Outcomes of acute myeloid leukemia patients who responded to venetoclax and azacitidine and stopped treatment.

Venetoclax-azacitidine is the standard of treatment for unfit acute myeloid leukemia patients. In the VIALE-A study, treatment was given until progression but there are no data on its optimal duration for responding patients who do not tolerate indefinite therapy. We retrospectively analyzed the outcome of patients who discontinued venetoclax or venetoclax-azacitidine due to poor tolerance. Sixty-two newly diagnosed (ND) AML patients and 22 patients with morphological relapse or refractory AML were included. In the ND cohort (n = 62), 28 patients stopped venetoclax and azacitidine and 34 patients continued azacitidine monotherapy. With a median follow-up of 23 months (IQR, 20-32), median overall survival and treatment-free survival were 44 (IQR, 16-NR) and 16 (IQR, 8-27) months, respectively. Patients who stopped both treatments and those who continued azacitidine monotherapy had the same outcomes. Negative minimal residual disease was associated with a 2-year treatment-free survival of 80%. In the RR cohort (n = 22), median overall survival and treatment-free survival were 19 (IQR, 17-31) and 10 (IQR, 5-NR) months, respectively. Prior number of venetoclax-azacitidine cycles and IDH mutations were associated with increased overall survival. The only factor significantly impacting treatment-free survival was the number of prior cycles. This study suggests that patients who discontinued treatment in remission have favorable outcomes supporting the rationale for prospective controlled trials.

Flow cytometric analysis of peripheral blood neutrophil myeloperoxidase expression in myelodysplastic neoplasms (MPO-MDS-Valid): protocol for a multicentre diagnostic accuracy study.

INTRODUCTION: Many patients referred for suspicion of myelodysplastic neoplasm (MDS) are subjected to unnecessary discomfort from bone marrow aspiration, due to the low disease prevalence in this population. Flow cytometric analysis of peripheral blood neutrophil myeloperoxidase expression could rule out MDS with sensitivity and negative predictive value estimates close to 100%, ultimately obviating the need for bone marrow aspiration in up to 35% of patients. However, the generalisability of these findings is uncertain due to the limited sample size, the enrolment of patients at a single study site, and the reliability issues associated with laboratory-developed tests and varying levels of operator experience. This study aims to validate the accuracy attributes of peripheral blood neutrophil myeloperoxidase expression quantified by flow cytometric analysis in an independent multicentre sample. METHODS AND ANALYSIS: The MPO-MDS-Valid project is a cross-sectional diagnostic accuracy study comparing an index test to a reference standard. Consecutive adult patients referred for suspicion of MDS are being recruited at seven university hospitals and one cancer centre in France. At each site, flow cytometric analysis of peripheral blood samples is performed by operators who are blinded to the reference diagnosis. A central adjudication committee whose members are unaware of the index test results will determine the reference diagnosis of MDS, based on cytomorphological evaluation of bone marrow performed in duplicate by experienced hematopathologists. The target sample size is 400 patients and the anticipated study recruitment completion date is 31 December 2025. ETHICS AND DISSEMINATION: An institutional review board (Comité de Protection des Personnes Nord-Ouest III, Caen, France) approved the protocol, prior to the start of the study. Participants are recruited using an opt-out approach. Efforts will be made to publish the primary results within 6 months after study completion. TRIAL REGISTRATION NUMBER: NCT05175469.

Comparative diagnostic accuracy between simplified and original flow cytometric gating strategies for peripheral blood neutrophil myeloperoxidase expression in ruling out myelodysplastic syndromes.

