Non-neoplastic Lymphocytes Research Articles

Effects of luteinizing hormone receptor activation in immortalized neoplastic canine T lymphocytes.

The expression of luteinizing hormone (LH) receptors has been reported in canine nonneoplastic and neoplastic lymphocytes. This study aimed to determine the effect of LH receptor activation in neoplastic canine T lymphocytes. Cell lines (CLC, CLK, EMA) derived from 3 dogs with primary T-cell lymphoma were used. Cell lines were previously phenotyped and evaluated for LH receptor expression with flow cytometry. Cell lines were stimulated with increasing concentrations (0, 4, 400, and 4,000 U/mL) of human chorionic gonadotropin (hCG; an LH receptor agonist), RNA was extracted, cDNA was synthesized, and gene expression was determined using quantitative PCR and the 2-ΔΔCt method. Cell lines were stimulated with the same increasing concentrations of hCG, and cell proliferation, adhesion, and transmigration were determined using commercial assays. The effects of LH receptor activation were compared between hCG concentrations using a one-way ANOVA. Significance was defined as P < .05. LH receptor stimulation increased LH receptor gene expression in the CLC cell line, and there was a trend for increased expression in the EMA cell line but no effect in the CLK cell line. Activation of LH receptors increased proliferation in all 3 cell lines, endothelium adhesion in 2 cell lines (CLC, CLK), and transmigration in 2 cell lines (CLK, EMA). LH receptor stimulation using hCG increased LH receptor gene expression in neoplastic canine T lymphocytes and increased cell proliferation, adhesion, and transmigration. These findings may provide a physiologic mechanism for the increased incidence of lymphoma reported in dogs with sustained supraphysiologic LH concentrations.

Lymphocyte migration and retention properties affected by ibrutinib in chronic lymphocytic leukemia.

The Bruton tyrosine kinase (BTK) inhibitor ibrutinib is widely used for treatment of patients with relapsed/refractory or treatment-naïve chronic lymphocytic leukemia (CLL). A prominent effect of ibrutinib is to disrupt the retention of CLL cells from supportive lymphoid tissues, by altering BTK-dependent adhesion and migration. To further explore the mechanism of action of ibrutinib and its potential impact on non-leukemic cells, we quantified multiple motility and adhesion parameters of human primary CLL cells and non-leukemic lymphoid cells. In vitro, ibrutinib affected CCL19-, CXCL12- and CXCL13-evoked migration behavior of CLL cells and non-neoplastic lymphocytes, by reducing both motility speed and directionality. De-phosphorylation of BTK induced by ibrutinib in CLL cells was associated with defective polarization over fibronectin and inability to assemble the immunological synapse upon B-cell receptor engagement. In patients' samples collected during a 6-month monitoring of therapy, chemokine-evoked migration was repressed in CLL cells and marginally reduced in T cells. This was accompanied by profound modulation of the expression of chemokine receptors and adhesion molecules. Remarkably, the relative expression of the receptors governing lymph node entry (CCR7) versus exit (S1PR1) stood out as a reliable predictive marker of the clinically relevant treatment-induced lymphocytosis. Together, our data reveal a multifaceted modulation of motility and adhesive properties of ibrutinib on both CLL leukemic cell and T-cell populations and point to intrinsic differences in CLL recirculation properties as an underlying cause for variability in treatment response.

Diagnostic utility of FOXO1 immunohistochemistry for rhabdomyosarcoma classification.

Rhabdomyosarcomas currently are classified into one of four subtypes (alveolar, embryonal, spindle cell/sclerosing, or pleomorphic) according to their morphological, immunohistochemical, and molecular genetic features. The alveolar subtype is characterised by a recurrent translocation involving PAX3 or PAX7 and FOXO1; identification of this translocation is important for appropriate classification and prognostication. In this study, we aimed to explore the diagnostic utility of FOXO1 immunohistochemistry for rhabdomyosarcoma classification. A monoclonal antibody targeting a FOXO1 epitope retained in the fusion oncoprotein was used to study 105 rhabdomyosarcomas. FOXO1 was positive for expression by immunohistochemistry in all 25 alveolar rhabdomyosarcomas, with 84% showing diffuse expression in greater than 90% of neoplastic cells; the remainder of alveolar rhabdomyosarcomas displayed at least moderate staining in a minimum of 60% of lesional cells. Apart from three spindle cell rhabdomyosarcomas showing heterogeneous nuclear immunoreactivity in 40-80% of tumour cells, the 80 cases of embryonal, pleomorphic, and spindle cell/sclerosing rhabdomyosarcoma were negative for FOXO1 expression (96.3% specific) when using a threshold of nuclear staining in 20% of neoplastic cells to determine positivity. Variable cytoplasmic staining was present in a fraction of all rhabdomyosarcoma subtypes. Nonneoplastic lymphocytes, endothelial cells, and Schwann cells also showed variably intense nuclear anti-FOXO1 immunoreactivity. Taken together, our findings suggest that FOXO1 immunohistochemistry is a highly sensitive and relatively specific surrogate marker of the PAX3/7::FOXO1 fusion oncoprotein in rhabdomyosarcoma. Cytoplasmic immunoreactivity, expression in nonneoplastic tissues, and limited nuclear staining of nonalveolar rhabdomyosarcomas represent potential pitfalls in interpretation.

