Multiple Nucleoli Research Articles

Secondary growth ovarian follicles of the pigmented sterlet sturgeon Acipenser ruthenus L. 1758 (Acipenseriformes, Chondrostei, Actinopterygii, Osteichthyes) – Microscopic study of oocytes, egg envelope and diversification of follicular cells

The individual ovarian follicle of sturgeons (Acipenseriformes, Acipenseridae) contains an oocyte surrounded by follicular cells (FCs), basal lamina, and thecal cells. The late stages of the secondary growth of follicles (mid- and advanced vitellogenic) are not fully explained in Acipenseriformes. To explore and discuss the ultrastructure of oocytes, FCs, an egg envelope, and explain how micropylar cells differentiate and the canals of a multiple micropyle are formed, the samples of ovaries of the mature sterlet sturgeon Acipenser ruthenus were examined. The oocytes are polarized, the nucleus is located in the animal hemisphere, contains lampbrush chromosomes and multiple nucleoli. In the ooplasm three regions are present: a perinuclear (contains the mitochondria), an endoplasm (contains the lipid droplets and yolk platelets), and a periplasm (contains the cortical granules, melanosomes, endocytotic and exocytotic vesicles). The melanosomes in animal hemisphere form two concentric rings separated by a lighter region between them. The FCs are differentiated into bright and dark cells that are both translationally and secretory active. Diversification of FCs involves repeated and cytoskeleton-dependent change of shape. In the advanced follicles the FCs are diversified into micropylar, the animal and vegetal regions cells, and the cells that delaminated from the epithelium in the animal region. The egg envelope is present in the perioocytic space and consists of three layers: (1) an inner layer or vitelline envelope, (2) a middle layer, and (3) an outer layer. The inner layer consists of four sublayers: (a) a filamentous sublayer composed of filaments released from the oocytes, (b) a trabecular 1 sublayer and (c) a trabecular 2 sublayer named due to the sequence of the deposition, and composed of filaments, fibres and trabecules, (d) a homogeneous sublayer located between the trabecular 1 and trabecular 2 sublayers composed of filaments that adhere to each other closely. The middle layer contains two sublayers: a porous 1 and a porous 2 (composed of granular material) which are released by the oocyte and FCs. The outer layer consists of fibrillar material released by the FCs. The egg envelope is pierced by radial canals formed around the microvilli of the oocyte and the microvilli-like processes of FCs. A micropylar field in the egg envelope that covers the animal pole of the oocyte contains 1 – 4 micropylar canals. Micropylar cells are involved in their formation. The shape of these cells is icicle-like and the cytoplasm is differentiated into two regions (a basal and apical bearing a projection) equipped with different sets of organelles.

Pathomorphological and Immunohistochemical Study of Fibrosarcoma in an Adult Dog: A Case Report

Aim: The objective of the present case report is to describe pathomorphological findings and immunohistochemical expression of pancytokeratin (PCK), vimentin, proliferating cell nuclear antigen (PCNA) and Ki67 in a case of fibrosarcoma in dog. Case Presentation: A large, round ball-like growth of approximately 14 centimetres in diameter was noticed on caudal aspect of left thigh region in ten years old male bull dog. Its surface was ulcerated. It was soft in consistency and cut surface appeared pinkish-white. Giemsa-stained smears revealed spindle cells with elongated nuclei containing granular chromatin, single to multiple nucleoli and scanty cytoplasm. Histopathological examination revealed moderately to well-differentiated neoplastic fibroblasts arranged in interwoven pattern with the presence of collagenous stroma. Neoplastic cells were characterized by pleomorphic, oval to elongated nuclei with single to multiple prominent nucleoli and moderate to scanty cytoplasm. Mitotic count was 1.2/high power field. The Immunohistochemical staining revealed intracytoplasmic positive immunoreactivity for vimentin and no immunoreactivity for PCK. Proliferating neoplastic cells revealed mild to strong nuclear immunostaining for PCNA and Ki67 with 56.7% PCNA index and 4.0% Ki67 index. Conclusion: Based on histopathological findings, the case was diagnosed as fibrosarcoma. Immunopositive reactivity for vimentin and no immunoreactivity for PCK indicated the mesenchymal origin of the tumour. Histopathological features, low mitotic count and low Ki67 index suggested the less aggressive behaviour of the tumour in the present case.