BACKGROUND: Flow cytometric analysis of peripheral blood neutrophil myeloperoxidase expression is accurate in ruling out myelodyplastic syndromes (MDS) but might not be suitable for implementation in busy clinical laboratories. We aimed to simplify the original gating strategy and examine its accuracy. METHODS: Using the individual data from 62 consecutive participants enrolled in a prospective validation study, we assessed the agreement in intra-individual robust coefficient of variation (RCV) of peripheral blood neutrophil myeloperoxidase expression and compared diagnostic accuracy between the simplified and original gating strategies. RESULTS: Cytomorphological evaluation of bone marrow aspirate confirmed MDS in 23 patients (prevalence, 37%), unconfirmed MDS in 32 patients (52%), and was uninterpretable in 7 patients (11%). Median intra-individual RCV for simplified and original gating strategies were 30.7% (range, 24.7-54.4) and 30.6% (range, 24.7-54.1), with intra-class correlation coefficient quantifying absolute agreement equal to 1.00 (95% confidence interval [CI], 0.99 to 1.00). The areas under the receiver operating characteristic (ROC) curves were 0.93 (95% CI, 0.82-0.98) and 0.92 (95% CI, 0.82-0.98), respectively (P = .32). Using simplified or original gating strategy, intra-individual RCV values lower than a pre-specified threshold of 30.0% ruled out MDS for 35% (19 of 55) patients, with both sensitivity and negative predictive value estimates of 100%. CONCLUSIONS: The simplified gating strategy performs as well as the original one for ruling out MDS and has the potential to save time and reduce resource utilization. Yet, prospective validation of the simplified gating strategy is warranted before its adoption in routine. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03363399 (First posted on December 6, 2017).

Flow cytometry lyophilised-reagent tube for quantifying peripheral blood neutrophil myeloperoxidase expression in myelodysplastic syndromes (MPO-MDS-Develop): protocol for a diagnostic accuracy study.

INTRODUCTION: Suspicion of myelodysplastic syndromes (MDS) is the most common reason for bone marrow aspirate in elderly patients. Peripheral blood neutrophil myeloperoxidase expression quantified by flow cytometric analysis might rule out MDS for up to 35% of patients referred for suspected disease, without requiring bone marrow aspiration. Yet laboratory-developed liquid antibody cocktails have practical limitations, because of lack of standardisation and poor stability. This research project aims to estimate the level of agreement and comparative accuracy between a single-use flow cytometry tube of lyophilised reagents (BD Lyotube Stain 468) and its laboratory-developed liquid reagent counterpart in quantifying peripheral blood neutrophil myeloperoxidase expression, among adult patients referred for suspected MDS. METHODS AND ANALYSIS: The MPO-MDS-Develop project is a cross-sectional diagnostic accuracy study of two index tests by comparison with a reference standard in consecutive unselected adult patients conducted at a single university hospital. Flow cytometry analysis of peripheral blood samples will be performed by independent operators blinded to the reference diagnosis, using either Lyotube Stain 468 or laboratory-developed liquid reagent cocktail. The reference diagnosis of MDS will be established by cytomorphological evaluation of bone marrow aspirate by two independent haematopathologists blinded to the index test results. Morphologic assessment will be complemented by bone marrow flow cytometric score, karyotype and targeted next-generation sequencing panel of 43 genes, where relevant. The target sample size is 103 patients. ETHICS AND DISSEMINATION: An institutional review board (Comité de Protection des Personnes Sud Est III, Lyon, France) approved the protocol prior to study initiation (reference number: 2020-028-B). Participants will be recruited using an opt-out approach. Efforts will be made to release the primary results within 6 months of study completion. TRIAL REGISTRATION NUMBER: NCT04399018.

CYCLON and NPM1 Cooperate within an Oncogenic Network Predictive of R-CHOP Response in DLBCL.

R-CHOP immuno-chemotherapy significantly improved clinical management of diffuse large B-cell lymphoma (DLBCL). However, 30-40% of DLBCL patients still present a refractory disease or relapse. Most of the prognostic markers identified to date fail to accurately stratify high-risk DLBCL patients. We have previously shown that the nuclear protein CYCLON is associated with DLBCL disease progression and resistance to anti-CD20 immunotherapy in preclinical models. We also recently reported that it also represents a potent predictor of refractory disease and relapse in a retrospective DLBCL cohort. However, only sparse data are available to predict the potential biological role of CYCLON and how it might exert its adverse effects on lymphoma cells. Here, we characterized the protein interaction network of CYCLON, connecting this protein to the nucleolus, RNA processing, MYC signaling and cell cycle progression. Among this network, NPM1, a nucleolar multi-functional protein frequently deregulated in cancer, emerged as another potential target related to treatment resistance in DLBCL. Immunohistochemistry evaluation of CYCLON and NPM1 revealed that their co-expression is strongly related to inferior prognosis in DLBCL. More specifically, alternative sub-cellular localizations of the proteins (extra-nucleolar CYCLON and pan-cellular NPM1) represent independent predictive factors specifically associated to R-CHOP refractory DLBCL patients, which could allow them to be orientated towards risk-adapted or novel targeted therapies.