Sensitivity of canine hematological cancers to BH3 mimetics.

Inhibition of antiapoptotic B-cell lymphoma 2 (BCL2) proteins by small molecule Bcl-2 homology 3 (BH3) mimetics causes rapid induction of apoptosis of human hematological cancers in vitro and in vivo. Assess in vitro sensitivity of non-neoplastic lymphocytes and primary hematological cancer cells from dogs to venetoclax (VEN) or the dual BCL2/ B-cell lymphoma-extra-large (BCLxL) inhibitor, navitoclax (NAV), and evaluate the association between BCL2 protein expression and VEN sensitivity. Nine client-owned dogs without cancer and 18 client-owned dogs with hematological cancer. Prospective, nonrandomized noncontrolled study. Lymphocytes isolated from peripheral blood, lymph node, or bone marrow from dogs were incubated with BH3 mimetics for 24 hours. Viable cells were counted using flow cytometry and half maximal effective concentration (EC50 ) was calculated. BCL2 protein from whole cell lysates was assessed via immunoblots. Nodal B and T lymphocytes were more sensitive to VEN than circulating lymphocytes (P=.02). Neoplastic T lymphocytes were sensitive to VEN (mean EC50 ± SD =0.023 ± 0.018 μM), whereas most non-indolent B cell cancers were resistant to killing by VEN (mean EC50 ± SD=288 ± 700 μM). Unclassified leukemias showed variable sensitivity to VEN (mean EC50 ± SD=0.49 ± 0.66 μM). Detection of BCL2 protein was not associated with VEN sensitivity. Neoplastic canine T lymphocytes are sensitive to VEN in vitro. Quantification of BCL2 protein alone is insufficient to predict sensitivity to VEN.

Clinical features, natural history and outcomes of pseudolymphoma of liver: A case-series and systematic review

BackgroundPseudolymphoma is a rare, benign, nonspecific condition that forms a mass-like lesion characterized by the proliferation of non-neoplastic lymphocytes. Lacking of specific clinical symptoms, serological markers, and imaging features, the diagnosis is difficult. We reporte five cases of hepatic pseudolymphoma and provide a systematic review of existing literatures to improve our understanding of this rare liver disease. MethodsWe followed-up five cases of hepatic pseudolymphoma in West China Hospital from January 2002 to January 2022. We also summarized the cases of hepatic pseudolymphoma from January 1981 to December 2021 through the PubMed database and comprehensively analyzed the characteristics of the cases. ResultsThe pathologic features of the five cases were characterized by benign lymphoid tissue hyperplasia, lymphoid follicle formation, and a polarized germinal center. Immunohistochemistry, in situ hybridization, and gene rearrangement revealed non-malignant lymphoma. Besides, a total of 116 cases have been reported in the PubMed database from 1981 to 2021. The incidence of hepatic pseudolymphoma is higher in middle-aged and elderly women and has been reported more frequently in Asia. All cases were pathologically diagnosed, among which 85.95% of the patients were treated by surgery. ConclusionsHepatic pseudolymphoma is an extremely rare benign disease, mainly in middle-aged and elderly women. Without distinctive clinical and imaging characteristics, pathological diagnosis is the highly reliable method at present. Thus, in the absence of risk factors for a primary liver tumor or metastatic tumor in middle-aged and elderly women, the possibility of pseudolymphoma should be considered to avoid extensive treatments.

Pax5 and CD3 immunophenotyping of lymphoma in 2 central bearded dragons.

