Amomum subulatum Induces Apoptosis in Tumor Cells and Reduces Tumor Burden in Experimental Animals via Modulating Pro-Inflammatory Cytokines.

This study investigates the anticancer potential of methanolic extract of A. subulatum dry fruits (MEAS) in Dalton's Lymphoma Ascites (DLA) cells in vitro and on DLA induced ascitic and solid tumor-bearing mice. MEAS induced apoptosis in DLA cells and MEAS administration effectively reduced tumor burden, and increased life span via modulating pro-inflammatory cytokines and regulating NF-κB pathway. MEAS seemed to be much safer than the standard drug cyclophosphamide, as the latter was associated with adverse effects such as body weight loss, depletion of hemoglobin level and hepatotoxicity, suggesting A. subulatum as a potential nutraceutical against cancer.

Utility of a new notation to visualize flow cytometry analysis results: first preliminary comparison with immunohistochemistry to detect CD30 expression on T-cell lymphoma cells.

BACKGROUND: It is important to confirm CD30 expression in T-cell lymphoma cases, but immunohistochemical staining for CD30 is not commonly performed and no comparison has been done between the results of flow cytometry (FCM) and immunohistochemical staining for CD30. Therefore, we devised a notation that we termed proportion of immunoreactivity/expression for FCM (PRIME-F notation), based on the cellular proportion showing different antigen-antibody reactivity. METHODS: We retrospectively compiled 211 cases of T-cell lymphoma, assessed via FCM, from major hospitals in Miyagi Prefecture from January 2012 to January 2019, and compared 52 of these cases with the immunohistochemical immunoreactive (IR) pattern of CD30 (PRIME-I notation). The PRIME-F notation was divided into five levels: notations starting with "-" followed by 3, 2, and 1 ">" correspond to level-I, level-II, or level-III; notations starting with "(dim)+" correspond to level-IV; and those starting with "+" or "(bright)+" correspond to level-V. RESULTS: The 52 cases of PRIME-F notation with "+" included 16 cases of peripheral T-cell lymphoma (PTCL/NOS), 3 of follicular T-cell lymphoma (FTL), 3 of angioimmunoblastic T-cell lymphoma (AITL), 6 of extranodal NK/T-cell lymphoma/nasal type (ENKL), 18 of adult T-cell lymphoma (ATL), and 6 cases of anaplastic large cell lymphoma (ALCL). Eight of the 52 cases were immunohistochemically CD30-negative. In the PRIME-F level-I to III group (excluding false-positive cases), 21.7% (5 out of 23 cases) were < 10% positive for CD30 upon immunohistochemistry (IHC). Contrarily, in the level-IV & -V group, no CD30 positivity rate of < 10% upon IHC was found (0%) (p = 0.0497). In level-IV, 42.9% of cases presented a CD30 negative rate > 1/3 upon IHC, while in level-V, only 7.1% (one out of 14 cases) did. The CD30 negative rate tended to be low (p = 0.0877) in level-V. CONCLUSIONS: To our knowledge, this is the first report describing the correspondence between FCM and immunohistochemistry findings for CD30 through newly proposed notations. The PRIME-F and PRIME-I notations for CD30 showed a minor positive correlation. The PRIME notation is considered universally applicable to antibodies, and notations of both FCM and IHC show great potential for big data.

Soluble programmed cell death protein 1 (sPD-1) and the soluble programmed cell death ligands 1 and 2 (sPD-L1 and sPD-L2) in lymphoid malignancies.

