Extracellular vesicles as biomarkers for AIDS-associated non-Hodgkin lymphoma risk.

Introduction: Extracellular vesicles are membrane-bound structures secreted into the extracellular milieu by cells and can carry bioactive molecules. There is emerging evidence suggesting that EVs play a role in the diagnosis, treatment, and prognosis of certain cancers. In this study, we investigate the association of EVs bearing PD-L1 and molecules important in B-cell activation and differentiation with AIDS-NHL risk. Methods: EVs were isolated from archived serum collected prior to the diagnosis of AIDS-NHL in cases (N = 51) and matched HIV+ controls (N = 52) who were men enrolled in the Los Angeles site of the MACS/WIHS Combined Cohort Study (MWCCS). Serum specimens of AIDS-NHL cases were collected at a mean time of 1.25 years (range of 2 to 36 months) prior to an AIDS-NHL diagnosis. The expression of PD-L1 and other molecules on EVs (CD40, CD40L, TNF-RII, IL-6Rα, B7-H3, ICAM-1, and FasL) were quantified by Luminex multiplex assay. Results and discussion: We observed significantly higher levels of EVs bearing PD-L1, CD40, TNF-RII and/or IL-6Rα in AIDS-NHL cases compared with controls. Using multivariate conditional logistic regression models adjusted for age and CD4+ T-cell count, we found that EVs bearing PD-L1 (OR = 1.93; 95% CI: 1.10 - 3.38), CD40 (OR = 1.97, 95% CI: 1.09 - 3.58), TNF-RII (OR = 5.06; 95% CI: 1.99 - 12.85) and/or IL-6Rα (OR = 4.67; 95% CI: 1.40 - 15.53) were significantly and positively associated with AIDS-NHL risk. In addition, EVs bearing these molecules were significantly and positively associated with non-CNS lymphoma: PD-L1 (OR = 1.94; 95% CI: 1.01 - 3.72); CD40 (OR = 2.66; 95% CI: 1.12 - 6.35); TNF-RII (OR = 9.64; 95% CI: 2.52 - 36.86); IL-6Rα (OR = 8.34; 95% CI: 1.73 - 40.15). These findings suggest that EVs bearing PD-L1, CD40, TNF-RII and/or IL-6Rα could serve as biomarkers for the early detection of NHL in PLWH.

Elevated serum TARC levels precede classic Hodgkin lymphoma diagnosis by several years.

Tumor cells in classic Hodgkin lymphoma produce high quantities of the thymus- and activation-related chemokine (TARC). We measured TARC levels in prediagnostic serum samples and found strikingly increased values in the vast majority of patients, as early as 6 years before diagnosis.

Predictive Value of Serum Biomarkers for Response of Limited-Stage AIDS-Associated Kaposi Sarcoma to Antiretroviral Therapy With or Without Concomitant Chemotherapy in Resource-Limited Settings.

BACKGROUND: Guidelines for limited-stage human immunodeficiency virus-associated Kaposi sarcoma (AIDS/KS) recommend antiretroviral therapy (ART) as initial treatment. However, many such individuals show worsening KS and require additional chemotherapy. Methods to identify such patients are lacking. SETTING: We studied whether serum levels of biomarkers associated with angiogenesis, systemic inflammation, and immune activation, which are elevated in HIV-infected individuals and implicated in the development of KS, could prospectively identify individuals with limited-stage AIDS-KS who would benefit from chemotherapy administered with ART. METHODS: Serum specimens were obtained from participants in a randomized trial evaluating the value of adding oral etoposide chemotherapy to ART in treatment-naïve people with limited-stage AIDS-KS in resource-limited settings. Serum biomarkers of inflammation (C-reactive protein [CRP], interleukin [IL]-6, IL-8, IL-10, granulocyte colony stimulating factor, soluble tumor necrosis factor receptor-2), immune system activation (soluble IL-2 receptor alfa, C-X-C motif chemokine ligand 10/interferon gamma-induced protein 10, C-C motif ligand 2/monocyte chemoattractant protein 1), and angiogenesis (vascular endothelial growth factor, matrix metalloproteinase-2, -9, endoglin, hepatocyte growth factor) were measured at entry to determine whether baseline levels are associated with KS response. On-treatment changes in biomarker levels were determined to assess how etoposide modifies the effects of ART. RESULTS: Pretreatment CRP and IL-10 were higher in those whose KS progressed, and lowest in those who had good clinical responses. Pretreatment CRP, IL-6, and soluble tumor necrosis factor receptor-2 showed significant associations with KS progression at the week-48 primary endpoint. Immediate etoposide led to lower inflammation biomarker levels compared with ART alone. Early KS progression was associated with elevated pretreatment levels of inflammation-associated biomarkers and increasing levels post-treatment. CONCLUSIONS: Quantifying serum biomarkers, especially CRP, may help identify persons with AIDS-KS who would benefit from early introduction of chemotherapy in addition to ART.