Two central bearded dragons (Pogona vitticeps), a 3-y-old male and a 5-y-old female, were diagnosed with different manifestations of lymphoma at the Kansas State Veterinary Diagnostic Laboratory between 2019 and 2020. The 3-y-old male was presented for postmortem evaluation and was in poor body condition. Microscopically, nearly all examined organs contained variable numbers of neoplastic round cells. Neoplastic cells in the stomach and liver had moderate immunoreactivity to CD3 consistent with multicentric T-cell lymphoma, and non-neoplastic lymphocytes infiltrating the stomach mass had strong immunoreactivity to Pax5. The 5-y-old female had an ulcerated oral mass located in the right lingual gingiva submitted as an excisional biopsy. Microscopically, the mass was composed of large numbers of neoplastic round cells in the epithelium and connective tissue that were strongly and diffusely positive for CD3 and frequently positive for Pax5, consistent with a dual-positive, localized, epitheliotropic T-cell lymphoma. Neoplastic and non-neoplastic lymphocytes did not stain with CD20 or CD79a. Neoplasms are increasingly reported as a cause of morbidity and mortality in reptiles. Our 2 cases illustrate various presentations of T-cell lymphoma and the effectiveness of CD3 and Pax5 immunohistochemistry in bearded dragons.

Small-sized type A thymoma with pulmonary metastasis: a case report

BackgroundType A thymomas comprise a homogenous population of neoplastic epithelial cells that are characterized by a spindle/oval shape without nuclear atypia. They may be accompanied by few non-neoplastic lymphocytes. Most type A thymomas are detected in the earlier Masaoka stages. Compared to other thymoma subtypes, they rarely metastasize or recur. There have been some reports of patients with type A thymomas with pulmonary metastasis; however, these thymomas were 20 mm or more in size. Herein, we report the case of a patient who underwent surgical resection for a small-sized type A thymoma (12 mm) with pulmonary metastasis.Case presentationA 62-year-old patient presented with an abnormal shadow in the left lung on plain chest radiography during a medical checkup. Chest computed tomography revealed a 12-mm tumor in the anterior mediastinum and a 13-mm nodule in the left lower lobe. 18F-fluorodeoxyglucose positron emission tomography/computed tomography revealed uptake in the anterior mediastinal tumor, but did not show a significant uptake in the pulmonary nodule. The patient underwent surgical resection on two separate occasions, and was diagnosed with an atypical type A thymoma and pulmonary metastasis. The TNM classification was p-T1aN0M1b stage IVb, and it was stage IVb according to the Masaoka staging system. No recurrence was observed during the follow-up.ConclusionsWe report a case of the smallest type A thymoma with pulmonary metastasis. Pulmonary metastasis secondary to a type A thymoma should be considered even if the thymoma is small in size (< 20 mm).

Neutrophil to T-Lymphocyte Ratio (NLR) and Neutrophil to Monocyte Ratio (NMR) Are Promising Biomarkers in Chronic Lymphocytic Leukemia Prognostic Assessment