BACKGROUND: The programmed cell death protein 1 (PD-1) and its ligand 1 and 2 (PD-L1/PD-L2) regulate the immune system, and the checkpoint pathway can be exploited by malignant cells to evade anti-tumor immune response. Soluble forms (sPD-1/sPD-L1/sPD-L2) exist in the peripheral blood, but their biological and clinical significance is unclear. METHOD: Time-resolved immunofluorometric assay (TRIFMA) and enzyme-linked immunosorbent assay (ELISA) were used to measure sPD-1, sPD-L1, and sPD-L2 levels in serum from 131 lymphoma patients and 22 healthy individuals. RESULTS: Patients had higher sPD-1 and sPD-L2 levels than healthy individuals. In diffuse large B-cell lymphoma, patients with high International Prognostic Index score had higher sPD-1 levels and sPD-L2 levels correlated with subtype according to cell of origin. Compared to other lymphoma types, follicular lymphoma displayed higher sPD-1 and lower sPD-L1 levels along with lower ligand/receptor ratios. CONCLUSION: This is the first study to simultaneously characterize pretherapeutic sPD-1, sPD-L1, and sPD-L2 in a variety of lymphoma subtypes. The relation between higher sPD-1 levels and adverse prognostic factors suggests a possible biological role and potential clinical usefulness of sPD-1. Moreover, the reverse expression pattern in follicular lymphoma and T-cell lymphoma/leukemia may reflect biological information relevant for immunotherapy targeting the PD-1 pathway.

Characterizing circulating nucleosomes in the plasma of dogs with lymphoma.

BACKGROUND: Nucleosomes consist of DNA wrapped around a histone octamer core like beads on a string so that DNA can be condensed as chromatin into chromosomes. Diseases such as cancer or inflammation lead to cell death where chromatin is fragmentated and released as mononucleosomes into the blood. The Nu.Q™ H3.1 assay measures total nucleosome concentration in plasma of humans and has been used to detect and identify cancer even at early stages. The objectives of this study were to determine if nucleosome levels could be used to distinguish between healthy dogs and dogs with various stages of lymphoma (LSA) using the Nu.Q™ H3.1 assay. A total of 126 dogs diagnosed with LSA and 134 healthy controls were recruited for this study. Plasma was collected from each dog and stored in K2-EDTA tubes. The LSA patient samples were recruited from TAMU or purchased from various biobanks. All control cases were recruited from TAMU. RESULTS: Dogs with LSA had an approximately 7-fold increase in their plasma nucleosome concentrations compared to controls (AUC 87.8%). Nucleosome concentrations increased with cancer stage and dogs with B cell lymphomas had significantly higher nucleosome concentrations than dogs with T cell lymphomas. CONCLUSIONS: The Nu.Q™ H3.1 assay was able to reliably detect elevated nucleosome concentrations in the plasma of dogs with LSA. Furthermore, it appears that nucleosomes are useful for differentiating cancer from healthy individuals in canines.

Delayed diagnosis of Angioimmunoblast T-cell lymphoma presenting with type II Cryoglobulinemia and acute kidney injury: a case report and narrative review of the literature.

BACKGROUND: Angioimmunoblastic T cell lymphoma (AITL) is an infrequent hematological malignancy with variable and often atypical presentations. The presence of dysproteinemia, autoantibodies and systemic involvement in AITL has often led to a delay in diagnosis or even misdiagnosis in practice. We herewith present a case of AITL that primarily presented with acute kidney injury associated with type II Cryoglobulinemia, the underlying cause was only identified 8 months after the emergence of initial symptoms. CASE PRESENTATION: A 67-year old woman presented with 2-month history of intermittent joint pain and a 3-day history of bilateral lower limb edema and acute kidney injury. Initial laboratory investigations showed marked hypocomplementemia with positive autoantibodies of ANA, anti-cardiolipin-IgM and direct antiglobulin. The serum and urinary Immunofixation and serum cryoglobulin tests were negative, while the serum free κ to λ light chain ratio was 0.231. A renal biopsy showed a diffuse proliferative glomerulonephritis with intracapillary pseudothrombi formation. There were orderly arranged microtubular structures of 20-35 nm in diameter in the subendothelial and mesangial area on electron microscopy. Shortly afterwards, the patient developed tingling affecting her finger tips and weak hands and legs. A diagnosis of cryoglobulinemia complicated with cryoglobulinemic glomerulonephritis and polyneuropathy was made. She responded well to methylprednisolone, plasma exchange and rituximab. However, 3 months later, she presented with generalized pruritic rash, weight loss, and inguinal lymphadenopathy. A subsequent inguinal excisional lymph node biopsy at month 8 revealed AITL as the underlying disease. CONCLUSIONS: AITL and its associated B cell dysregulation can give rise to autoimmunity and cryoglobulinemia which may conceal itself as the underlying disorder. In various clinical scenarios of auto-immune diseases, it is advisable that the clinicians should take into consideration the multi-faceted lymphoma.