Ocular squamous cell carcinoma: A patho-morphological and AgNOR based diagnosis

A fifteen years old female buffalo was referred to the Teaching Veterinary Clinical Complex (TVCC), RS Pura, Jammu, India with a history of purulent discharge and protruded mass from the right eye with an impaired vision for 30 days. On physical examination, there was ulcerated vesicular growth at the sclera corneal junction. Protruded mass was removed surgically and was taken for histopathological examination and AgNOR count. On histopathological studies, cells displayed severe anaplasia, pleomorphism, hyperchromatic nuclei, multiple nucleoli, and frequent mitotic figures and it was diagnosed as ocular squamous cell carcinoma. The value of mean and proliferative AgNOR for ocular squamous cell carcinoma was found to be 2.83±0.87 and 32%, respectively. Based on patho-morphological examination and AgNOR count, the protruded mass was led to the final diagnosis of ocular squamous cell carcinoma.

Acute Myeloid Leukemia in a Three-Year-Old Girl Mimicking Chronic Myeloid Leukemia in Blast Crisis, Challenge in Diagnosis and Treatment in Limited-Resourced Health Care Facility: A Case Report

Introduction: : Acute myeloid leukemia (AML) is the second most common hematologic cancer in childhood. Clinically, AML has similar symptoms to other types of leukemias, and diagnosis remains a challenge due to limited access to pediatric hemato-oncology diagnostic examination. This study aims to report AML in a 3-year-old girl who was first suspected of Chronic Myeloid Leukemia (CML) in blast crisis, using a combination of clinical-hematology parameters and conventional morphology examination in a limited-resourced healthcare facility. Case Presentation: A 3-year-old girl was referred from a rural Eastern Indonesia hospital with symptoms of prolonged fever, leucocytosis, severe anemia, and severe thrombocytopenia. In the previous hospital, the patient had a history of hyperleukocytosis and peripheral blood smear suspected of CML in blast crisis. A peripheral blood smear was repeated in our hospital and showed findings of Auer rod cells suggesting AML. Two bags of packed red cells and 2 thrombocyte concentrations were administered before referral for further diagnostic evaluation with bone marrow aspiration. Bone marrow aspiration revealed multiple nucleoli suggested for AML with FAB classification as M4 subtypes, and she received chemotherapy in a tertiary hospitalConclusions: With limited resources, suspicion based on persistent clinical symptoms, routine hematology tests, and peripheral blood smear examination are important to distinguish AML from CML in blast crisis. Understanding clinical hematology parameters and peripheral blood smears is the first step in AML diagnosis pathway and decision for further diagnostic referral. Supportive therapy and early recognition of oncologic emergency must be done before referral to a tertiary referral hospital.

Asymmetry in previtellogenic and early vitellogenic oocytes, ultrastructure of follicular cells and egg envelope in the pigmented sterlet, Acipenser ruthenus L. 1758 (Chondrostei, Acipenseriformes).

Ovarian follicles of sterlets (Acipenser ruthenus) are composed of a single oocyte surrounded by follicular cells (FCs), basal lamina, and thecal cells. Previtellogenic oocytes are polarized. Homogeneous ooplasm (contains ribosomes) and granular ooplasm (comprises nuage aggregations of nuclear origin, rough endoplasmic reticulum (RER), Golgi complexes, ribosomes, and mitochondria) are distinguished. Granular ooplasm is initially located near the nucleus, contacts the plasma membrane of the oocyte (oolemma) and forms a thin layer underneath its entire perimeter. Next, a ring that surrounds the nucleus is formed and sends strands directed toward the oolemma. The lipid body composed of lipid droplets forms adjacent to this ring. Later, the granular ooplasm and strands enlarge toward the oolemma, lipid body disperses, and homogeneous ooplasm is no longer present. A thin cortical ooplasm is formed underneath the oolemma and does not contain any organelles. The oocyte nucleus moves to the center. The nucleoplasm contains lampbrush chromosomes, nuclear bodies, and multiple nucleoli. Early vitellogenic oocytes are polarized, too. Three regions in the ooplasm are distinguished: the perinuclear (contains lipid droplets near the nuclear envelope), the endoplasm (contains yolk platelets and lipid droplets), and the periplasm (contains yolk spheres, pigment granules, and microtubules). In all these regions the RER, Golgi complexes, nuage, and mitochondria are present. Micropinocytotic vesicles, Golgi vesicles and precursors of the internal layer of the egg envelope are in the cortical ooplasm. Some FCs delaminate from the follicular epithelium, degenerate and vesicles are released into the perioocytic space. They may contain precursors of egg envelope and may be involved in "cell-cell" communication. The egg envelope (zona radiata, zona pellucida) is made up of three layers: the vitelline envelope (inner layer), the middle layer, and the outer layer. In its deposition, both the oocyte and FCs are engaged.