Development of targeted drugs produced a significant changes in the management of patients with Chronic Lymphocytic Leukemia (CLL). The recent advances in molecular diagnostic techniques has provided a better prediction of disease progression but they are not easily accessible to all Institutions.CLL cells proliferation and survival are sustained by a complex microenvironmental network of cells interactions that are at least partially mediated through B cell receptor signaling.An increased number of studies describe the role of neutrophils and monocytes as markers of the inflammation and antitumor immunity that support tumorigenesis. Recently, authors published the increased NLR is associated to a prognosis in different cancer type.In our study we retrospectively evaluated the prognostic significance of peripheral blood neutrophils, monocytes and non-neoplastic lymphocytes in a unicentric, unselected, CLL cohort of 400 CLL patients at diagnosis (median age 64; stage Binet A 76%,B 18%,C 6%).Using blood count and the flow cytometric analysis reports, absolute neutrophil count (ANC), absolute monocyte count (AMC) and absolute T-lymphocyte count (ALC-CD3+) were evaluated and the neutrophil to T-lymphocyte ratio (NLR), the monocyte to T-lymphocyte ratio (MLR) and the neutrophil to monocyte ratio (NMR) were calculated at diagnosis, follow up and at relapse. Clinical and biological data from all patients were also retrieved to make a better comparison with the major clinical and prognostic markers commonly used.Compared to normal controls, CLL patients showed an increase AMC value (788±65 vs 469±51 cells/μL, p=0.005) and a slightly increased in the ALC-CD3+ value (2514±63 vs 2189±45 cells/μL, p=0.04). No difference in the ANC. The median NLR ratio was higher in CLL patients compared to healthy individuals (2.32 vs 1.7, p=0.02) and appeared more increased in early stage (2.65, 2.14, 1.95 mean value in Binet A, B, C respectively, p=0.0015). NMR median value was also significantly higher in early stage compared to advanced stage (8.0, 6.7, 5.1 in Binet A, B, C, p=0.0008).Conversely, MLR showed the opposite trend resulting more elevated in advanced stage at diagnosis, although with a low statistical significance.We did not found any correlation between these ratio and the IGVH status or genetic abnormalities.NLR and NMR higher level are associated with the absence of serum prognostic markers, such as CD38, and CD49d (p=0.002).160 out of 400 patients of our CLL cohort received a treatment during the course of disease. In our analysis, NLR and NMR are significantly higher in untreated patients than treated ones (median NLR 2.42 vs 1.95, p=0.0021; NMR 8.0 vs 7.0, p=0.0002). Among treated patients NLR and NMR significantly correlate with the time to treatment initiation (TTFT): lower NLR and NMR ratio correspond to a shorter TTFT (NLR p=0.03; NMR p=0.0035). For MLR the trend is opposite.A subgroup analysis conducted only on Rai 0/Binet A patients (n 219, 55%) showed that NLR and specially NMR are significantly associated with the risk of progression: lower NLR and NMR ratio correspond to an increased risk to symptomatic progression (p=0.0017), strengthening the hypothesis that high ratios are associated with a more indolent form of disease.In the 0/A population, analysis showed that a higher AMC and at a lesser extend a high ALC-CD3+ correlate with the progression rate and treatment initiation (Kaplan-Meier survival curve, p= 0.0001).Univariate analysis suggested that NLR ≥ 3.0, NMR ≥ 8.0 and AMC <900/mmc predicted a good prognosis in CLL patients. However, only NMR and AMC appeared as independent prognostic factors in multivariable analysis in Rai 0/Binet A patients.Our in vitro experiments supported these finding confirming: the non “immunosuppressive” role for neutrophils in CLL and the increased ability for monocytes to suppress T-lymphocytes activation and expand T-regulatory cell.In our study NLR ratio showed a positive prognostic role in CLL. A strongest protective meaning is displayed by NMR value. These combined value and AMC count provide more prognostic information at diagnosis and are associated with clinical outcome in CLL patients.These simple, cheap and reliable markers could be easily integrated into clinical practice reflecting both inflammatory status, immune response and microenviroment that contribute to disease progression. DisclosuresNo relevant conflicts of interest to declare.

Managing Metastatic Thymoma With Metabolic and Medical Therapy: A Case Report

Thymomas consist of neoplastic thymic cells intermixed with variable numbers of non-neoplastic lymphocytes. Metastatic thymomas are typically managed with non-curative chemotherapy to control tumor-related symptoms; no prolonged survival is expected. Metabolic-based approaches, such as fasting and ketogenic diets, target cancer cell metabolism by creating an increased reliance on ketones while decreasing glucose, glutamine, and growth factor availability, theoretically depriving cancer cells of their metabolic fuels while creating an unfavorable environment for cancer growth, which may be beneficial in metastatic thymoma. We report the case of a 37-year-old woman with myasthenia gravis, diagnosed with an inoperable type AB, stage IVA thymoma, who pursued a metabolic intervention consisting of periodic fasting (7-day, fluid-only fasts every 1–2 months), combined with a modified ketogenic diet on feeding days, for 2 years. Fasting-related adverse effects included cold intolerance, fatigue, and generalized muscle aches, all of which resolved during the second year. She experienced two myasthenia relapses, each associated with profoundly reduced oral intake, marked weight loss, and tumor regression-the first relapse was followed by a 32% decrease in tumor volume over 4 months, the second relapse by a dramatic 96% decrease in tumor volume over 4 months. The second relapse also required prednisone to control the myasthenia symptoms. We hypothesize that 2 years of fasting and ketogenic diet therapy metabolically weakened the neoplastic thymic cell component of the thymoma, “setting the stage” for immune activation and extreme energy restriction to destroy the majority of cancer cells during both relapses, while prednisone-induced apoptosis eradicated the remaining lymphocytic component of the thymoma during the second relapse. This case is unique in that a metabolic-based fasting and ketogenic diet intervention was used as the primary management strategy for a metastatic cancer in the absence of surgery, chemotherapy, or radiotherapy, culminating in a near-complete regression. Nearly 3 years after being diagnosed with inoperable metastatic cancer, our patient shows no signs of disease and leads a full and active life.