Immunoglobulinopathies in patients with angioimmunoblastic T-cell lymphoma.

AIM: The aim of the study was to characterize quantitative and qualitative immunoglobulinopathies in patients with AITL at the onset of the disease. MATERIALS AND METHODS: 55 patients with newly diagnosed AITL were enrolled in the study, the male/female ratio was 30/25; median age was 61 (29-81) years. Diagnosis was based on standard WHO criteria. Immunochemical studies of blood serum included serum protein electrophoresis/immunofixation, nephelometric quantification of total immunoglobulins, serum free light chain assay. RESULTS: Quantitative and qualitative immunoglobulinopathies were determined in 49 (89,1%) of 55 pts. Quantitative immunoglobulinopathies were revealed in 47 (85.5%) of 55 cases, qualitative - in 14 (25,5%). Combination quantitative and qualitative immunoglobulinopathies was observed in 12 (21,8%) of 55 pts. The detected immunoglobulinopathies were divided into 4 groups: polyclonal hypergammaglobulinaemia, hypogammaglobulinaemia, oligoclonal gammapathy, and monoclonal gammapathy. Polyclonal hypergammaglobulinaemia was marked in 41 (74.5%) of 55 pts, elevated level of IgG was determined in 27 (49,15%) of 55 cases, IgM - in 18 (32,7%) and IgA - in 21 (38.2%). Interestingly, polyclonal IgE hypergammaglobulinaemia was detected in 12 (48,0%) of 25 cases of performed studies. Hypogammaglobulinaemia was detected in 8 (14,5%) of 55 cases. Oligoclonal gammapathy was determined in 4 (7.3%) of 55 pts. Monoclonal gammapathy was revealed in 11 (20,0%) of 55 cases. The amount of monoclonal immunoglobulin varied from 2.6 to 14.1 g/l. Monoclonal immunoglobulin Gk was detected in 5 of 11 pts, Gλ - in 2, Mλ - in 2, Mk - in 2. Monoclonal gammapathy was accompanied by polyclonal hypergammaglobulinaemia in 9 of 11 cases, hypogammaglobulinaemia - in 2. CONCLUSION: Quantitative and qualitative immunoglobulinopathies are observed in most patients at the onset of AITL. Quantitative abnormalities were determined more often than qualitative. Monoclonal gammapathy can be a manifestation of lymphoproliferation and other concomitant disorders. The prognostic value of immunochemical parameters is still unclear and requires dynamic observation and study.

Detection of the circulating tumor DNAs in angioimmunoblastic T- cell lymphoma.

Recent genetic studies identified that the disease-specific G17V RHOA mutation, together with mutations in TET2, DNMT3A, and IDH2, is a hallmark of angioimmunoblastic T cell lymphomas (AITL). The diagnostic value of these mutations is now being investigated. Circulating tumor DNAs (ctDNAs) may offer a non-invasive testing for diagnosis and disease monitoring of cancers. To investigate whether these mutations are useful markers for ctDNAs in AITL and its related lymphomas, we performed targeted sequencing for TET2, RHOA, DNMT3A, and IDH2 in paired tumors and cell-free DNAs from 14 patients at diagnosis. Eighty-three percent of mutations detected in tumors were also observed in cell-free DNAs. During the disease course, mutations were detectable in cell-free DNAs in a refractory case, while they disappeared in a chemosensitive case. These data suggest that the disease-specific gene mutations serve as sensitive indicators for ctDNAs and may also be applicable for non-invasive monitoring of minimal residual diseases in AITL.

Comprehensive serum cytokine analysis identifies IL-1RA and soluble IL-2Rα as predictors of event-free survival in T-cell lymphoma.