c-MYC and p53 expression highlight starry-sky pattern as a favourable prognostic feature in R-CHOP-treated diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is a clinically heterogeneous entity, in which the first-line treatment currently consists of an immuno-chemotherapy regimen (R-CHOP). However, around 30% of patients will not respond or will relapse. Overexpression of c-MYC or p53 is frequently found in DLBCL, but an association with prognosis remains controversial, as for other biomarkers previously linked with DLBCL aggressivity (CD5, CD23, or BCL2). The aim of this study was to explore the expression of these biomarkers and their correlation with outcome, clinical, or pathological features in a DLBCL cohort. Immunohistochemical (c-MYC, p53, BCL2, CD5, and CD23), morphological ('starry-sky' pattern [SSP]), targeted gene panel sequencing by next-generation sequencing (NGS), and fluorescence in situ hybridisation analyses were performed on tissue microarray blocks for a retrospective cohort of 94 R-CHOP-treated de novo DLBCL. In univariate analyses, p53 overexpression (p53high ) was associated with unfavourable outcome (p = 0.04) and with c-MYC overexpression (p = 0.01), whereas c-MYC overexpression was linked with an SSP (p = 0.004), but only tended towards an inferior prognosis (p = 0.06). Presence of a starry-sky morphology was found to be correlated with better survival in p53high DLBCL (p = 0.03) and/or c-MYC-positive DLBCL (p = 0.002). Furthermore, NGS data revealed that these three variables were associated with somatic mutations (PIM1, TNFRSF14, FOXO1, and B2M) involved in B-cell proliferation, survival, metabolism, and immune signalling. Taken together, these results show that the SSP pattern seems to be a protective factor in high-risk DLBCL subgroups and highlight cell death as a built-in failsafe mechanism to control tumour growth.

Flow cytometric analysis of peripheral blood neutrophil myeloperoxidase expression for ruling out myelodysplastic syndromes: a prospective validation study.

Suspicion of myelodysplastic syndromes (MDS) is the most common reason for bone marrow aspirate in elderly patients. This study aimed to prospectively validate the accuracy for flow cytometric analysis of peripheral blood neutrophil myeloperoxidase expression in ruling out MDS. We enrolled 62 consecutive patients who were referred for suspected MDS, based on medical history and peripheral blood cytopenia. The accuracy of intra-individual robust coefficient of variation (RCV) for peripheral blood neutrophil myeloperoxidase expression was assessed with a prespecified 30% threshold. Cytomorphological evaluation of bone marrow aspirate performed by experienced hematopathologists confirmed MDS in 23 patients (prevalence, 37%), unconfirmed MDS in 32 patients (52%, including 3 patients with idiopathic cytopenia of undetermined significance (ICUS)), and was uninterpretable in 7 patients (11%). The median intra-individual RCV values for neutrophil myeloperoxidase expression in peripheral blood were 37.4% (range, 30.7-54.1), 29.2% (range, 28.1-32.1), and 29.1% (range, 24.7-37.8) for patients with confirmed suspicion of MDS, ICUS, and unconfirmed suspicion of MDS, respectively (P<0.001). The area under the ROC curve was 0.92 (95% confidence interval, 0.86-0.99). An intra-individual RCV value lower than 30% ruled out MDS for 35% (i.e., 19/55) patients referred for suspected disease, with 100% sensitivity (95% CI, 85-100%) and 100% negative predictive value (95% CI, 82-100%) estimates. This study shows that flow cytometric analysis of peripheral blood neutrophil myeloperoxidase expression might obviate the need for bone marrow aspirate for 35% of patients with suspected MDS. Trial registration: ClinicalTrials.gov identifier: NCT03363399 (first posted on December 6, 2017).

