomodulators such as adalimumab, a tumor necrosis factor (TNF) inhibitor and azathioprine, a purine analogue. These medications have risks associated with severe illness from infectious etiologies as well as lymphoma development. This is due to the immunosuppressive effect of the medications as well as a chronic inflammatory state(1). Anaplastic large-cell lymphoma (ALCL) is a subtype of non-Hodgkin's lymphoma (NHL). We present a rare case of sepsis and ALCL in a patient on immunomodulator therapy. CASE DESCRIPTION A 31-year-old G1P0 female at 19 weeks gestation with history of Crohn’s disease presents with abdominal pain. Her medications are adalimumab and azathioprine which were started one year ago due to poorly controlled CD resulting in anal fistulas. She was recently treated for right thigh folliculitis. This progressed to abdominal pain and right inguinal lymphadenopathy. She met SIRS criteria on presentation and was admitted to the intensive care unit for respiratory failure requiring intubation. Despite broad-spectrum antibiotics for pneumonia, she remained febrile with an increasing leukocytosis. Blood, lower respiratory, and fungal cultures, acid-fast staining, viral cultures and smears, and urine tests were negative for a source of infection. Commuted tomography (CT) of the abdomen and pelvis was significant for extensive retroperitoneal lymphadenopathy but found no infectious source. An excisional biopsy of the right groin lymph node was a preliminary read as Hodgkin's lymphoma (HL), but the initial immunohistochemical stain was consistent with NHL. Her immunomodulator therapy was stopped due to sepsis and her risk of lymphoma. She subsequently developed septic shock, acute renal failure requiring continuous renal replacement therapy, atrial fibrillation with rapid ventricular response, and suffered a spontaneous vaginal delivery of a 20-week stillborn baby. A final diagnosis of ALCL was confirmed by pathology. The molecular studies were significant for positivity for CD45, CD30, EMA, CD2, CD4, CD25, ALK1, granzyme B, perforin, and TIA1. She was started on brentuximab, cyclophosphamide, and prednisone. DISCUSSION ALCL is a rare subtype of NHL that accounts for roughly one percent of all NHL (3). The development of lymphomas occurs when the TNF alpha subunit binds to the cell surface. This inhibits both the apoptosis pathway and cytokine-mediated inhibition of CD30+ lymphoma cells resulting in cell proliferation (4). There is limited documentation of adalimumab or azathioprine associated ALCL and this association should be studied further. The patient suffered from severe consequences of her immunosuppression from her chronic immunomodulator therapy and immunosuppression from her malignancy resulting in septic shock with multiorgan failure. Right inguinal lymph node biopsy showing atypical large lymphoid infiltrate, background small lymphocytes, plasma cells, and histiocytes. The large atypical cells are characterized by large irregular nuclei with occasional bilobed nuclei and horseshoe-shaped nuclei, multiple small nucleoli, and abundant amphophilic cytoplasm.

A Rare Case of Cutaneous Anaplastic Large Cell Lymphoma in an Adolescent Female

Primary cutaneous anaplastic large cell lymphoma (C-ALCL) is a CD30 positive lymphoproliferative disorder, which is the second most common group of cutaneous T-cell lymphomas. It is common in adults 50-70 years of age with a male to female ratio 2:1. Here, we report a 14-year-old Caucasian female who presented with a painless, growing, friable, hemorrhagic nodule on her right medial buccal cheek of 2 weeks’ duration. The lesion was traumatized during a basketball game and significant bleeding was noted. A shave biopsy was performed on the 1.7 cm x 1.3 cm lesion. Histopathology demonstrated epidermal necrosis, acanthosis, large atypical lymphocytes with a number mitoses, prominent eosinophils, and smaller lymphocytes. Immunohistochemical staining revealed CD30 positivity of much of the tumor infiltrate, but was not diffusely positive. Rare cells were granzyme positive. ALK-1, TdT, EBER, CD1a were negative. The diagnosis was confirmed by a second dermatopathologist. The patient was referred to oncology. Labs were within normal limits and PET/CT scan was negative for metastasis. The lesion started to spontaneously regress after the shave biopsy. Spontaneous regression of C-ALCL occurs in 20-42% of cases and usually has an indolent course, therefore, aggressive treatment is not appropriate. In most cases, solitary or localized lesions are treated with surgical excision or radiotherapy. Although, about half of cases reoccur, but are not life threatening. This unusual case of C-ALCL provides an additional example of a rare clinical presentation in an adolescent female and reiterates the important histopathological findings for diagnosis.

What is your diagnosis? Perineal swelling in a dog.