BACKGROUND: T-cell malignancies are heterogeneous in their clinical presentation and pathology, and have a poor prognosis. New biomarkers are needed to predict prognosis and to provide insights into signal pathways used by these cells. The goal of this study was to evaluate pretreatment serum cytokines in patients with newly diagnosed T-cell neoplasms and correlate with clinical outcome. PATIENTS AND METHODS: We evaluated 30 cytokines in pretreatment serum from 68 untreated patients and 14 normal controls. Significantly elevated cytokines were correlated with patterns of abnormalities, event-free survival (EFS) and overall survival (OS). RESULTS: Our data demonstrated significantly elevated levels (versus controls) of seven cytokines-epidermal growth factor (EGF), IL-6, IL-12, interferon gamma-induced protein (IP)-10, soluble interleukin (sIL)-2Rα, monokine induced by gamma interferon (MIG), and IL-1RA-in all T-cell neoplasms (P < 0.05). In the angioimmunoblastic subset, all seven cytokines except IP-10 and in the peripheral T-cell lymphoma (TCL)-not otherwise specified subset, only IP-10, sIL-2Rα, MIG, and IL-8 were statistically elevated compared with control. Of these, elevated cytokines all but EGF were predictive of an inferior EFS; IL-1RA, sIL-2Rα, and MIG predicted an inferior OS. In a multivariate analysis, sIL-2Rα [hazard ratio (HR) = 3.95; 95% confidence interval (CI) 1.61-8.38] and IL-1RA (HR = 3.28; 95% CI 1.47-7.29) levels remained independent predictors of inferior EFS. TCL cell lines secreted high levels of sIL-2Rα and expressed the IL-2Rα surface receptor. CONCLUSIONS: This report describes the cytokines relevant to prognosis in patients with untreated TCL and provides the rationale to include serum IL-1RA and sIL-2Rα as biomarkers in future trials. Inhibition of these cytokines may also be of therapeutic benefit.

A case of angioimmunoblastic T-cell lymphoma with high serum VEGF preceded by RS3PE syndrome.

We report the case of a 76-year-old man diagnosed with angioimmunoblastic T-cell lymphoma (AITL) with high serum vascular endothelial growth factor (VEGF) preceded by Remitting seronegative symmetrical synovitis with pitting edema syndrome. He suffered respiratory discomfort caused by large amounts of pleural effusion. Interestingly, changes in serum VEGF measured over time were similar to changes in pleural effusion. Whether VEGF is related to the pathological condition of AITL is a very important question.

Evaluation of lymphocytes inactivation by extracorporeal photopheresis using tetrazolium salt based-assay.

Extracorporeal photopheresis (ECP) is accepted as a second-line therapy for the treatment of acute and chronic steroid-refractory graft versus host disease (GvHD), cutaneous T-cell lymphoma and solid organ transplantation. ECP should be validated: we compared in parallel apoptosis and proliferation analysis of patient lymphocytes treated with 8-MOP ECP using respectively Annexin V/7-aminoactinomycin D (7-AAD) and CFSE with a tetrazolium salt (WST-1) method. Using WST-1 assay we found a significant decrement (p < 0.01) of metabolic activity at 4 days between ECP-treated and untreated cells. This finding was confirmed by the significant decrease of cell proliferation and increase of cell death observed by CFSE and 7AAD-Annexin V, respectively. Accordingly, once validated against a reference method, WST-1 could represent a rapid and easy assay for routinely quality control of ECP.

Dose-Effect Relationship of Alkylating Agents on Testicular Function in Male Survivors of Childhood Lymphoma.