Transformation of a low-grade follicular lymphoma into a composite lymphoma combining a high-grade B-cell lymphoma and a lymphoblastic neoplasm expressing Terminal deoxynucleotidyl Transferase: a case report.

BACKGROUND: High-grade B-cell lymphoma with rearrangements of MYC and BCL2 and/or BCL6 is an aggressive mature B-cell neoplasm, whereas B-lymphoblastic lymphoma is immature cell proliferation, with a frequent positivity for terminal deoxynucleotidyl transferase. The transformation of a low-grade follicular lymphoma into a lymphoblastic neoplasm expressing terminal deoxynucleotidyl transferase is a very rare event. CASE PRESENTATION: A 55-year-old Caucasian man was followed for a grade 1-2 follicular lymphoma carrying a t(14;18) IGH/BCL2+ and was initially treated with R-CHOP. The follicular lymphoma presented two relapses. In the third relapse, the patient had multiple lymphadenopathy and ascites, which motivated a retroperitoneal biopsy and an ascitic tap. These samples were analyzed by histological, cytological, flow cytometric, cytogenetic, and molecular assessments. The patient died of a multiple organ dysfunction syndrome 2 weeks after his third relapse. The biopsy revealed a diffuse proliferation made up of two types of tumor cells: centroblasts (Bcl-6-positive) and immature cells (terminal deoxynucleotidyl transferase-positive). Flow cytometric analysis confirmed the immature phenotype, with an expression of terminal deoxynucleotidyl transferase, combined with a loss of membrane immunoglobulins. The cytogenetic analysis performed on the ascites revealed a clonal evolution characterized by a t(8;22)(q24;q11) MYC+ translocation not previously detected in follicular lymphoma. Fluorescence in situ hybridization confirmed the double rearrangement of the BCL2 and MYC genes. Polymerase chain reactions and sequencing were used to study the clonal relationship between follicular lymphoma and the secondary tumors. The IGVH gene rearrangement revealed a unique clonal rearrangement involving an IGVH4-59 subset in all three specimens. CONCLUSION: These findings suggest a clonal relationship between the two types of lymphoma cells. Furthermore, they support the transformation of an acute follicular lymphoma into a composite lymphoma combining a high-grade B-cell lymphoma and a lymphoblastic neoplasm expressing terminal deoxynucleotidyl transferase. This case report highlights the possible transformation of follicular lymphoma into a highly aggressive and immature proliferation.

Flow cytometric analysis of neutrophil myeloperoxidase expression in peripheral blood for ruling out myelodysplastic syndromes: a diagnostic accuracy study.

Suspicion of myelodysplastic syndromes (MDS) is one of the commonest reasons for bone marrow aspirate in elderly patients presenting with persistent peripheral blood (PB) cytopenia of unclear etiology. A PB assay that accurately rules out MDS would have major benefits. The diagnostic accuracy of the intra-individual robust coefficient of variation (RCV) for neutrophil myeloperoxidase (MPO) expression measured by flow cytometric analysis in PB was evaluated in a retrospective derivation study (44 MDS cases and 44 controls) and a prospective validation study (68 consecutive patients with suspected MDS). Compared with controls, MDS cases had higher median RCV values for neutrophil MPO expression (40.2% vs 30.9%; P<0.001). The area under the receiver operating characteristic curve estimates were 0.94 [95% confidence interval (CI): 0.86-0.97] and 0.87 (95%CI: 0.76-0.94) in the derivation and validation studies, respectively. A RCV lower than 30% ruled out MDS with 100% sensitivity (95%CI: 78-100%) and 100% negative predictive value (95%CI: 83-100%) in the prospective validation study. Neutrophil MPO expression measured by flow cytometric analysis in PB might obviate the need for invasive bone marrow aspirate and biopsy for up to 29% of patients with suspected MDS.