What is your diagnosis? Perineal swelling in a dog.

Intrapancreatic accessory spleen

Introduction: Intrapancreatic accessory spleen (IPAS) is a congenital focus of healthy splenic tissue present outside of the splenic parenchyma. There are numerous locations for IPAS and they are usually found incidentally. The location of ectopic splenic tissue is important as it can often mimic as a neoplasm, most commonly neuroendocrine tumor (NET) due to similar imaging characteristics on nuclear imaging and computed tomography (CT) scan. We report on the finding of an IPAS in the tail of the pancreas for which fine needle aspiration was performed. Patient concerns: A 66-year-old man with past medical history of prostate cancer managed with brachytherapy presented to his urologist for workup of persistently elevated prostate specific antigen. He underwent a nuclear medicine whole body scan which revealed uptake in the pancreatic tail for which follow up CT was recommended. The patient was seen by the gastroenterology and a CT scan was obtained. CT revealed a 4 cm × 3 cm isoenhancing mass in the tail of the pancreas without invasion to surrounding structures nor communication with the pancreatic ducts. No lymphadenopathy was noted. Intervention: The patient subsequently underwent endoscopic ultrasound (EUS) with fine needle aspiration (FNA) of the mass. EUS revealed a hypoechoic, oval, mass-like structure in the distal pancreatic tail measuring ~20 mm × 20 mm in maximal cross-sectional diameter. FNA of the mass was performed. Diagnosis: Cytopathology of the specimen revealed small mature lymphocytes and vessels suggestive of IPAS. Outcome: The patient remained asymptomatic through the duration of the workup. Lessons: All enhancing pancreatic lesions require further workup to rule out malignancy. Typically, patients undergo scintigraphy for further evaluation or FNA biopsy via EUS for confirmation. Currently, there are no established serum markers or radiographic findings with adequate specificity for diagnosing IPAS. It is important to recognize the impact of incidental findings as they frequently require further testing and often result in procedural intervention. IPAS is often confused with NET, frequently necessitating tissue sampling. While the prevalence of intrapancreatic accessory spleen is higher than initially thought, contrary to NET it carries no inherent risk unless its location compromises vital structures. This highlights the importance of recognizing this anomaly.

Waldenstrom macroglobulinemia with 11q deletion: A rarely diagnosed entity with review of literature

Waldenstrom macroglobulinemia (WM) is a rare, chronic, and indolent B-cell lymphoproliferative disorder characterized by bone marrow infiltration by small lymphocytes, lymphoplasmacytoid cells, and plasma cells along with the presence of a detectable monoclonal immunoglobulin M. It represents 1%–2% of hematological malignancies with an overall incidence of 3–4 cases/million persons/year. Some deletions are associated with a more aggressive IgM gammopathy and have a high probability of symptomatic transformation. 6q deletion, the most common cytogenetic abnormality, which is present in 42% of cases whereas 11q deletion is rare in WM and is present in only 8% of cases. We are presenting a case of a 70-year-old male patient diagnosed as WM with 11q deletion.

B-cell small lymphocytic lymphoma in a free-ranging South American fur seal (Arctocephalus australis).

Lymphomas are malignant neoplasms of the hematopoietic system arising from lymphocytes with highly variable biologic behavior. B-cell small lymphocytic lymphoma (B-SLL) is a non-Hodgkin lymphoma infrequently described in domestic and wild animals. The present study describes a case of B-SLL in a free-ranging adult male Arctocephalus australis in Brazil. The main necropsy findings included poor body condition, generalized lymphadenomegaly, severe and diffuse splenomegaly, and multiple, white to yellow nodules in the kidneys and small intestine. Histologically, these organs were partially or totally effaced by neoplastic small lymphocytes arranged in sheets, with moderate anisocytosis and anisokaryosis and a low mitotic count. These cells diffusely immunolabeled for CD79α and CD20, and were negative for CD3. A diagnosis of multicentric B-SLL was established and to the authors' knowledge, it has not been previously described in this genus.

High-throughput Raman spectroscopy allows ex vivo characterization of murine small intestinal intra-epithelial lymphocytes (IEL).