The purpose of our study was to assess the gonadal function in male survivors of childhood lymphoma. We studied 171 male survivors of childhood lymphoma (83 with B-cell non-Hodgkin lymphoma [B-NHL], 32 with T-cell non-Hodgkin lymphoma [T-NHL], 50 with Hodgkin lymphoma [HL], and 6 with anaplastic large-cell lymphoma [ALCL]), measuring follicle-stimulating hormone [FSH] and luteinizing hormone [LH] levels at a median age of 21.1 (17-30.4) years after a median delay of 9.3 (2-22.4) years from treatment. FSH levels were above normal range (≥10 IU/L) in 42.1% and LH levels ≥8 IU/L in only 8.9% of survivors. In multivariate analysis, only the following chemotherapeutic agents were associated with higher FSH or LH levels: cyclophosphamide (P < .0001, .04), lomustine (CCNU; P = .002, 0.04), and procarbazine (P < .0001, .07). No significant correlation was found between FSH or LH levels and age or pubertal status at diagnosis. Mean FSH level was significantly lower in NHL survivors treated more recently: 6 ± 5.1 IU/L in B-NHL survivors treated since 1986 versus 12.3 ± 5.4 IU/L for those treated before 1981 (P = .0001), and 6.8 ± 9.6 IU/L in T-NHL survivors treated since 1989 versus 9.4 ± 5.7 IU/L for those treated before 1989 (P = .035). In HL, mean FSH level was 12.4 ± 9.9 IU/L following procarbazine containing chemotherapy versus 3.4 ± 1.9 IU/L in the absence of procarbazine and increased significantly with the number of MOPP/OPPA (mechlorethamine, Oncovin [vincristine], procarbazine, and prednisone/Oncovin, procarbazine, and prednisone, and Adriamycin [doxorubicin]) courses received, from 6.8 ± 5.7 IU/L for 1-2 MOPP/OPPA to 12.6 ± 7.5 for 3-4 MOPP/OPPA and 19.6 ± 13.3 for more than 4 MOPP/OPPA (P for trend = .006). Testicular toxicity of alkylating agents on childhood lymphoma survivors is dose dependent and not correlated to diagnosis, age, or pubertal status at diagnosis.

Peripheral blood sCD3⁻ CD4⁺ T cells: a useful diagnostic tool in angioimmunoblastic T cell lymphoma.

Angioimmunoblastic T cell lymphoma (AITL) belongs to the subgroup of mature T cell lymphomas according to the World Health Organization and is one of the common T cell lymphomas in Western countries. Particularly in cases in which histological confirmation cannot be easily achieved, immunophenotyping of peripheral blood can give important information for the differential diagnosis of AITL. sCD3⁻ CD4⁺ T cells are a typical feature of AILT in flow cytometry of peripheral blood. In this retrospective study, the diagnostic value of flow cytometry for the diagnosis 'AITL' was assessed by comparing the frequency of sCD3⁻ CD4⁺ T cells in leukemic AITL patients and in patients with other leukemic CD4⁺ T cell lymphomas. Immunophenotyping of peripheral blood by flow cytometry was performed in a lymphocyte gate using fluorochrome-labelled antibodies against CD3, CD2, CD4, CD5, CD7, CD8, CD10, CD14, CD16, CD19, CD56, CD57 and T cell receptor. In 17/17 leukemic AITL patients, a small but distinct population of sCD3⁻ CD4⁺ T cells was detected (mean percentage of sCD3⁻ CD4⁺ T cells in the lymphocyte gate: 11.9 ± 15.4%, range 0.1-51.8%). In contrast, sCD3⁻ CD4⁺ T cells were found in only 1/40 patients with other leukemic CD4⁺ T cell lymphomas (one patient with mycosis fungoides). sCD3⁻ CD4⁺ T cells have a high positive predictive value (94%) for the diagnosis 'AITL'. Flow cytometry is particularly useful in the differential diagnosis of AITL, even if the aberrant T cell population has a very low frequency. Further biological characterization of this subfraction of lymphoma cells is warranted.

Serum IgA level, monocyte count, and international prognostic index are independently associated with overall survival in patients with HTLV-I-negative nodal peripheral T cell lymphoma.

Peripheral T cell lymphomas (PTCL) account for 10-15 % of non-Hodgkin's lymphomas and are associated with poor prognosis. Although many prognostic factors for PTCL have been proposed, the heterogeneity of PTCL seems to be an obstacle in the establishment of clinically useful prognostic system, such as the International Prognostic Index (IPI) in diffuse large B cell lymphoma. PTCL with nodal manifestation include the HTLV-I-negative histologic subtypes of PTCL not otherwise specified (PTCL-NOS), angioimmunoblastic T cell lymphoma (AITL), and anaplastic large cell lymphoma (ALCL). As PTCL-NOS encompasses a group of similar tumors and mostly shares their clinical pictures, we retrospectively analyzed clinical data from 77 patients diagnosed with ALCL, AITL, and PTCL-NOS at Kobe City Medical Center General Hospital from May 1994 to February 2012 to identify the prognostic factor for nodal PTCL. The median age of patients was 64 years, ranging from 23 to 83 years. With a median follow-up of 50 months, 5-year overall survival (OS) was 43 %. Multivariate analysis identified high-risk IPI (hazard ratio (HR), 4.04; P = 0.015), absolute monocyte count > 0.8 × 10(9)/L (HR, 3.44; P = 0.001), and serum concentration of IgA > 410 mg/dL (HR, 2.31; P = 0.013) as poor prognostic factors for OS. Thus, we have identified novel prognostic factors of monocyte count and serum IgA level for nodal PTCL. Although conventional prognostic models mainly reflect both tumor characteristics and host factors, the present model indicates the importance of host immune response as the unfavorable prognosis.