Heat shock protein 90α in thymic epithelial tumors and non-thymomatous myasthenia gravis

ABSTRACT Background Thymic epithelial tumors (TETs) are rare malignancies with unique association to the autoimmune disease myasthenia gravis (MG). Heat shock proteins (HSPs) harbor great potential as cancer biomarkers and HSP inhibitors approach clinical cancer therapy. Methods To explore HSP pathophysiology, we assessed sera (immunoassays) and tissues (immunohistochemistry) of TETs (and thymic tissues) for HSP27, phosphorylated (p)HSP27, HSP70 and HSP90α expression in 114 TETs and 26 non-thymomatous MG patients undergoing extended thymectomy. Results Serum concentrations of HSP90α were significantly increased in patients with thymic carcinomas, thymomas, thymic neuroendocrine tumors and non-thymomatous MG compared to patients who underwent thymectomy revealing regular thymic morphology or controls. In thymoma patients, high serum HSP90α represented a significantly worse prognostic factor for free-from-recurrence, and complete tumor resection led to decreased levels. The expression of HSP90 in nuclei and cytoplasm of tumor cells and non-neoplastic lymphocytes varied with WHO histological subtype. HSP90 was expressed in centroblasts of thymic germinal centers in MG patients. Higher pHSP27 serum concentrations were observed in seropositive MG and those not treated with steroids. Conclusions HSP data suggest high potential for HSPs as TET cancer biomarkers or as candidates for targeted therapy. Caution is warranted in TET patients with associated MG overexpressing HSPs.

Prognostic role of non-neoplastic lymphocytes in lymph node aspirates from dogs with diffuse large B-cell lymphoma treated with chemo-immunotherapy

Dogs with Diffuse Large B-Cell Lymphoma (DLBCL) benefit from the addition of active immunotherapy to traditional chemotherapy. We hypothesized that immune cells within neoplastic lymph nodes (LNs) may play a role in the tumor pathobiology and treatment response.The present study describes the composition and prognostic role of non-neoplastic lymphocytes in LNs of 59 dogs with treatment-naive DLBCL receiving chemo-immunotherapy.The percentage of small non-neoplastic cells and of CD5+, CD21+, CD4+ and CD8+ small cells was recorded via flow cytometry. CD4+/CD8+ and CD5+/large CD21+ cell ratios were calculated.The likelihood of progression significantly diminished with increasing percentage of small cells, CD5+ and CD8+ small cells, and CD5+/large CD21+ cell ratio, with decreasing CD4+/CD8+ ratio and in non-anemic dogs.Active immunotherapy is more effective in dogs with higher percentage of non-neoplastic lymphocytes at diagnosis. We lay the ground for future studies assessing the role of the immune system in the pathobiology of canine DLBCL.

Luteinizing hormone receptor expression by nonneoplastic and neoplastic canine lymphocytes.

To investigate luteinizing hormone (LH) receptor expression in canine nonneoplastic and neoplastic lymph nodes, circulating nonneoplastic lymphocytes, and T-cell lymphoma (TCL) cell lines. Formalin-fixed, paraffin-embedded lymph nodes (5 neoplastic and 3 nonneoplastic) from 6 dogs, circulating lymphocytes from venous blood specimens obtained from 12 healthy dogs, and 3 TCL cell lines derived from 3 dogs with primary lymphoma. Lymph node specimens were immunohistochemically stained for determination of LH receptor expression. Circulating nonneoplastic lymphocytes and TCL cell lines were evaluated for LH receptor expression by use of flow cytometry; circulating lymphocytes were also immunophenotyped. The mean percentage of cells positive for LH receptors was determined for each type of specimen. For the healthy dogs, percentages of circulating B and T lymphocytes that expressed LH receptors were assessed on the basis of sex and reproductive status. The mean percentage of LH receptor-positive cells in canine neoplastic and nonneoplastic lymph nodes was 12.4% and 4.1%, respectively. For the healthy dogs, the mean percentage of circulating LH receptor-positive T lymphocytes was significantly higher in gonadectomized dogs (16.6%) than in sexually intact dogs (10.5%); the percentages of circulating LH receptor-positive B lymphocytes did not significantly differ by reproductive status. Among the 3 canine TCL cell lines, LH receptor expression ranged from 10% to 45%. In this study, LH receptor expression by canine neoplastic and nonneoplastic lymphocytes was detected. Research into the effects of downregulation of LH receptor activation in dogs with lymphoma is warranted.

Cytological tumour cell characteristics and reactive small lymphocytes influence patient prognosis in acute and lymphoma type adult T-cell leukaemia/lymphoma.