T cells are considered to be critical drivers of intestinal inflammation in mice and people. The so called intra-epithelial lymphocyte (IEL) compartment largely consist of T cells. Interestingly, the specific regulation and contribution of IELs in the context of inflammatory bowel disease remains poorly understood, in part due to the lack of appropriate analysis tools. Powerful, label-free methods could ultimately provide access to this cell population and hence give valuable insight into IEL biology and even more to their disease-related functionalities. Raman spectroscopy has demonstrated over the last few years its potential for reliable cell characterization and differentiation, but its utility in regard to IEL exploration remains unknown. To address this question experimentally, we utilized a murine, T cell-driven experimental model system which is accepted to model human gut inflammation. Here, we repopulated the small intestinal IEL compartment (SI IELs) of Rag1-deficient mice endogenously lacking T cells by transferring naïve CD4+ T helper cells intraperitoneally. Using multivariate statistical analysis, high-throughput Raman spectroscopy managed to define a cell subpopulation ex vivo within the SI IEL pool of mice previously receiving T cells in vivo that displayed characteristic spectral features of lymphocytes. Raman data sets matched flow cytometry analyses with the latter identifying T cell receptor (TCR)αβ+ CD4+ T cell population in SI IELs from T cell-transferred mice, but not from control mice, in an abundance comparable to the one detected by Raman spectroscopy. Hence, in this study, we provide experimental evidence for high-throughput Raman spectroscopy to be a novel, future tool to reliably identify and potentially further characterize the T cell pool of small intestinal IELs ex vivo.

Diffuse Variant of Nodular Lymphocyte-Predominant Hodgkin Lymphoma in A 39-Year-Old Female: A Rare Case Report from Syria

Introduction: Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) represents a rare subtype of Hodgkin Lymphoma that constitutes approximately 5% of all HL cases, with an annual incidence of 1-2 per 1,000,000. Histological diagnosis requires detecting the lymphocyte-predominant (LP) cells, characterized by their popcorn-like contours and distinct immunophenotypic features, including the expression of the B cell surface marker CD20, with negative expression of CD15 and CD30, within a characteristic tumoral microenvironment. Case Presentation: A 39-year-old Syrian female was admitted to our hospital due to a painless, palpable mass in the left axilla, accompanied by no other symptoms. Three months earlier, the patient noticed the mass and visited an external clinic, where she received a ten-day course of antibiotics without experiencing any improvement. Physical examination revealed a firm, round mass devoid of signs of inflammation. An ultrasonography was performed and identified six enlarged lymph nodes in the left axilla displaying signs indicative of malignancy. These nodes were surgically excised, and the subsequent microscopic examination unveiled a biphasic nodular and diffuse proliferation of small lymphocytes, follicular dendritic cells, and epithelioid histiocytes, mixed with large distinct tumor cells recognized as LP cells, that are characterized by multilobulated nuclei with finely granular chromatin and prominent nucleoli. Immunohistochemical examination demonstrated positive expression for CD20 and CD45 in the LP cells, along with CD57 in the background cells, with negative expression for CD15, CD30, and BCL6, while CD10, CD3, BCL2, and Cyclin D1 markers yielded inconclusive results. Consequently, the final diagnosis was confirmed as NLPHL-Diffuse variant. Conclusion: In our manuscript, we presented a diagnostically challenging case of NLPHL diffuse variant in a middle-aged otherwise healthy woman. This case emphasizes on the importance of thorough microscopic examination with the use of immunostaining to make an accurate histopathological diagnosis and thus to provide the best possible medical care in similar cases.

HISTOLOGICAL AND MORPHOLOGICAL CHANGES IN THE LYMPHOID STRUCTURES OF THE GASTRIC MUCOUS MEMBRANE IN WHITE RATS WITH THE ADMINISTRATION OF SODIUM GLUTAMATE.