Accurate quantification of fourteen normal bone marrow cell subsets in infants to the elderly by flow cytometry.

BACKGROUND: Studies of normal bone marrow (BM) cell composition by flow cytometry are scarce. Presently, we aimed to quantify 14 cell subsets from infants to elderly patients. METHODS: Cell subsets in BM samples from 180 individuals without morphologically abnormal leukocytes were analyzed using a single combination of eight antibodies: CD3/CD10/CD38/CD19/CD36/CD16/CD34/CD45. RESULTS: By comparison with the Holdrinet score, we first validated the immature granulocyte/neutrophil (IGRA/N) ratio as a readily obtainable criterion of BM sample purity in 145 cases. Then, the 115 highly pure samples were selected (IGRA/N ≥ 1.2) and analyzed according to age group. CD34+ myeloblasts became progressively more infrequent with age: median 1.4% in infancy to 0.5% in the elderly. Neutrophils increased: 10.7% to 22.8%; all other myeloid subsets, IGRA, eosinophils, basophils and monocytes remained stable: respectively 40.3% to 46.7%, 2.0% to 2.8%, 0.2% to 0.3%, and 4.4% to 5.0% throughout life. Erythroblasts were lower in children (8.4% to 10.3%) than in adults (12.5% to 15.1%). For lymphoid cells, hematogones and transitional B-cells decreased: 15.5% to 0.6% and 3.6% to 0.1%, respectively; mature lymphocytes remained stable: B-cells: 1.4% to 2.8%, T-cells: 5.8% to 8.7%, and NK-cells: 0.7% to 1.4%. Plasma cells varied slightly: 0.1% to 0.5%. Differences of about 40% were seen in moderately pure (IGRA/N: 0.5 to 1.2) BM samples. CONCLUSION: We thus provide the first values for 14 myeloid and lymphoid subsets characterizing BM cell composition in 5 age ranges. They should provide important information when screening patients for hematological disorders or abnormal bone marrow development. © 2018 International Clinical Cytometry Society.

Cooperation of adenosine and prostaglandin E2 (PGE2) in amplification of cAMP-PKA signaling and immunosuppression.

INTRODUCTION: We hypothesize that adenosine and PGE2 could have a complementary immunosuppressive effect that is mediated via common cAMP-PKA signaling. MATERIALS AND METHODS: To test this hypothesis, the effect of adenosine and PGE2 on the cytotoxic activity and cytokine production of lymphokine activated killer (LAK) cells was investigated. RESULTS: PGE2 and adenosine inhibited LAK cells cytotoxic activity and production of INF-gamma, GM-CSF and TNF-alpha. In combination they showed substantially higher inhibition than each modality used alone. Using agonists and antagonists specific for PGE2 and adenosine receptors we found that cooperation of PGE2 and adenosine in their inhibitory effects are mediated via EP2 and A2A receptors, respectively. LAK cells have 35-fold higher expression of EP2 than A2A. Combined PGE2 and adenosine treatment resulted in augmentation of cAMP production, PKA activity, CREB phosphorylation and inhibition of Akt phosphorylation. Wortmannin and LY294002 enhanced the suppressive effects of adenosine and PGE2. In contrast, Rp-8-Br-cAMPS, an inhibitor of PKA type I blocked their immunosuppressive effects, suggesting that the inhibitory effects of PGE2 and adenosine are mediated via common pathway with activation of cAMP-PKA and inhibition of Akt. CONCLUSION: In comparison to other immunosuppressive molecules (TGF-beta and IL-10), adenosine and PGE2 are unique in their ability to inhibit the executive function of highly cytotoxic cells. High intratumor levels of adenosine and PGE2 could protect tumor from immune-mediated destruction by inactivation of the tumor infiltrating functionally active immune cells.

Inhibition of cytokine production and cytotoxic activity of human antimelanoma specific CD8+ and CD4+ T lymphocytes by adenosine-protein kinase A type I signaling.