Longitudinal Changes in Immune Activation Serum Biomarkers Prior to Diagnosis and Risk of B-cell NHL Subtypes.

BACKGROUND: To examine the contribution of B-cell activation molecules to B-cell follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL), a prospective study was conducted using pre-diagnosis serial serum samples from the US Department of Defense Serum Repository. METHODS: Each case (n = 142 FL, n = 211 DLBCL) was matched to two controls on age, gender, race, military branch, and blood collection dates. Immune activation molecules (IL1ß, IL2, IL4, IL5, IL6, IL10, IL12, CXCL13, IL8, TNFα, IFNγ, GM-CSF, VEGF, sCD30, IgE) were quantified using ELISA or multiplex immunometric (Luminex) assay. Longitudinal data were analyzed using linear mixed modeling. As serial specimens were collected over several years before diagnosis, we evaluated the temporal dynamics of these markers. RESULTS: Increased serum levels of sCD30, CXCL13, and to a lesser extent IL10, were associated with both FL and DLBCL in cases compared with controls, with a median follow-up of 5.5 years from the earliest specimen collection to diagnosis date. Significant increasing sCD30 and CXCL13 trajectories for FL and DLBCL subtypes were noted starting at the earliest time points and with IL10 levels increasing significantly at time points closer to diagnosis. CONCLUSIONS: These results suggest that sCD30, CXCL13, and IL10 may contribute to the etiology of FL and DLBCL and are potential biomarkers for these non-Hodgkin lymphoma subtypes. IMPACT: The increasing trajectories of the B-cell activation molecules, sCD30, CXCL13, and to a lesser extent IL10, may indicate early disease-induced effects or reflect the chronic stimulation of B-cells that promotes the development of FL and DLBCL subtypes.

Circulating immune markers and risks of non-Hodgkin lymphoma subtypes: A pooled analysis.

Although prediagnostic circulating concentrations of the immune activation markers soluble CD27 (sCD27), sCD30 and chemokine ligand-13 (CXCL13) have been associated with non-Hodgkin lymphoma (NHL) risk, studies have been limited by sample size in associations with NHL subtypes. We pooled data from eight nested case-control studies to investigate subtype-specific relationships for these analytes. Using polytomous regression, we calculated odds ratios (ORs) with 95% confidence intervals (CIs) relating study-specific analyte tertiles to selected subtypes vs controls (n = 3310): chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL; n = 623), diffuse large B cell lymphoma (DLBCL; n = 621), follicular lymphoma (FL; n = 398), marginal zone lymphoma (MZL; n = 138), mantle cell lymphoma (MCL; n = 82) and T cell lymphoma (TCL; n = 92). We observed associations with DLBCL for elevated sCD27 [OR for third vs first tertile (ORT3 ) = 2.2, 95% CI = 1.6-3.1], sCD30 (ORT3  = 2.0, 95% CI = 1.6-2.5) and CXCL13 (ORT3  = 2.3, 95% CI = 1.8-3.0). We also observed associations with sCD27 for CLL/SLL (ORT3  = 3.3, 95% CI = 2.4-4.6), MZL (ORT3  = 7.7, 95% CI = 3.0-20.1) and TCL (ORT3  = 3.4, 95% CI = 1.5-7.7), and between sCD30 and FL (ORT3  = 2.7, 95% CI = 2.0-3.5). In analyses stratified by time from phlebotomy to case diagnosis, the sCD27-TCL and all three DLBCL associations were equivalent across both follow-up periods (<7.5, ≥7.5 years). For other analyte-subtype comparisons, associations were stronger for the follow-up period closer to phlebotomy, particularly for indolent subtypes. In conclusion, we found robust evidence of an association between these immune markers and DLBCL, consistent with hypotheses that mechanisms related to immune activation are important in its pathogenesis. Our other findings, particularly for the rarer subtypes MZL and TCL, require further investigation.

Plasma extracellular vesicles bearing PD-L1, CD40, CD40L or TNF-RII are significantly reduced after treatment of AIDS-NHL.