Detection of Leptomeningeal Carcinomatosis by Cerebrospinal Fluid in a Dog with a Negative MRI

ABSTRACT An 11 yr old female French bulldog was presented for acute onset of seizures and a 2 wk history of disorientation. On physical examination, a nodular mass at the fourth mammary gland level was observed. Neurological evaluation showed obtundation and compulsive behavior. Brain MRI study did not reveal any abnormalities. Cerebrospinal fluid (CSF) collected from the cerebellomedullary cistern showed a marked increase of total nucleated cell count (400 cells/μL). Cytological evaluation identified the presence of a monomorphic round cell population characterized by large cell bodies, a single eccentrical located nucleus with high nuclear:cytoplasmatic ratio, and marked atypia with anisocytosis, anisokaryosis, and multiple nucleoli. Leptomeningeal carcinomatosis (LC) was suspected. The dog was euthanatized for worsening of clinical signs. Post-mortem examination identified an anaplastic mammary carcinoma in the nodular mammary mass. Infiltration by neoplastic cells exhibiting the same morphological features was detected along leptomeninges of the telencephalon and cerebellum associated with cortical and subcortical parenchymal micrometastases. To our knowledge, this is the first case of LC in a dog detected by CSF evaluation but without any MRI abnormalities. This finding emphasizes the usefulness of CSF cytology in patients with suspected LC even in the absence of any MRI identifiable lesions.

Incidental Findings of Duodenal-Type Follicular Lymphoma by Endoscopic Screening: A Case Report

Introduction: Follicular Lymphoma (FL) is a B-cell neoplasm arising from the germinal center. Gastrointestinal FL is a rare case, accounting for less than 4% of all primary lymphoma of the gastrointestinal tract. Duodenal-type Follicular Lymphoma (DTFL) was included in an entity of primary intestinal FL. However, in the 2017 World Health Organization (WHO) Classification, this entity was included in a specific variant called DTFL. In this article, we report a case of DTFL in a 56-year-old man which was incidentally found during upper gastrointestinal endoscopy Case Presentation: : We report a case of incidentally found DTFL in a 56-year-old man during an endoscopy. The endoscopic finding showed a small, hypertrophic, or nodular lesion on part two duodenum. Microscopic evaluation of duodenal mucosa consists of proliferative atypical lymphoid cells arranged in follicular architecture. The lesion was confined to the lamina propria. The atypical lymphoid cells were dominated by monotonous centrocyte-like cells, small-sized, with scant cytoplasm, and cleaved nuclei. There were some scattered larger centroblast-like cells with multiple nucleoli. No mantle zone and tingible body macrophage were found. The immunohistochemical evaluation showed positivity for CD 20, CD 10, Bcl-6, and Bcl-2 in the follicular structure. CD 21 was dominantly stained at the periphery of the follicular structure, and Ki-67 was low. Conclusions: According to clinical data, endoscopic findings, and histopathological and immunohistochemical findings, the patient was diagnosed with DTFL.

Re-organization of nucleolar architecture in myogenic differentiation.

Myogenesis, the process of muscle differentiation, requires an extensive remodeling of the cellular transcriptome and proteome. Whereas the transcriptional program underpinning myogenesis is well characterized, the required adaptation in protein synthesis is incompletely understood. Enhanced protein synthesis necessitates ribosome biogenesis at the nucleolus. Nucleolar size and activity are inextricably linked with altered gene expression. Here, we report changes in nucleolar morphology and function during myogenic differentiation. Immunofluorescence analysis revealed alterations in nucleolar morphology that were dependent on the cellular state - proliferative or quiescent myogenic progenitors (myoblasts or reserve cells) contained multiple small nucleoli with a characteristic spherical shape, whereas multinucleated myotubes typically contained one large, often irregularly shaped nucleolus. These morphological alterations are consistent with changes to nucleolar phase separation properties. Re-organization of the nucleolar structure was correlated with enhanced rRNA production and protein translation. Inhibition of mTOR signaling with rapamycin perturbed nucleolar re-organization. Conversely, hyperactivated mTOR enhanced alterations in nucleolar morphology. These findings support the idea that there is an mTOR dependent re-organization of nucleolar structure during myogenesis, enhancing our understanding of myogenesis and possibly facilitating new approaches to therapeutic interventions in muscle pathologies.