Elevated monoclonal and polyclonal serum immunoglobulin free light chain as prognostic factors in B- and T-cell non-Hodgkin lymphoma.

The serum immunoglobulin free light chain (FLC) assay quantitates free kappa (κ) and lambda (λ) light chains. FLC elevations in patients with diffuse large B-cell lymphoma (DLBCL), Hodgkin lymphoma (HL), and chronic lymphocytic leukemia (CLL) are associated with an inferior survival. These increases in FLC can be monoclonal (as in myeloma) or polyclonal. The goal was to estimate the frequency of these elevations within distinct types of B-cell and T-cell non-Hodgkin lymphoma (NHL) and whether the FLC measurements are associated with event-free survival (EFS). We studied serum for FLC abnormalities using normal laboratory reference ranges to define an elevated κ or λ FLC. Elevations were further classified as polyclonal or monoclonal. Four hundred ninety-two patients were studied: 453 B-cell and 34 T-cell NHL patients. Twenty-nine % (142/453) of patients had an elevated FLC of which 10% were monoclonal elevations. Within B-cell NHL, FLC abnormalities were most common in lymphoplasmacytic (79%), mantle cell (68%), and lymphomas of mucosa associated lymphoid tissue (31%); they were least common in follicular (15%). The hazard ratio (HR) for EFS in all patients was 1.41 (95% CI; 1.11-1.81); in all B-cell NHL the HR was 1.44 (95% CI 1.11-1.96); in all T-cell NHL the HR was 1.17 (95% CI 0.55-2.49). FLC abnormalities predicted an inferior OS (HR = 2.75, 95% CI: 1.93-3.90, P < 0.0001). The serum FLC assay is useful for prognosis in both B-cell and T-cell types of NHL. In B-cell NHL further discrimination between a monoclonal and polyclonal elevation may be helpful and should be analyzed in prospective clinical trials.

Absolute lymphocyte count is a novel prognostic indicator in extranodal natural killer/T-cell lymphoma, nasal type.

BACKGROUND: extranodal natural killer (NK)/T-cell lymphoma (ENKL) is a heterogeneous entity with poor survival, requiring risk stratification in affected patients. We proposed absolute lymphocyte count (ALC) as a new prognostic factor in ENKL. PATIENTS AND METHODS: we retrospectively analyzed 128 patients newly diagnosed with ENKL. Independent prognostic factors of survival were determined by Cox regression analysis. RESULTS: patients with low ALC (<1.0 × 10(9)/l) at diagnosis tended to have more adverse clinical features. Patients with high ALC (≥1.0 × 10(9)/l) at diagnosis had better overall survival (OS; P < 0.0001) and progression-free survival (PFS; P<0.0001), and achieved higher complete remission rates (P=0.001). Multivariate analysis with known prognostic factors showed that ALC, B symptoms and advanced stage were independent predictors for OS and PFS. Using the International Prognostic Index, Prognostic Index for Peripheral T-cell lymphoma unspecified, or Korean Prognostic Index for nasal NK/T-cell lymphoma, the majority of patients were in the low-risk category (with no or one adverse factor). ALC was helpful to differentiate the low-risk patients with different survival outcomes (P < 0.0001). CONCLUSIONS: our data suggest that ALC at diagnosis is a novel, powerful predictor of prognosis in ENKL. Immune status at diagnosis might have an important influence on survival in patients with ENKL.