Emerging evidence shows that tumor cells secrete extracellular vesicles (EVs) that carry bioactive cell surface markers, such as programmed death-ligand 1 (PD-L1), which can modulate immune responses and inhibit anti-tumor responses, potentially playing a role in lymphomagenesis and in promoting the growth of these cancers. In this study, we investigated the role of EVs expressing cell surface molecules associated with B cell activation and immune regulation. We measured levels of EVs derived from plasma from 57 subjects with AIDS-related non-Hodgkin lymphoma (AIDS-NHL) enrolled in the AIDS Malignancies Consortium (AMC) 034 clinical trial at baseline and post-treatment with rituximab plus concurrent infusional EPOCH chemotherapy. We found that plasma levels of EVs expressing PD-L1, CD40, CD40L or TNF-RII were significantly reduced after cancer treatment. AIDS-NHL patients with the diffuse large B cell lymphoma (DLBCL) tumor subtype had decreased plasma levels of EVs bearing PD-L1, compared to those with Burkitt's lymphoma. CD40, CD40L and TNF-RII-expressing EVs showed a significant positive correlation with plasma levels of IL-10, CXCL13, sCD25, sTNF-RII and IL-18. Our results suggest that patients with AIDS-NHL have higher levels of EVs expressing PD-L1, CD40, CD40L or TNF-RII in circulation before cancer treatment and that levels of these EVs are associated with levels of biomarkers of microbial translocation and inflammation.

Targeting TfR1 with the ch128.1/IgG1 Antibody Inhibits EBV-driven Lymphomagenesis in Immunosuppressed Mice Bearing EBV+ Human Primary B-cells.

Epstein-Barr virus (EBV) is a human gammaherpesvirus associated with the development of hematopoietic cancers of B-lymphocyte origin, including AIDS-related non-Hodgkin lymphoma (AIDS-NHL). Primary infection of B-cells with EBV results in their polyclonal activation and immortalization. The transferrin receptor 1 (TfR1), also known as CD71, is important for iron uptake and regulation of cellular proliferation. TfR1 is highly expressed in proliferating cells, including activated lymphocytes and malignant cells. We developed a mouse/human chimeric antibody targeting TfR1 (ch128.1/IgG1) that has previously shown significant antitumor activity in immunosuppressed mouse models bearing human malignant B-cells, including multiple myeloma and AIDS-NHL cells. In this article, we examined the effect of targeting TfR1 to inhibit EBV-driven activation and growth of human B-cells in vivo using an immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl /SzJ [NOD/SCID gamma (NSG)] mouse model. Mice were implanted with T-cell-depleted, human peripheral blood mononuclear cells (PBMCs), either without EBV (EBV-), or exposed to EBV in vitro (EBV+), intravenously via the tail vein. Mice implanted with EBV+ cells and treated with an IgG1 control antibody (400 µg/mouse) developed lymphoma-like growths of human B-cell origin that were EBV+, whereas mice implanted with EBV+ cells and treated with ch128.1/IgG1 (400 µg/mouse) showed increased survival and significantly reduced inflammation and B-cell activation. These results indicate that ch128.1/IgG1 is effective at preventing the growth of EBV+ human B-cell tumors in vivo, thus, indicating that there is significant potential for agents targeting TfR1 as therapeutic strategies to prevent the development of EBV-associated B-cell malignancies. SIGNIFICANCE: An anti-TfR1 antibody, ch128.1/IgG1, effectively inhibits the activation, growth, and immortalization of EBV+ human B-cells in vivo, as well as the development of these cells into lymphoma-like tumors in immunodeficient mice.

Immune Activation and Microbial Translocation as Prognostic Biomarkers for AIDS-Related Non-Hodgkin Lymphoma in the AMC-034 Study.