Images in haematology: Primary cutaneous diffuse large B cell lymphoma, leg type with an unusual presentation: A diagnostic challenge.

An 83-year-old woman with a history of arterial hypertension treated with amlodipine, and a well-differentiated cutaneous squamous cell carcinoma completely excised during the past year, was referred to dermatology consultations for the progressive appearance of indurated nodular skin lesions over the last two months, which began on the right lower extremity and progressed to the upper extremities, trunk and buttocks, with no other symptoms associated. With the differential between cutaneous lymphoma versus subcutaneous metastases, a punch biopsy of one of the lesions was performed. Histopathological examination demonstrated a dense hypodermal infiltrate composed of atypical large lymphocytes showing a round-shaped nucleus with vesicular chromatin and prominent single or multiple nucleoli. The immunohistochemical study showed positivity in neoplastic cells for CD79a, CD20, Bcl-2, MUM-1, c-myc and IgM, along with a high expression of Ki-67; and negativity for CD3, CD10, Bcl-6, CD30, p53, PD-L1 and Epstein-Barr Virus Encoded RNA. Molecular analyses were then performed without detecting BCL-2, BCL-6 or c-MYC rearrangements by fluorescence in situ hybridization, as well as the absence of p.L265P mutation in MYD88 through a quantitative polymerase chain reaction. Taking into account both histopathological and clinical findings, a primary cutaneous diffuse large B cell lymphoma, leg type (PCDLBCL-LT) and skin involvement in the course of a systemic DLBCL were considered, so blood tests and a positron emission tomography (18-FDG PET/CT) were then solicited. Blood tests showed a haemoglobin value of 12.5 g/dl with a normal white blood cell count and a slight β-2-microglobulin elevation (3.33 mg/L) in the context of a decreased renal function (54.38 ml/min/1.73m2). 18-FDG PET/CT revealed 23 hypermetabolic subcutaneous nodules distributed predominantly in the right arm and lower extremities, with no sign of disease in other locations (Figure 1), confirming the primary cutaneous origin of the disease and the final diagnosis of a PCDLBCL-LT. The patient underwent treatment with 6 cycles of rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone, with progressive improvement of the skin lesions and complete resolution of them, and is currently being followed up by haematology and dermatology departments. Although the majority of PCDLBCL-LT, arise in the lower limbs (80%–85%), some patients can develop the disease in other areas, but in those cases, the lesions are usually individual or few in number, contrary to our case. PET imaging tests have a major role in the initial staging of the disease and treatment response evaluation, and they could even avoid bone marrow biopsy in selected patients. The double expression of bcl-2 and c-myc is frequent in PCDLBC-LT, although the prognostic value of this dual expression is yet unclear, whereas the absence of the MYD88 mutation could be associated with both better response to lenalidomide as well as better prognosis. For these reasons, immunohistochemical and molecular techniques can be helpful in defining prognosis and deciding therapeutic options in patients with PCDLBCL-LT and should be performed in routine practice. In conclusion, we present an extremely rare 18-FDG PET/CT image of a PCDLBCL-LT with 23 subcutaneous nodules, underlying the importance of this test for the study of these patients. Lucía Prieto-Torres – Attending the case and performing clinical research and manuscript revision. Pablo Villagrasa-Boli – Attending the case, revising existing literature and manuscript writing. Luis Ignacio Sancho-Val – Attending the case and manuscript revision. Pedro Paul – Attending the case and manuscript revision. Ana Leticia Tardin-Cardoso – Performing the imaging tests and their clinical interpretation. Mar García-García – Pathology diagnosis, taking microscopic images and manuscript revision. Mariano Ara-Martín – Proof checking. The authors have nothing to report. The authors declare that they have no conflict of interest. No funding has been received for this article.

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.