The aim: To determine the histological and morphological changes of the lymphoid structures of the stomach in male rats under the influence of oral sodium glutamate at the rate of 15 mg/kg of body weight. Materials and methods: The scientific experiment was performed on 20 white non-linear male rats of reproductive age (4-5 months). The experimental animals were divided into two groups (10 rats in each group), which were orally received monosodium glutamate at a dose of 15 mg/kg body weight every day. We studied the effect of 2 and 4 weekly administration of monosodium glutamate at a dose of 15 mg/kg body weight, respectively, in the I and II groups of experimental animals (depending on the week of their decapitation). Rats of the control groups (n=10) were injected with a placebo for 2 and 4 weeks, namely 0.5 ml of dechlorinated tap water at room temperature. Intact control animals were also divided into two groups, 5 rats each, depending on the week of decapitation: respectively, III group - decapitation on the 2nd week of the experiment; IV group - decapitation on the 4th week of the experiment. After the experiments were completed, animals were decapitated under light ether anesthesia. According to the purpose of the study, pieces of rat stomach measuring 1.0 x 1.0 cm were taken from the front wall of the bottom of the stomach near the great curvature, cardiac and portal parts of the organ. Histological preparations were examined using a MICROmed SEO SСAN light microscope and a Vision CCD Camera. Morphometric studies were carried out according to the method of S. B. Stefanov, using grids No. 3/16. For electron microscopic examination, pieces of the stomach wall of rats were fixed in a 2.5% solution of glutaraldehyde in a 0.1 M phosphate buffer (pH 7.2-7.4) with subsequent fixation in a 2.0% solution of osmium tetroxide. After dehydration in alcohols and acetone, the material was embedded in eponaraldite. Sections were made on an LKB-8800-III ultramicrotome and studied using a JEM - 100-V microscope. To study the structural components of the lymphoid formations of the mucous membrane of different parts of the stomach of rats, semi-thin sections were made for the purpose of sharpening the blocks, which were stained with methylene blue. Results: The analysis of the obtained data of the conducted experiment indicates that the administration of monosodium glutamate in a dose of 15 mg/kg of body weight to rats already after 14 days leads to an increase in the density and size of the lymphoid structures of the GMM. The number of immunocompetent cells between the fundus of the gastric glands and the muscle plate increases in the diffuse lymphoid tissue of the gastrointestinal tract of rats in all its parts, both in the I and II groups of experimental animals. These changes are most pronounced in the cardiac and portal parts of the stomach. In both groups of experimental animals, the migration of interepithelial lymphocytes, macrophages, plasma cells, and tissue basophils to the surface epithelium increases. In both groups of experimental animals (and the II group of rats), lymphoid nodules and lymphoid pre-nodules of the gastric mucous membrane (GMM) are located between the bottom of the gastric glands and the muscular plate of the GMM. A gradual increase of medium lymphocytes in the GMM was established both in animals of I and II groups, while large lymphocytes increased in almost the same amount in experimental animals of both groups. Similar changes occur in the characteristics of the number of plasma cells, macrophages and tissue basophils in the lymphoid pre-nodules of GMM. Conclusions: Administering monosodium glutamate to rats at a dose of 15 mg/kg of body weight for 2 weeks leads to an increase in the density and size of lymphoid structures of the mucous membrane in all parts of the stomach with a predominant increase in the number of immunocompetent cells between the bottom of the gastric glands and the muscle plate. At the same time, more pronounced changes were found in the number of small lymphocytes, which tend to decrease by the 2nd week of the experiment, and vice versa - their density increases by the 4th week of monosodium glutamate administration.

A Case of Fluorine-18-Fluorodeoxyglucose Positron Emission Tomography/Computed Tomography Findings of Mycosis Fungoides with Large Cell Transformation Involving Cutaneous Lesions, and Metastasis to Lymph Node and Viscera

Large cell transformation (LCT) of mycosis fungoides (MF) is a histopathological transformation of neoplastic small lymphocytes with a clonally identical large cell phenotype, which is found in 20%–50% of cases of advanced MF. Some few cases and studies have been reported about the role of fluorine-18-fluorodeoxyglucose positron emission tomography/computed tomography (18FFDG PET/CT) in diagnosing MF-LCT. This is a case of a 55-year-old man who had a left neck mass for 4–5 months. He underwent excisional biopsies on the skin and soft tissue. The histopathologic report showed MF-LCT. The 18F-FDG PET/CT findings revealed hypermetabolic lesions in the skin, cervical lymph node, and visceral organ. We introduce a rare case of MF-LCT involving both cutaneous and extracutaneous lesions.

Peripheral blood cells of native pig (Zovawk) of Mizoram, India: Light and scanning electron microscopy analysis

The literature on the blood cells of Zovawk pigs is scanty; thus, this study was designed to elaborate the light microscopy (LM) and scanning electron microscopy (SEM) characterization of blood cells of Zovawk pigs by LM and SEM. Twelve different blood samples were aseptically obtained from adult healthy Zovawk pigs, irrespective of sex. To determine the cytomorphological and cytochemical structures of the many produced constituents of blood, blood smears were stained with various stains. The blood samples were treated with various substrates for cytoenzymatic research, and the alterations were noted. A 1000× magnification Olympus Trinocular Research microscope was used to examine the smears. The blood samples were prepared for electron microscopy according to the standard procedure. The prepared samples were delivered to the Sophisticated Analytical Instrumentation Facility (SAIF), All India Institute of Medical Sciences (AIIMS), New Delhi, for SEM imaging. On LM, pig erythrocytes were spherical and nonnucleated. The cytoplasm of the neutrophils was spherical and included cytoplasmic granules. The eosinophils had prominent cytoplasmic granules and were round. Basophils were infrequently present and had cytoplasmic granules that were clear blue. The sizes of small, medium and large lymphocytes were noted. The monocytes were oval or circular. The platelets ranged in form from asymmetric to round. The blood samples were stained for cytochemical analyses using acid ferrocyanide stain for iron, Sudan black blue stain for lipids, toluidine blue stain for mucopolysaccharides, and periodic acid Schiff's stain for glycogen. The cytoenzymatic characteristics were evaluated and compared with substrates treated with acid phosphatase, alkaline phosphatase, peroxidase, arylsulfatase, cytochrome oxidase, beta-glucuronidase, and succinate dehydrogenase. Erythrocytes appeared as biconcave disks under SEM. Two forms of leukocytes were observed, having a rough and pointed cell surface like a flower. It can be concluded that the LM and SEM morphology of blood cells of Zovawk pigs resembled other domestic animals, however, few differences were observed among the discussed animals.