PURPOSE: AIDS-related non-Hodgkin lymphoma (ARL) is the most common cancer in HIV-infected individuals in the United States and other countries in which HIV-positive persons have access to effective combination antiretroviral therapy (cART). Our prior work showed that pretreatment/postdiagnosis plasma levels of some cytokines, such as IL6, IL10, and CXCL13, have the potential to serve as indicators of clinical response to treatment and survival in ARL. The aims of this study were to identify novel prognostic biomarkers for response to treatment and/or survival in persons with ARL, including biomarkers of microbial translocation and inflammation. EXPERIMENTAL DESIGN: We quantified plasma levels of several biomarkers (sCD14, LBP, FABP2, EndoCab IgM, IL18, CCL2/MCP-1, sCD163, IP-10/CXCL10, TARC/CCL17, TNFα, BAFF/BLyS, sTNFRII, sCD44, and sIL2Rα/sCD25) by multiplexed immunometric assays (Luminex) or ELISA in plasma specimens obtained from ARL patients enrolled in the AMC-034 trial, which compared infusional combination chemotherapy (EPOCH: etoposide, vincristine, doxorubicin, cyclophosphamide, and prednisone) with concurrent or sequential rituximab. Plasma was collected prior to the initiation of therapy (n = 57) and after treatment initiation (n = 55). RESULTS: We found that several biomarkers decreased significantly after treatment, including TNFα, sCD25, LBP, and TARC (CCL17). Moreover, pretreatment plasma levels of BAFF, sCD14, sTNFRII, and CCL2/MCP-1 were univariately associated with overall survival, and pretreatment levels of BAFF, sTNFRII, and CCL2/MCP-1 were also associated with progression-free survival. CONCLUSIONS: Our results suggest that patients with ARL who responded to therapy had lower pretreatment levels of inflammation and microbial translocation as compared with those who did not respond optimally.

Circulating Biomarkers of Inflammation and Ovarian Cancer Risk in the Nurses' Health Studies.

BACKGROUND: Chronic inflammation is a well-established mechanism of ovarian carcinogenesis; however, the specific immunogenic processes influencing ovarian tumor development remain unclear. In a case-control study nested within the Nurses' Health Study (NHS) and the NHSII, we examined the association between six inflammatory chemokines and cytokines [B-cell activating factor (BAFF), C-X-C motif chemokine ligand 13 (CXCL13), IL8, soluble(s)IL2-receptor-α(Rα), sIL6Rα] and epithelial ovarian cancer risk. METHODS: Among 299 epithelial ovarian cancer cases and 334 matched controls, six inflammatory biomarkers were measured in plasma collected 1-24 years before diagnosis or index date using two custom multiplex Luminex panels. ORs and 95% confidence intervals (CI) were estimated for the association between each biomarker and risk using multivariable conditional logistic regression with adjustment for relevant confounders. We additionally assessed heterogeneity in the risk associations by histotype [high-grade serous carcinoma (HGSC) vs. non-HGSC], body mass index, smoking status, menopausal status, and aspirin use. RESULTS: Women with the highest versus lowest quartile (Q) levels of CXCL13 had a 72% increased ovarian cancer risk (OR = 1.72; 95% CI = 1.04-2.83; P trend = 0.007). The positive association with CXCL13 was stronger in magnitude for non-HGSC, overweight or obese women, and postmenopausal women, although only menopausal status demonstrated statistically significant heterogeneity (P interaction = 0.04). The remaining biomarkers were not associated with risk. CONCLUSIONS: This first evidence that prediagnostic CXCL13, a B-cell chemoattractant, is associated with an increased risk of epithelial ovarian cancer expands current understanding of the role of inflammation in ovarian carcinogenesis. IMPACT: CXCL13 may represent a novel biomarker for ovarian cancer.

Elevated Serum Levels of sCD30 and IL6 and Detectable IL10 Precede Classical Hodgkin Lymphoma Diagnosis.

Background: We investigated whether an immune system environment characterized by elevated serum levels of B-cell activation molecules was associated with the subsequent development of classical Hodgkin lymphoma (cHL).Methods: We measured serum levels of B-cell-stimulatory cytokines, IL6 and IL10, soluble CD30 (sCD30), and total IgE prior to cHL diagnosis in 103 cases and 206 matched controls with archived specimens in the DoD Serum Repository.Results: Prediagnosis serum sCD30 and IL6 levels had strong positive associations with risk of a cHL diagnosis 0 to 1 year prior to diagnosis [sCD30 OR = 5.5; 95% confidence interval (CI), 3.4-9.0; IL6 OR = 4.6; 95% CI, 2.9-7.5] and >1 year to 2 years pre-cHL diagnosis (sCD30 OR = 3.3; 95% CI, 1.6-6.7; IL6 OR = 2.9; 95% CI, 1.3-6.5). We observed similar, albeit not consistently significant positive associations, over 4 or more years preceding diagnosis. We did not observe a clear association with IgE levels. Of note, detectable IL10 levels were significantly associated with Epstein-Barr virus (EBV)-positive cHL cases compared with EBV-negative cases.Conclusion: In this prospective analysis, elevated sCD30 and IL6 levels and detectable IL10 preceded cHL diagnosis.Impact: The associations of these cytokines with cHL risk may reflect the production of these molecules by proliferating nascent cHL tumor cells, or by immune cells responding to their presence, prior to clinical detection. The stable elevation in cHL risk, 4 or more years prediagnosis, also suggests that a B-cell-stimulatory immune system milieu precedes, and may promote, lymphomagenesis. Cancer Epidemiol Biomarkers Prev; 26(7); 1114-23. ©2017 AACR.