Heterogeneity of nucleolar morphology in four‐cell mouse embryos after IVF: association with developmental potential

AbstractIn mammals, around fertilization, the nucleolus of embryos transforms into the nucleolus precursor bodies (NPBs), which continue to mature until the blastocyst stage, leading to distinct morphological changes. In our study, we observed two types of nucleolar morphology in mouse in vitro fertilized embryos at the four‐cell stage, which we refer to single nucleolus (SN) and multiple nucleoli (MN). To visualize nucleolar morphology, four‐cell embryos were immunostained with anti‐NOPP140 antibody. These embryos were categorized into five types based on the number of blastomeres carrying SN: SN4/MN0, SN3/MN1, SN2/MN2, SN1/MN3, and SN0/MN4, with percentages of 13, 27, 21, 23 and 9, respectively. Next, using a light microscope, we divided the four‐cell in vitro fertilized embryos without fixation into two groups: those with at least two blastomeres displaying SN (SN embryos) and those without (MN embryos). Notably, significantly more SN embryos developed into blastocysts and offspring at 18.5 dpc compared with MN embryos. Furthermore, SN embryos displayed a higher NANOG‐positive cell number at the blastocyst stage, significantly lower body and placental weights, resulting in a higher fetal/placental ratio. These findings suggest a close association between nucleolar state at the four‐cell stage and subsequent developmental potential.

A Rare Case of Anaplastic Lymphoma Kinase-Positive Large B-Cell Lymphoma Involving Epidural Tissue

Abstract Introduction/Objective Anaplastic lymphoma kinase-positive large B-cell lymphoma (ALK-positive LBCL) is a rare, aggressive type of non-Hodgkin’s B-cell lymphoma with characteristic rearrangements and overexpression of ALK. Diagnosis of ALK-positive LBCL is challenging because of its rarity and uncharacteristically low expression of B- lineage antigens usually needed for diagnosis of B-cell lymphomas. In this report, we describe a case of ALK-positive large B cell lymphoma involving epidural soft tissue in a young adult. Methods/Case Report 29 year old presented to emergency department with a two week history of back pain and a three day history of worsening numbness and weakness in the lower extremities. MRI of the thoracic spine showed an epidural soft tissue mass posterior to T5, T6 and T7 vertebral bodies with abnormal signal in the T6 spinous process. Histological examination from the epidural mass revealed sheets of atypical lymphoid cells with abundant pale cytoplasm and round vesicular nuclei with prominent single to occasional multiple nucleoli. Immunohistochemical stains demonstrated neoplastic cell positivity for ALK, VS38c, CD138, EMA, MUM1, CD45, PAX5 and MCK. They were negative for CD3, CD20, CD19, CD79a, CD30, CD2, CD117, SOX10, HMB45, cytokeratin, synaptophysin and chromogranin. These overall findings were consistent with ALK-positive large B-cell lymphoma. Results (if a Case Study enter NA) NA. Conclusion ALK-positive large B-cell lymphoma is a non-Hodgkin lymphoma associated with high morbidity and mortality. Absence of reliable B-cell marker expression in this B-cell lymphoma requires recognition of morphological findings to order confirmatory testing such as immunohistochemical stains for CD138 and ALK. This particular case also showed an unusual anatomic presentation in epidural tissue.

Cancer cells corrupt normal epithelial cells through miR-let-7c-rich small extracellular vesicle-mediated downregulation of p53/PTEN

Tumor volume increases continuously in the advanced stage, and aside from the self-renewal of tumor cells, whether the oncogenic transformation of surrounding normal cells is involved in this process is currently unclear. Here, we show that oral squamous cell carcinoma (OSCC)-derived small extracellular vesicles (sEVs) promote the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of normal epithelial cells but delay their apoptosis. In addition, nuclear-cytoplasmic invaginations and multiple nucleoli are observed in sEV-treated normal cells, both of which are typical characteristics of premalignant lesions of OSCC. Mechanistically, miR-let-7c in OSCC-derived sEVs is transferred to normal epithelial cells, leading to the transcriptional inhibition of p53 and inactivation of the p53/PTEN pathway. In summary, we demonstrate that OSCC-derived sEVs promote the precancerous transformation of normal epithelial cells, in which the miR-let-7c/p53/PTEN pathway plays an important role. Our findings reveal that cancer cells can corrupt normal epithelial cells through sEVs, which provides new insight into the progression of OSCC.

Burkitt leukaemia with B-cell precursor immunophenotype.