Acute and lymphoma type adult T-cell leukaemia/lymphoma (ATLL) patients show an aggressive clinical course. While some clinical signs indicate good prognosis, definitive cytohistological prognostic factors have yet to be described. We classified 65 ATLL patients into three groups by tumour cell size and nuclear pleomorphism on fine-needle aspiration and tumour touch smear samples. Semi-quantitative analysis of background small lymphocytes, reactive CD20-positive B cells and CD8-positive T cells was performed. Thirty-one patients had pleomorphic lymphoma with predominantly medium-sized cells and coarse granular nuclei. Another 24 patients showed pleomorphic large cell lymphoma with stippled chromatin. The remaining 10 demonstrated monomorphic large lymphoma cells with fine granular chromatin. Patients with pleomorphic lymphoma with medium-sized cells showed significantly higher serum lactate dehydrogenase and lower CD30 and C-MYC expression in lymphoma cells than the other two groups (P = .0216, P < 0.01, respectively). Patients with pleomorphic medium-sized ATLL had few usual small lymphocytes observed on routine morphological examination and showed less concurrent detection of CD20-positive B cells and CD8-positive T cells, both of which were lower than in the other two groups (P = .006, P = .019, respectively). Furthermore, ATLL patients with predominantly medium-sized lymphocytes exhibited a worse prognosis than patients with pleomorphic large cells (P = .0197). Background small lymphocytes and concurrent detection of CD20-positive B cells and CD8-positive T cells may thus be good prognostic factors (P = .011, P = .021, respectively). Morphological features, size of neoplastic cells and background non-neoplastic lymphocyte (B cells and CD8-positive T cells) volume appear to influence the prognosis of patients with aggressive-type ATLL.

Volume, conductance, and scatter parameters of neoplastic and nonneoplastic lymphocytes using Coulter LH780.

PURPOSE:Automated hematology analyzers yield a complete hematological profile including a complete blood count and a differential white blood cell count. The differential count is based on analyses of three parameters, namely, volume, conductance, and scatter (VCS). We aimed to evaluate the VCS parameters, histograms, and scatterplots of neoplastic and nonneoplastic lymphocytes.MATERIAL AND METHODS:Patients were grouped into four categories, namely, acute lymphoblastic leukemia (ALL), chronic systemic disorders, chronic lymphocytic leukemia (CLL), and acute viral disease. Lymphocytes from all four groups were compared with lymphocytes from normal participants.RESULTS AND CONCLUSIONS:The histogram for acute viral disease showed a trough at T1, which was slightly obliterated, and the F1 curve mildly extended to the right. The T1 for ALL was replaced with a peak at >40% of the preset limit. The F1 peak was shifted to left for CLL. The scatterplot for viral disease showed lymphocytes extending to the variant lymphocyte window. The lymphocytes of ALL extended to the blast window, with both increase in volume and mild increase in scatter. The lymphocytes in CLL were smaller and located below the normal lymphocyte region. Mean lymphocyte volume was significantly increased in ALL and was significantly decreased in CLL. Mean lymphocyte conductance was significantly increased in CLL and significantly decreased in both acute viral disease and ALL. Mean lymphocyte scatter was significantly decreased in acute viral disease and significantly increased in ALL.

Mitochondrial and glycolytic metabolic compartmentalization in diffuse large B-cell lymphoma

Metabolic heterogeneity between neoplastic cells and surrounding stroma has been described in several epithelial malignancies; however, the metabolic phenotypes of neoplastic lymphocytes and neighboring stroma in diffuse large B-cell lymphoma (DLBCL) is unknown. We investigated the metabolic phenotypes of human DLBCL tumors by using immunohistochemical markers of glycolytic and mitochondrial oxidative phosphorylation (OXPHOS) metabolism. The lactate importer MCT4 is a marker of glycolysis, whereas the lactate importer MCT1 and TOMM20 are markers of OXPHOS metabolism. Staining patterns were assessed in 33 DLBCL samples as well as 18 control samples (non-neoplastic lymph nodes). TOMM20 and MCT1 were highly expressed in neoplastic lymphocytes, indicating an OXPHOS phenotype, whereas non-neoplastic lymphocytes in the control samples did not express these markers. Stromal cells in DLBCL samples strongly expressed MCT4, displaying a glycolytic phenotype, a feature not seen in stromal elements of non-neoplastic lymphatic tissue. Furthermore, the differential expression of lactate exporters (MCT4) on tumor-associated stroma and lactate importers (MCT1) on neoplastic lymphocytes support the hypothesis that neoplastic cells are metabolically linked to the stroma likely via mutually beneficial reprogramming. MCT4 is a marker of tumor-associated stroma in neoplastic tissue. Our findings suggest that disruption of neoplastic-stromal cell metabolic heterogeneity including MCT1 and MCT4 blockade should be studied to determine if it could represent a novel treatment target in DLBCL.