The goal of this study was to investigate the effects of adenosine and its stable analogue 2-chloroadenosine (CADO) on the cytotoxic activity and cytokine production by human antimelanoma specific CD8+ and CD4+ T-helper type 1 (Th1) clones. The cytotoxic activity of CD8+ T cells was inhibited by adenosine and CADO. Using Lab MAP multiplex technology, we found that adenosine inhibits production of various cytokines and chemokines by CD8+ and CD4+ T cells. Studies with CGS21680, a specific agonist of adenosine A2A receptor (AdoRA2A), and ZM241385, an AdoRA2-selective antagonist, indicate that the inhibitory effects of adenosine are mediated via cyclic AMP (cAMP)-elevating AdoRA2A, leading to protein kinase A (PKA) activation. Using cAMP analogues with different affinities for the A and B sites of the regulatory subunits of PKAI and PKAII, we found that activation of PKAI, but not of PKAII, mimicked the inhibitory effects of adenosine on T-cell cytotoxic activity and cytokine production. Inhibitors of the PKA catalytic subunits (H89 and PKA inhibitor peptide 14-22) failed to abrogate the inhibitory effects of CADO. In contrast, Rp-8-Br-cAMPS that antagonizes binding of cAMP to the regulatory I subunit and PKA activation was efficient in blocking the inhibitory effect of adenosine on the functional activity of T cells. Our findings on the ability of adenosine to inhibit the effector function of antimelanoma specific T cells suggest that intratumor-produced adenosine could impair the function of tumor-infiltrating T lymphocytes. Thus, blocking the inhibitory activity of tumor-produced adenosine might represent a new strategy for improvement of cancer immunotherapy.

Adenosine-mediated inhibition of the cytotoxic activity and cytokine production by activated natural killer cells.

Adenosine is an important signaling molecule that regulates multiple physiologic processes and exerts major anti-inflammatory actions. Tumors have high concentrations of adenosine, which could inhibit the function of tumor-infiltrating lymphoid cells. We investigated the ability of adenosine and its stable analogue 2-chloroadenosine (CADO) to inhibit cytokine production and cytotoxic activity of lymphokine-activated killer (LAK) cells and determined whether both these effects are initiated via a common pathway. CADO strongly inhibited cytotoxic activity of LAK cells and attenuated the production of IFN-gamma, granulocyte macrophage colony-stimulating factor, tumor necrosis factor alpha, and macrophage inflammatory protein-1alpha by LAK cells stimulated by cross-linking of the Ly49D receptor. These inhibitory effects were associated with the ability of CADO to stimulate cyclic AMP (cAMP) production and activate protein kinase A (PKA). Using cAMP analogues with different affinities for the A and B sites of the regulatory subunits of PKA types I and II, we found that activation of PKA I, but not PKA II, mimicked the inhibitory effects of CADO on LAK cell cytotoxic activity and cytokine production. Inhibitors of the PKA catalytic subunits (H89 and PKI(14-22) peptide) failed to abrogate the inhibitory effects of CADO whereas Rp-8-Br-cAMPS, an antagonist of the RI subunit, blocked the inhibitory effects of CADO. We conclude that the inhibitory effects of adenosine are probably mediated via cAMP-dependent activation of the RI subunits of PKA I but are independent of the catalytic activity of PKA. Tumor-produced adenosine could be a potent tumor microenvironmental factor inhibiting the functional activity of tumor-infiltrating immune cells.

Adenosine-mediated inhibition of cytotoxic activity and cytokine production by IL-2/NKp46-activated NK cells: involvement of protein kinase A isozyme I (PKA I).

Adenosine suppresses the production of various cytokines/ chemokines and inhibits the cytotoxic activity of murine and human NK cells activated with IL-2 or Ly49D, NKp46-receptor crosslinking, respectively. These effects are mediated by the type A2A adenosine receptor via stimulation of adenylyl cyclase, increased production of cAMP, and activation of PKA. PKA I, but not PKA II, participates in the inhibitory effects of adenosine. Blocking regulatory, but not catalytic, subunits of PKA I abrogates the inhibitory effects of adenosine. These findings suggest that tumor-produced adenosine inhibits the activity of NK and other effector cells and thereby protects tumors from immune-mediated destruction.