A 64-year-old male presented with malaise and B symptoms. An automated full blood count showed haemoglobin 124 g/L, white blood cells 40 × 109/L, platelet 51 × 109/L. Positron emission tomography/computed tomography indicated widespread activity above and below the diaphragm with bone marrow, peritoneal, pleural, hepatic, renal, small bowel and possible adrenal involvement. The blood film (Figure 1A, May-Grünwald-Giemsa stain ×100 objective) showed numerous large cells with basophilic cytoplasm and cytoplasmic vacuolation. By flow cytometry (Beckman Coulter Duraclone) (Figure 1E–H) these cells (red colour population) were CD45 low (Figure 1E). They were positive for surface CD19, CD10, HLADR, CD38 and cytoplasmic CD79a, terminal deoxynucleotidyl transferase (TdT) (Figure 1H); negative for surface CD34, CD20 (panel F), CD22, light chains (Figure 1G) and cytoplasmic CD3, myeloperoxidase (MPO). Bone marrow aspirate (Figure 1B, May-Grünwald-Giemsa stain x100 objective) was packed with medium to large-sized cells with basophilic cytoplasm, and frequent cytoplasmic vacuolation. No abnormalities were detected by reverse transcription-polymerase chain reaction screening for common leukaemic fusion genes using the Q30 leukaemia assay (QuanDX). Fluorescence in situ hybridization (FISH) analysis of the liquid sample detected immunoglobulin heavy chain/MYC translocation and gain of 1q. Next-generation sequencing (Archer VariantPlex) identified NRAS p.Gln61Arg variant (VAF 36%). Bone marrow histology (Figure 1C, haematoxylin and eosin stain x40 objective) revealed effacement by large cells with finely dispersed chromatin, with frequent mitoses and some with multiple small nucleoli. A classical ‘starry sky’ pattern was not seen. Immunohistochemical staining was strongly positive for CD45, CD20 (most positive) and TdT (Figure 1D, x40 objective); positive for paired box 5 (PAX5), CD19, CD79a, CD10, multiple myeloma 1; and negative for B-cell lymphoma 2 (BCL2), BCL6, CD5, cyclin D1, CD1a, CD99, CD117, CD34, MPO and light chains. Staining for Epstein-Barr virus ribonucleic acid was negative. The MIB1 proliferation factor was near 100%. FISH confirmed the MYC translocation but neither BCL6 nor BCL2 translocations were detected. A diagnosis of “Burkitt leukaemia (BL) with a B-cell precursor immunophenotyped”. The patient's medical comorbidities made him unfit for chemotherapy and he received palliative corticosteroid treatment. BL with precursor B cell immunophenotype is rare in adults. The WHO Classification of Tumours of Haematopoietic and Lymphoid Tissue (revised 4th edition) notes approximately 2% of paediatric cases of BL that “have a phenotype of precursor” B-cells, with an expression of TdT, and sometimes CD34, and absence of CD20 and surface immunoglobulin expression. The reason for this aberrant phenotype remains unknown [1]. A small study reported IG-MYC+ neoplasms with precursor B cell immunophenotype to resemble precursor B-cell acute lymphoblastic leukaemia/ lymphoblastic lymphoma rather than BL in genomic, epigenomic profiling, the mutational landscape, and the DNA methylation pattern, with frequent activation of the RAS pathway [2]. Further studies are needed for a better understanding of this rare subgroup of disease (see Figure 1). Ke Xu wrote the manuscript. Ke Xu, Anna Childerhouse and Rajeev Gupta critically revised the final version of the manuscript.

A challenging case of intravascular large B-cell lymphoma presenting as pyrexia of unknown origin

Abstract Introduction: Intravascular large B-cell lymphoma (IVLBCL) is a rare subtype of large cell lymphoma, characterized by the growth of neoplastic cells within the lumina of small blood vessels, without an obvious extravascular tumor mass or presence of circulating lymphoma cells in the peripheral blood. Patient concern: A 68-year-old woman presented with fever of unknown origin and abdominal pain. Diagnosis: Bone marrow aspiration showed abnormally large cells with increased hemophagocytic activity. Trephine biopsy showed moderate to large neoplastic cells with single and multiple prominent nucleoli sequestered in the blood vessel lumen. Immunohistochemistry was positive for CD20, PAX5, CD79a, MUM1, and BCL2 and showed a high proliferative fraction of 80% confirming the diagnosis of IVLBCL. Interventions: The patient received six cycles of R-CHOP chemotherapy. Outcomes: The patient has been in remission for nearly two years after completing the treatment. Conclusion: Clinical recognition of IVLBCL remains a challenge. Standard staging and therapeutic approaches need to be addressed to further elucidate the characterization and management of this rare disease.