Clinical features and prognosis of eight patients with splenic diffuse red pulp small B-cell lymphoma

Objective: To investigate the clinical characteristics, response, and prognosis of splenic diffuse red pulp small B-cell lymphoma (SDRPL) . Methods: Eight cases of SDRPL were diagnosed and treated at Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, between May 2017 and April 2022. Data on the clinical features, laboratory results, bone marrow and spleen biopsy results, response, and prognosis were collected and analyzed. Results: The median age at diagnosis was 54 (42-69) years. Splenomegaly and lymphocytosis were present in all cases, and PET/CT revealed normal to slightly elevated splenic FDG uptake. All cases were in stage Ⅳ, with spleen, peripheral blood, and bone marrow but no proximal lymph nodes involved. The cytoplasm of neoplastic villous cells was abundant, and splenic pathology showed that small homogenous lymphocytes permeated the splenic sinus and splenic cord, and the white pulp atrophied. Immunohistochemistry was not typical, and B-cell markers including CD19, CD20 and CD79α were positive. After a median follow up of 35.5 (4-60) months, 7 cases were alive after splenectomy with or without chemoimmunotherapy. The patient with CCND3 P284A and MYC S146L mutation developed to B-cell prolymphocytic leukemia (B-PLL) 1 month after splenectomy and died at 16 months of follow-up. Conclusion: A rare indolent B-cell lymphoma that primarily affects the elderly, SDRPL. Most patients achieved long-term survival, but the prognosis of patients who progress to B-PLL was poor.

Lymphoplasmacytic Lymphoma with Paraprotein IgG

Lymphoplasmacytic lymphoma (LPL) is a rare indolent mature B-cell lymphoma. LPL secreting immunoglobulins other than IgM are rare. There are very few case series on non-IgM LPL, and little is known about the clinical features and outcomes of patients with this disease. We report the case of a 65-year-old-male who was referred to our hospital for further investigations of persistent chronic anaemia and was diagnosed with IgG-LPL based on the presence of M protein from serum electrophoresis. Bone marrow morphology exhibit a spectrum of B-cell differentiation ranging from small mature lymphocytes to plasma cells. The patient underwent treatment with a combination of bortezomib, dexamethasone and rituximab and showed positive response. LPL with paraprotein IgG is a rare indolent disease and has a heterogeneous clinicopathological presentation with limited literature reviews. Our case report and literature review provide insights and knowledge in the description of the clinicopathological features of IgG LPL.

Unique Presentation of Bing-Neel Syndrome With Co-existing Chronic Lymphocytic Leukemia.

Unique Presentation of Bing-Neel Syndrome With Co-existing Chronic Lymphocytic Leukemia.

Insights from a patient with chronic lymphocytic leukemia complicating ALK+ anaplastic large cell lymphoma.

Chronic lymphocytic leukemia (CLL) that transforms into a more aggressive lymphoma has been termed Richter syndrome (RS). CLL with T-cell neoplasia is rarely reported; those with ALK+ anaplastic large cell lymphoma (ALCL) are also exceedingly rarely reported. A 63-year-old woman from the south of China presented with generalized lymphadenectasis and fever; she already had a prior diagnosis of CLL 9 years ago. As per her current diagnosis, it was CLL with ALK+ ALCL. The two-lymph node and bone marrow biopsies presented two types of cellular groups: i) left cervical lymph node biopsy suggested CLL (Ki67: 10%), along with bone marrow biopsy exhibited enhancement of the small lymphocytes (30%) with scant cytoplasm, round or irregular cell nuclei, and massive amounts of chromatin. Large cells (< 1%) that expressed CD30 and ALK were visible; The results of immunohistochemistry were as follows: CD20 (weak positive); PAX5 (positive); CD23 and CD5 (weak positive); and CD3, CD10, and CyclinD1 (negative); ii) left supraclavicular lymph node biopsy suggested ALK+ ALCL (Ki67: 70%). The final diagnosis was CLL with ALCL. The mechanisms of this condition are not fully understood, which might be associated with chronic stimulation of T cells by CLL cells along with immune dysfunction.