The Combined Evaluation of Neutrophil, T-Lymphocyte and Monocyte Counts Provides New Prognostic Information in CLL Patients

The recent advances in cytogenetic and molecular diagnostic techniques has provided a better understanding of biology of CLL and a better prediction of disease progression and refractoriness but they are not easily accessible to all Institutions.CLL cells depend on microenvironmental interactions for proliferation and survival that are at least partially mediated through B cell receptor (BCR) signaling. An increased number of studies describe the emerging role of neutrophils and monocytes as mediators of the inflammation process and antitumor immunity modulation that support tumorigenesis and tumor progression. The neutrophil to lymphocyte ratio (NLR), has been recently published as new marker of systemic inflammation associated with outcome in different cancer types.In our study we retrospectively evaluated the prognostic significance of peripheral blood neutrophils, monocytes and non-neoplastic lymphocytes in a unicentric, unselected, CLL cohort of 400 CLL patients (160 were treated). The study was approved by the institutional board review;informed consent was obtained from patients.Using the blood count and the Flow cytometric analysis reports, peripheral blood absolute neutrophil count (ANC), absolute monocyte count (AMC) and absolute T-lymphocyte count (ALC-CD3+) were evaluated and the neutrophil to T-lymphocyte ratio (NLR), the monocyte to T-lymphocyte ratio (MLR) and the neutrophil to monocyte ratio (NMR) were calculated. Clinical and biological data from all patients were also retrieved. Serial count and ratio were evaluated at diagnosis, during follow up and at relapse to make a better comparison with the major clinical and prognostic markers commonly used.The median ratio for NLR 2.67 and patients with pre-treatment NLR > 3 had a shorter time from diagnosis to treatment initiation. CLL patients showed an increase in the absolute number of monocytes compared to normal controls (788±65 cells/μL vs 469±51 cells/μL, p=0.005) and MLR appeared higher in advanced stage (stage Binet C) and bulky disease (p=0.06). High level of both NLR and MLR (cut off >3) correlated with the presence of a complex karyotype detected by FISH (p=0.03, p=0.016). NLR ratio was associated with the absence of serum prognostic markers, such as the expression of CD38, ZAP-70 and CD49d. This result, apparently conflicting, could strengthen the NLR as an independent prognostic factor.NMR was higher in asymptomatic patients (absence vs. presence of B symptoms, p=0.02) and this data resulted independent from disease stage. NMR median value was significantly higher in untreated patients than in patients who received treatment (p=0.01), strengthening the hypothesis that this ratio is associated with a more indolent form of disease. In this contest, the subset of patients with CD49d positive disease showed the lowest NMR value. This data seems of relevance, being CD49d a recently discovered and validated prognostic marker.ANC/ALC and AMC/ALC ratio significantly increased at relapse compare to baseline (NLR average onset 2.3±0.4 vs 3.4±0.6 at relapse). The median NMR value showed conversely the opposite trend: NMR is reduced in first relapse (NMR average onset 7.2±0.4 vs. 5.4±0.5 at relapse).These combined ratios reflect both the inflammatory status, the immune response and the microenviromental network that contribute to aggressive tumor biology and disease progression. They are simple, cheap, easily measured and reproducible and can be integrated into daily clinical practice as new prognostic markers for CLL. DisclosuresChiarenza:Gilead: Consultancy; Janssen: Consultancy; Roche: Consultancy.

Case report of unusual presentation of invasive thymoma in A 49 years old male

Invasive thymomas are rare tumours in the anterior mediastinum, representing 50% of anterior mediastinal masses and about 20-30% of all mediastinal tumours. They are of unknown etiology; about 50% of patients with thymomas are diagnosed incidentally with chest radiography. Thymoma is classified into different stages, which determine the prognosis and type of management, the standard primary treatment for these tumours is Thymectomy. This case study presents a 49 year-old man with unusual presentation of thymoma. On the non-contrast CT images, there was a welldefined heterogeneous anterior mediastinal mass adjacent to the right border of the heart. There was evidence of infiltrations into the anterior mediastinal fat but no mediastinal lymphadenopathy after contrast enhancement, the mass showed heterogeneous enhancement. CT guided trucut tissue biopsy and histological analysis of the mass showed that the tumour consists of neoplastic epithelial cells and non-neoplastic lymphocytes. The findings were consistent with invasive lymphoepithelial thymoma.