Seminoma e sertolioma em cão não criptorquida – relato de caso

The most common testicular neoplasms in dogs are seminoma, leydigocytoma and sertolioma, affecting middle--aged and elderly dogs, where cryptorchidism is a predisposing factor, as well as some breeds. It can occur alone or, less fren-quently, concurrently, generally affecting the same testicle. This study aimed to report the case of a non-cryptorchid 14-year--old mixed breed dog diagnosed with seminoma and sertolioma, each in a testicle. The animal showed an increase in scrotal volume, with no changes in other clinical parameters on physical examination. On ultrasound examination, it was possible to observe alterations suggestive of neoplasia in both testicles and prostatic alteration suggestive of benign prostatic hyperplasia. Complementary blood count and biochemical tests were performed and, as treatment, orchiectomy was performed. Fragments were collected from both testicles and sent for histopathological examination. Microscopy of the left testicle showed the presence of round cells, multiple and evident nucleoli, cells in different phases of mitosis and binucleation, these changes being compa-tible with seminoma. In contrast, in the right testicle, spindle and elongated cells (pseudo-lobular) were observed, presence of long cytoplasmic projections with rounded ovoid nucleus, spindle cells and degeneration of seminiferous tubules, compatible with sertolioma. It was observed that physical examination associated with ultrasound was efficient to detect the presence of neoplasms, being validated by histopathological examination. Orchiectomy was an assertive treatment indicated for this case.

Nuclear Organization during Hepatogenesis in Zebrafish Requires Uhrf1.

Acquisition of cellular fate during development is initiated and maintained by well-coordinated patterns of gene expression that are dictated by the epigenetic landscape and genome organization in the nucleus. While the epigenetic marks that mediate developmental gene expression patterns during organogenesis have been well studied, less is known about how epigenetic marks influence nuclear organization during development. This study examines the relationship between nuclear structure, chromatin accessibility, DNA methylation, and gene expression during hepatic outgrowth in zebrafish larvae. We investigate the relationship between these features using mutants that lack DNA methylation. Hepatocyte nuclear morphology was established coincident with hepatocyte differentiation at 80 h post-fertilization (hpf), and nuclear shape and size continued to change until the conclusion of outgrowth and morphogenesis at 120 hpf. Integrating ATAC-Seq analysis with DNA methylation profiling of zebrafish livers at 120 hpf showed that closed and highly methylated chromatin occupies most transposable elements and that open chromatin correlated with gene expression. DNA hypomethylation, due to mutation of genes encoding ubiquitin-like, containing PHD and RING Finger Domains 1 (uhrf1) and DNA methyltransferase (dnmt1), did not block hepatocyte differentiation, but had dramatic effects on nuclear organization. Hepatocytes in uhrf1 mutants have large, deformed nuclei with multiple nucleoli, downregulation of nucleolar genes, and a complete lack of the nuclear lamina. Loss of lamin B2 staining was phenocopied by dnmt1 mutation. Together, these data show that hepatocyte nuclear morphogenesis coincides with organ morphogenesis and outgrowth, and that DNA methylation directs chromatin organization, and, in turn, hepatocyte nuclear shape and size during liver development.

NODULAR SCLEROSIS HOGDKINS LYMPHOMA GRADE 2 MIMICKING ANAPLASTIC LARGE CELL LYMPHOMA - A CASE REPORT

INTRODUCTION: Hodgkin's lymphoma (HL) is a lymphoproliferative neoplasm constituting less than 1% of all neoplasms. It has been categorized into- nodular lymphocyte predominant HL and classical type with subtypeslymphocyte rich, lymphocyte depleted, nodular sclerosis and mixed cellularity. Nodular sclerosis Hodgkin's lymphoma (NSHL) is difcult to diagnose on FNA because of the lack of Reed Sternberg (RS) cells, low cellularity and difculty in identifying the counterpart of lacunar cells. CASE A 16-year-old female presented with anterior chest wall swelling si REPORT: nce 6 months measuring 5x4cm along with incidentally noted cervical lymph nodes measuring 2x2cm and 1x1cm. On examination, multiple papular skin lesions were observed. FNAC smears from cervical lymph node showed high cellularity, with sheets of binucleated and multinucleated cells having multiple nucleoli, some showing pale bluish abundant cytoplasm whereas others showing dense cytoplasm; in a background of reactive lymphoid population with few neutrophils. Few giant cells showed hyperchromatic nuclei. Abnormal mitosis seen. No typical RS cell was seen. Peripheral blood smear showed neutrophilic leukocytosis with no atypical cell. Possibility of NSHL was considered and biopsy advised to rule out ALCL. Histological sections showed near total effacement of lymph node architecture by nodules separated by broad brotic bands. These nodules exhibit proliferating lymphocytes with clustered lacunar cells along with some mononucleate, binucleate and multinucleated cells. Prominent mitotic activity, angiogenesis, focal micro abscesses were noted. Immunohistochemistry showed CD 15+ and CD 30+ expression in the giant cells, conrming diagnosis of NSHL grade 2. NSHL poses a cytological diagnostic dilemma specially in abse CONCLUSION: nce of sclerosis on FNA, making histological examination and IHC mandatory