Single Cell RNA-Seq of Extranodal Marginal Zone Lymphoma (MZL) Identifies Site-Specific and Shared Features in Both B and T Cell Components

Background. Extranodal MZL of MALT type (MALT lymphomas) represents 5-8% of all human B-cell lymphomas can occur at any anatomical site. We and others have previously highlighted the genetic, biological and clinical peculiarities of this tumor, differences due in part to the diverse primary sites of disease. Almost by definition, the cellular composition of MALT lymphomas can be very heterogenous, encompassing small B cells resembling MZ cells, monocytoid cells, and small lymphocytes, in addition to scattered immunoblasts, centroblast-like cells and plasma cells. This issue and the possible presence of anatomical site-specific nonmalignant cell biopsies make single cell (sc) RNA-Seq ideal for studying MALT lymphomas. scRNA-Seq has the potential to provide detailed information on the transcriptome of non-neoplastic cells and give insight on the heterogeneity of the lymphoma cell population. Methods. Cells were analyzed with the 10× Chromium Chip and the Single Cell 3′ Reagent Kit v3. Libraries were run on an Illumina Nextseq 500. A minimum of 50'000 reads per cell were sequenced. FASTQ files were mapped to human reference genome (HG38) using the cellranger pipeline. Data went through quality control and downstream analysis in R (v3.6.1) using Seurat (v3.0). Cell clusters were generated with the FindNeighbors/FindClusters functions and cells annotated with a mixed approach considering known biomarkers and unsupervised computational methods (celldex, SingleR). Non-linear dimensional reduction (UMAP) was used to visualize clustering results and Monocle 2.0 for pseudo-time analysis. Results. ScRNA-seq was performed on pulmonary (n=2), gastric and parotid (n=1, each one) MZLs. Publicly available data for a second parotid case were included in the analyses. A total of ~19K cells passed QC with a median of 1,368 detected genes per cell. Each specimen contained mostly B cells (median 76%; range 49-85%), followed by T cells (median 21%; 12-47%), plasma cells (median 2%; 1.6-2.7%), epithelial and myeloid cells (median <=1%). Each sample contained both neoplastic B cells (median percentage 49%; range 8-79%), defined as those bearing a monoclonality for κ (n.=4) or λ (n.=1), and normal B cells (median 26%; 6-41%). The latter mainly comprised naïve and memory B cells. When we merged the five cases together, neoplastic B cells had highly patient-specific profiles, whereas nonmalignant B cells and plasma cells shared similar gene expression profiles across different cases. CD4+T cells also showed patient specific-signatures, while CD8+ cells co-clustered, indicating a more homogenous phenotype across patients. Transcripts expressed across MZL cells were enriched for IRF4 and BCL6 targets, genes higher in splenic MZL vs normal spleen, genes expressed in memory B cells, plasma cells, ABC-DLBCL with high STAT3, and in OxPhos DLBCL. Examples of genes expressed in MZL cells vs normal B cells were PAX5 and CXCR4 (pulmonary MZLs), TNFRSF1B and KRAS in parotid MZL, PIK3CD in gastric MZL. SPIBand SOX5, among other genes, showed high expression levels in malignant cells from all individuals compared to the other cells. MZL cells showed high inter- and intra-tumoral heterogeneous expression of potential therapeutic targets (for example, MS4A1/CD20, CD19, BCL2, MCL1). Pseudo-temporal analysis identified a main trajectory that ordered naïve B cells and healthy memory B cells to plasma cells, while the differentiation toward malignant B cells was inferred as a distinct branch. Analysis of immune-related ligand-receptor (L-R) pair interactions showed that most of the B cell subclusters provided costimulatory signals to CD4+ or CD8+ T cells via CD22-PTPRC interactions. Another suggestive costimulatory interaction occurred through CD70-CD27, involving malignant B cells, CD4+, CD8+ cells and also Tregs. Regarding coinhibitory pairs, the CD86-CTLA4 axis was frequently identified between CD4+/Tregs subclusters and malignant B cells but not between non-malignant B and T cells. Conclusions. scRNA-Seq highlighted peculiar inter- and intra-tumoral behaviors of B and T cells in MALT lymphomas. The results indicate that although each anatomical site can have a different molecular portrait, common gene expression programs are found in malignant cell populations of MALT as well as in cells of the tumor microenvironment. Acknowledgments: supported by Barletta foundation and EASI-Genomics.