Reactive Lymphoid Hyperplasia Treated with Radiofrequency Ablation

Reactive lymphoid hyperplasia (RLH) is a rare benign liver mass that is characterized by proliferation of non-neoplastic lymphocytes extranodally. To date, only 43 cases have been reported in the English literature and 2 cases in the Korean literature. We report a case of hepatic RLH in a 36-year-old woman who had been diagnosed two years previously with an intrahepatic tumor that measured 0.6 × 1.0 cm on abdominal ultrasonography. Her medical history was otherwise unremarkable. On follow-up ultrasonography, the hepatic mass had increased in size. We biopsied the liver mass and the histopathologic findings confirmed reactive lymphoid hyperplasia. Radiofrequency ablation (RFA) was performed instead of surgical resection. The patient is currently doing well with no sign of relapse 1 year and 8 months after the RFA. (Korean J Med 2015;88:177-181)

Corpuscular thymoma, case report and review of literature

Aim Hassall corpuscles are concentric arrangement of keratinizing epithelial cells that are restricted to the medulla of the normal thymus. Hassall corpuscles in thymomas are unusual findings and are seen in thymomas WHO type B1-3 with medullary differentiation, but rare cases have been documented. Thymoma with abundant Hassall corpuscles (corpuscular thymoma) must be differentiated from thymic carcinoma, well differentiated keratinizing squamous cell carcinoma type. Case report We report a case of a 73-year-old male patient, presenting with cough for one year. Computed tomography (CT) scan showed a well defined anterior superior mediastinal mass. Surgical excision of the mass was performed. Gross examination revealed a large, oval shaped, well circumscribed, encapsulated, firm brown mass measuring 10 × 6 × 4 cm, and weighing 219 g, with heterogeneous pale white solid, small cystic spaces, and necrotic cut surfaces. Light microscopic examination revealed two alternating components, the first forming a delicate loose network of neoplastic epithelium equally admixed with non-neoplastic lymphocytes, and the second forming vague solid sheets with paucity of lymphocytes. Both components show abundant Hassall corpuscles, extensive areas of coagulative tumor necrosis, small cystic spaces, and cholesterol clefts. Immunohistochemical studies showed that the neoplastic epithelium is strongly and diffusely immunoreactive for HMWCK, panCK, and EMA, and focally positive for CAM5.2, and negative for CD5, and C-KIT immunohistochemical stains. Conclusion Corpuscular thymoma reported in the literature as WHO type B2 or B3. Our presented case is of type B3, Modified Masoaka stage 1. We believe that thymoma with corpuscular differentiation is a rare distinct variant of organotypical thymomas, although it is not mentioned in the 2004 WHO classification. Recognition of this rare variant of thymomas is very important to avoid confusion with thymic carcinoma and squamous cell carcinoma type. Careful microscopic examination and immunohistochemical stains are mandatory to differentiate between these entities.

Immunohistochemical detection of arginase-I expression in formalin-fixed lung and other tissues

Arginases are a family of enzymes that convert l-arginine to l-ornithine and urea. Alterations in expression of the isoform arginase-I are increasingly recognized in lung diseases such as asthma and cystic fibrosis. To define expression of murine arginase-I in formalin-fixed tissues, including lung, an immunohistochemical protocol was validated in murine liver, a tissue that has distinct zonal arginase-I expression, making it a useful control. In the lung, arginase-I immunostaining was observed in airway surface epithelium and this decreased from large to small airways, with a preferential staining of ciliated epithelium versus Clara cells and alveolar epithelia. In submucosal glands, the ducts and serous acini had moderate immunostaining, which was absent in mucous cells. Focal immunostaining was observed in alveolar macrophages, endothelial cells, pulmonary vein cardiomyocytes, pulmonary artery smooth muscle, airway smooth muscle, and neurons of ganglia of the lung. Arginase-I immunostaining was also detected in other tissues including salivary glands, pancreas, liver, skin, and intestine. Differential immunostaining was observed between sexes in submandibular salivary glands; arginase-I was diffusely expressed in the convoluted granular duct cells of females, but was rarely noted in males. Strain specific differences were not detected. In a mouse with an incidental case of lymphoma, neoplastic lymphocytes lacked arginase-I immunostaining, in contrast to immunostaining detected in non-neoplastic lymphocytes of lymphoid tissues. The use of liver tissue to validate arginase-I immunohistochemistry produced consistent expression patterns in mice, and this approach can be useful to enhance consistency of arginase-I immunohistochemical studies.