Croatia’s First Experiences in Cytological Diagnosis of High-Grade Ductal Carcinoma in Situ of the Breast: Case Reports and a Review of Literature

Cases here presented are also first Croatia’s experiences in the cytological diagnosis of breast ductal carcinoma in situ (DCIS). The first patient was a 64-year-old woman, by imaging methods with a wide area of suspect microcalcifications in the left breast lower medial quadrant and abundant, dense, yellow-greyish discharge of the same mammilla and second was a 65-year-old woman with an equivocal lesion in left breast lower lateral quadrant. Morphological findings of both aspirates and nipple discharge from the first patient were practically the same - moderately to highly cellular, with 3D solid aggregates, monolayer sheets and many scattered single clearly malignant cells of large - mainly >5x the diameter of an erythrocyte, round-to-ovaloid, manifestly irregular nuclei, dark blue, polymorphous, often multiple (macro)nucleoli, irregular nuclear outlines and dense, more basophilic, mostly scant cytoplasms arranged in syncytium–like structures. “Dirty” background was overfull of comedo necrosis and dark grayish, sharp, irregular microcalcifications. Cytological diagnosis in both cases was DCIS, high-grade, comedo type. Histopathologically first tumor was big, 6 cm large, estimated as comedo carcinoma with microinvasion focuses not bigger than 1 mm, but without signs of angioinvasion, while the second tumor was smaller, 0.6 cm with wide ducts fully with large polymorphic malignant epithelial cells, central comedo necrosis, cancerisation of some lobules but with the intact basement membrane. It was pure high-grade DCIS, comedo type. Presented cases completely reflect to date knowledge about cytological diagnostic of high-grade DCIS; include necessary morphological criteria - highgrade nuclear atypia, an abundance of comedo necrosis and microcalcifications, confirm our limitation in the presumption of invasion status with large lesion extent, but also prove that cytology is the unquestionably reliable in breast morphological diagnostic, even in such sophisticated and demanding pathological issue like DCIS.

Atypical hemograms of the commercial duck

A description of standard and atypical heterophils, lymphocytes, and 2 types of giant cells found in the circulation of 17 wk commercial ducks (N = 24) in apparent good health is the subject. Heterophils were sorted as either “classic” (HC) having red rod-shaped cytoplasmic granules, “typical” (HT) having weakly stained granules providing a reticular cytoplasmic appearance, or rarely as “variant” types (HV) having orange spherical granules. Atypical HT's and HC's were in 14 of 24 (58%) of the ducks.Small lymphocytes (Ls), reactive lymphocytes and plasmacytes (Lm) were routinely found. Giant cells, also present, were placed with Lm or monocytes (Mn) depending on cytology. Two counts of 200 leukocytes gave the total white count (TWBC) and 2 heterophil/lymphocyte ratios. H/L 1 = (HT + HC +HV) / Ls; and H/L 2 = (HT + HC + HV) / (Ls + Lm). The results showed that TWBC were normal (~ 23,000 /μL) but both H/L ratios were highly variable. HT were differentiated from HC on nuclear and cytoplasmic criteria. Many HT and HC exhibited signs of deterioration.Some giant cells likely represented developmental stages. Multiple nucleoli were evident in others suggesting polyploidy. The more common lymphoid giants were usually round whereas monocyte types were irregular. Mn types were actively phagocytic often consuming thrombocytes or rarely erythrocytes (RBC). Giant cells of either type were in 13 of 24 (54%) of the duck hemograms.Conidiospores were detected in the blood smears of 4 ducks and bacteria in 2 with 1 duck having both.As all ducks were in apparent good health the blood born microorganisms likely represented low grade infections. Presumably the atypical cells were a response to the presence of toxins of bacterial and fungal origin.The presence of atypical heterophils and lymphocytes complicates interpretation of H/L ratios traditionally used to establish stress. As atypical cells can be found in the context of normal TWBC or nonstress H/L values cytological observations attain additional importance. Moreover, giant cells may be useful indicators of infection even without direct microscopic observation or isolation of the offending organisms.