Identification of Nodular Lymphocyte-Predominant Hodgkin Lymphoma Variant Morphology Using Artificial Intelligence

Background Lymphocyte predominant (LP) cells located outside B-cell-rich nodules are the hallmark of variant morphologic patterns of nodular lymphocyte predominant Hodgkin lymphoma (NLPHL). In comparison with typical NLPHL (patterns A and B), cases with variant morpholoy (patterns C, D, E and F) are associated with significantly worse clinical behavior. Nevertheless, assessment of tissue architecture may be challenging in limited biopsy specimens, which are increasingly common in routine clinical practice. We sought to address this gap by developing artificial intelligence-assisted modules to accurately recognize NLPHL and distinguish patterns A/B from variant patterns, namely the most common of those, pattern C. Material & Methods The study group included whole slide images (WSI) of hematoxylin and eosin (H&E) stains and CD20 immunohistochemistry stains from 10 NLPHL patients; cases emcompassed patterns A, B & C (A/B: 5 cases; C: 5 cases) as determined by hematopathology review. We designed modules to assess the spatial relationship between LP cells and B cell-rich areas by first seeking to detect LP cells using high-power field (HPF) magnification of H&E WSI (Figure A1). We split the WSI into tiles (8000 x 8000 pixels) to mitigate the computational burden of WSI analysis. For each tile, we deployed the pretrained HoVer-Net for nuclear segmentation. To distinguish LP cells from surrounding small lymphocytes, we analyzed nuclear size, intensity, and gray-level co-courrance matrix (GLCM) features (correlation and dissimilarity). For each feature, we identified the optimal threshold based on feature histogram of LP in comparison to the remainder surrounding cells. Nuclei meeting all feature criteria were labeled as LP cells and validated morphologically by hematopathologists. All tiles were then stitched to illustrate the spatial distribution of LP cells on WSI. Next we deployed the active contour (AC) technique, an iterative region-growing image segmentation algorithm, to identify B-cell areas from CD20 stains (Figure A2). To reliably define the intial contours for the AC, we abided by the following criteria: 1) Red (R) channel intensity larger than both the green (G) and blue (B) channels, with a ratio of 1.25; 2) Including pixels in the bottom 10% of the intensity histogram after removal of slide background and converting the RGB image into gray scale. We merged the contours from these two techniques and used them as initial seeds for AC segmentation, after which we took the final evolved contour as the boundaries of B-cell areas. We then overlayed CD20 on H&E WSI to assess the spatial relationship between the detected LP cells and B-cell areas. Since tissue sections on the CD20 and H&E slides represent neighboring slices, only translation, rotation, and slight scaling were surmised to occurr between slides in this pilot study set. Accordingly, we employed the affine registration paradigm and optimized mutual information between the transformed CD20 and fixed H&E images until convergence then warped the detected B-cell areas via the obtained deformation field (Figure A3). Results The concordant positioning between two stains after registration allowed us to compare the spatial relationship of LP cells to B-cell areas (including assessment of the number of LP cells inside (922) and outside (639) B-cell areas) (Figure A4). We then segmented the final image into quadrants to simulate needle core biopsies and confirmed the efficiency of our modules in detecting LP cells and analyzing their relationship to B-cell areas in limited tissue, in all quadrants (Figure A5). Our model was successfully applied on 100% of 10 NLPHL cases in this pilot group. After proof of concept was established in a full tissue biopsy sample (Figure A1-5), we applied the same steps to an actual physical needle core biopsy of an NLPHL-pattern C case (Figure B1-3) and were able to identify 7 out of 29 LP cells located outside B-cell areas, in congruence with the previously rendered diagnosis (Figure B4). Conclusion This technique, using H&E staining and CD20 immunohistochemistry only, is especially promising to work-up cases when ordering a routine panel of immunohistochemical stains is not feasible due to limited tissue that may be exhausted. Further validation using an independent cohort is warranted in the future. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Focal Myositis with CD8+T-Cell predominance: an Inflammatory Myositis Mimicking a Soft Tissue Neoplasm