Combined antiangiogenic and immune therapy of prostate cancer.

Experimental studies of antiangiogenic or immune therapy of cancer have generated a great deal of optimism. However, the results of clinical testing of these therapies are below expectations. We hypothesized that the antitumor efficacy can be increased when immune destruction of tumor cell is combined with destruction of tumor vasculature by antiangiogenic drugs. In the present study the therapeutic efficacy of combined antiangiogenic and immune therapy has been tested against the highly aggressive, MHC class I negative murine RM1 prostate tumor. SU6668 was used as the antiangiogenic drug and recombinant murine B7.2-IgG fusion protein was used to stimulate T cell-mediated immune destruction of tumor cells. SU6668 is an inhibitor of the tyrosine kinase activity of three angiogenic receptors VEGFR2 (Flk-1/KDR), PDGFRbeta and FGFR1 that play a crucial role in tumor-induced vascularization. Our studies show that B7.2-IgG treatment of mice with established RM1 prostate tumors resulted in a significant inhibition of tumor growth. Both CD4+ and CD8+ T cells were responsible for this effect. SU6668 therapy substantially inhibited tumor vascularization and tumor growth. When tumor-bearing mice were treated with SU6668 in combination with B7.2-IgG, the antitumor effects were substantially higher than in mice treated separately with SU6668 or B7.2-IgG. Prolonged treatment of mice with SU6668 did not inhibit the immunoreactivity of T lymphocytes. On the contrary, T cells from mice treated with a combination of SU6668 and B7.2-IgG showed higher proliferative responses and cytokine production following anti-CD3 stimulation than T cells of mice treated separately with these modalities. These results indicate that antiangiogenic and immune therapies using SU6668 and B7.2-IgG are compatible and manifest complementary antitumor effects. Combined antiangiogenic and immune therapy might represent a new strategy for cancer treatment.

Gs protein-coupled adenosine receptor signaling and lytic function of activated NK cells.

The effect of adenosine and its analogues on the cytotoxic activity of IL-2-activated NK cells was investigated. Adenosine is an endogenous ligand for four different adenosine receptor (AdoR) subtypes (AdoRA1, AdoRA2A, AdoRA2B, and AdoRA3). Increased concentrations of adenosine were found in ascites of MethA sarcoma or in culture medium of 3LL Lewis lung carcinoma growing under hypoxic conditions. We hypothesize that intratumor adenosine impairs the ability of lymphokine-activated killer (LAK) cells to kill tumor cells. The effect of AdoR engagement on LAK cells cytotoxic activity was analyzed using AdoR agonists and antagonists as well as LAK cells generated from AdoR knockout mice. Adenosine and its analogues efficiently inhibited the cytotoxic activity of LAK cells. CGS21680 (AdoRA2A agonist) and 5-N-ethylcarboxamide adenosine (NECA) (AdoRA2A/ADoRA2B agonist) inhibited LAK cell cytotoxicity in parallel with their ability to increase cAMP production. The inhibitory effects of stable adenosine analog 2-chloroadenosine (CADO) and AdoRA2 agonists were blocked by AdoRA2 antagonist ZM 241385. Adenosine and its analogues impair LAK cell function by interfering with both perforin-mediated and Fas ligand-mediated killing pathways. Studies with LAK cells generated from AdoRA1-/- and AdoRA3-/- mice ruled out any involvement of these AdoRs in the inhibitory effects of adenosine. LAK cells with genetically disrupted AdoRA2A were resistant to the inhibitory effects of adenosine, CADO and NECA. However, with extremely high concentrations of CADO or NECA, mild inhibition of LAK cytotoxicity was observed that was probably mediated via AdoRA2B signaling. Thus, by using pharmacological and genetic blockage of AdoRs, our results clearly indicate the prime importance of cAMP elevating AdoR2A in the inhibitory effect of adenosine on LAK cell cytotoxicity. The elevated intratumor levels of adenosine might inhibit the antitumor effects of activated NK cells.