Abstract Introduction/Objective FM is a rare self-limiting T-cell rich lesion arising within muscles of young adults as a solitary lesion and can be confused with a variety of neoplastic/inflammatory conditions or lymphoma. Here we describe a rare T-cell rich variant of FM with CD8 predominance. Methods/Case Report A 51-year-old female presented with two-month history of left trapezius swelling. MRI showed an enhancing tumor within the muscle, suspicious of sarcoma, less likely myeloma. Results (if a Case Study enter NA) Biopsy showed diffuse infiltration of small lymphocytes in a fibrotic background and atrophic skeletal muscle. These lymphocytes stained positive for CD2, CD3, CD7, CD8, CD5 (partial), TIA1 (partial), CD43, CD57 and negative for CD4, CD30, CD56, CD57, granzyme B, perforin, BCL-6, BCL-2, Pax5, TCL1, and CD56. Ki-67 was (&amp;lt;10%) with few background CD20+ B cells. By flow cytometry, the CD8+ T-cells co-express CD2, CD3, CD7, with dim CD5 expression. TCR gene rearrangement study by PCR showed no clonal TCR Gamma gene rearrangement. Conclusion FM is extremely rare with only 22 cases well-described in the literature, all predominantly composed of CD4+ T-cells, clinically concerning for low-grade sarcomas, as inflammatory myofibroblastic tumor, inflammatory leiomyosarcoma, or liposarcoma. Careful analysis is essential for correct diagnosis. Mimics include other causes of myositis, such as polymyositis and inclusion body myositis. Depending on the extent of lymphocytic infiltrate, gene rearrangement studies might be necessary to rule out clonality. Recognition of this rare entity with excellent prognosis is crucial to provide appropriate management and avoid unnecessary, aggressive procedures.

Correlation of Cytokine Content in the Lymph with the Morphological Parameters of the Mesenteric Lymph Nodes in Adjuvant Therapy of Experimental Breast Cancer with the Use of Fragmented Human DNA.

We studied the relationship between the level of cytokines in the lymph of the thoracic duct and the morphometric parameters of the mesenteric lymph nodes after surgical treatment of breast cancer, chemotherapy, and administration of fragmented (double-stranded, dsDNA) human DNA. In comparison with surgical treatment and with chemotherapy alone, administration of a human dsDNA has a stimulating effect on the T-cell link of the immune response. In the paracortical zone, the relationship between the chemokine MCP-1 and increased content of small lymphocytes in this zone was revealed. Interrelations of IL-2 cytokines with small lymphocytes and of IL-4 with medium lymphocytes were revealed in germinal centers. We also observed interrelations of IL-7 with small lymphocytes and IL-4 with macrophages in the medullary cords, chemokine MIP-1α with immature and mature plasma cells (the number of these cells is reduced), and of MCP-1 with immunoblasts (the number of which is also reduced) in the medullary sinuses.

PSAT325 Unmasking the Effect of IgG4 Disease Surge on Thyroid Gland: A Case of Bilateral Sialadenitis in Patient with Past History of Graves.

Abstract Introduction Immunoglobulin G4-related disease is a fibrous-inflammatory disease characterized by lymphoplasmacytic tissue infiltration of IgG4 positive plasmablasts and small lymphocytes, that may be related to storiform fibrosis, obliterative phlebitis and modestly increased tissue eosinophils. This condition was recently recognized in 2013. It has unknown pathophysiology. The common characteristics shared by the different entities that make up the IgG4-related disease are: raised serum IgG4 levels, alterations in the imaging tests with neoplastic-like swelling of the affected organs, specific histopathological characteristics, and in immunostaining, as well as a good response to treatment with glucocorticoids. Clinical Case A 75-year-old male with a history of Grave's disease with ophthalmopathy, osteopenia, gastroesophageal reflux disease, obstructive sleep apnea, and benign prostatic hypertrophy presented to the clinic after a neck ultrasound and CT neck were ordered for investigation of bilateral "neck lumps". Neck ultrasound was evident for the heterogeneous appearance of submandibular glands and a hypoechoic nodule in the left neck with internal vascularity below the left jaw. CT neck with IV contrast showed evidence of possibly sialadenitis and lobular soft tissue thickening in the inferior aspect of the left submandibular gland measuring approximately 3.5 cm. Surgical pathology report of the left submandibular gland demonstrated a significant increase with IgG and IgG4 plasma cells and CD38 confirmed a dense plasmacytic infiltrate. IN the laboratory, IgG4 was elevated, 462 mg/dL (2-96 mg/dl) Conclusion As with many autoimmune diseases, it is unknown what triggers the Immunoglobulin G4-related disease. Given that the latter has just recently been diagnosed and recognized it is imperative to continue to record and develop data regarding it to better understand it. Multiple endocrine organs could be involved in the pathology of IgG4 related disease. The health care providers need to be cognizant of this condition and the wide variety of manifestations it could have. This disease can be first encountered by any medical field due to the broad spectrum of clinical findings that it could encompass. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m.