Predictive Value of Cytokines and Immune Activation Biomarkers in AIDS-Related Non-Hodgkin Lymphoma Treated with Rituximab plus Infusional EPOCH (AMC-034 trial).

PURPOSE: The aims of this study were to determine whether pretreatment plasma levels of cytokines and immune activation-associated molecules changed following treatment for AIDS-NHL with rituximab plus infusional EPOCH, and to determine whether pretreatment levels of these molecules were associated with response to treatment and/or survival. EXPERIMENTAL DESIGN: We quantified plasma levels of B-cell activation-associated molecules (sCD27, sCD30, and sCD23) and cytokines (IL6, IL10, and CXCL13) before and after the initiation of treatment in persons with AIDS-NHL (n = 69) in the AIDS Malignancies Consortium (AMC) 034 study, which evaluated treatment of AIDS-NHL with EPOCH chemotherapy and rituximab. RESULTS: Treatment resulted in decreased plasma levels of some of these molecules (CXCL13, sCD27, and sCD30), with decreased levels persisting for one year following the completion of treatment. Lower levels of CXCL13 before treatment were associated with complete responses following lymphoma therapy. Elevated levels of IL6 pretreatment were associated with decreased overall survival, whereas higher IL10 levels were associated with shorter progression-free survival (PFS), in multivariate analyses. Furthermore, patients with CXCL13 or IL6 levels higher than the median levels for the NHL group, as well as those who had detectable IL10, had lower overall survival and PFS, in Kaplan-Meier analyses. CONCLUSIONS: These results indicate that CXCL13, IL6, and IL10 have significant potential as prognostic biomarkers for AIDS-NHL.

The role of chemokine C-C motif ligand 2 genotype and cerebrospinal fluid chemokine C-C motif ligand 2 in neurocognition among HIV-infected patients.

OBJECTIVES: We examined interrelationships between chemokine C-C motif ligand 2 (CCL2) genotype and expression of inflammatory markers in the cerebrospinal fluid (CSF), plasma viral load, CD4 cell count and neurocognitive functioning among HIV-infected adults. We hypothesized that HIV-positive carriers of the 'risk' CCL2 -2578G allele, caused by a single nucleotide polymorphism (SNP) at rs1024611, would have a higher concentration of CCL2 in CSF, and that CSF CCL2 would be associated with both higher concentrations of other proinflammatory markers in CSF and worse neurocognitive functioning. DESIGN: A cross-sectional study of 145 HIV-infected individuals enrolled in the National NeuroAIDS Tissue Consortium cohort for whom genotyping, CSF and neurocognitive data were available. METHODS: Genomic DNA was extracted from peripheral blood mononuclear cells and/or frozen tissue specimens. CSF levels of CCL2, interleukin (IL)-2, IL-6, tumour necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), soluble tumor necrosis factor receptor 2, sIL-6Rα, sIL-2, sCD14 and B-cell activating factor were quantified. Neurocognitive functioning was measured using a comprehensive battery of neuropsychological tests. RESULTS: Carriers of the CCL2 -2578G allele had a significantly higher concentration of CCL2 in CSF. CSF CCL2 level was positively and significantly associated with other CSF neuroinflammatory markers and worse cognitive functioning. There was a significant association between genotype and plasma viral load, such that carriers of the CCL2 -2578G allele with high viral load expressed greater levels of CCL2 and had higher neurocognitive deficit scores than other genotype/viral load groups. CONCLUSION: Individuals with the CCL2 -2578G allele had higher levels of CCL2 in CSF, which was associated with increased pro-inflammatory markers in CSF and worse neurocognitive functioning. The results highlight the potential role of intermediate phenotypes in studies of genotype and cognition.

Serum levels of cytokines and biomarkers for inflammation and immune activation, and HIV-associated non-Hodgkin B-cell lymphoma risk.

BACKGROUND: HIV infection is associated with a marked increase in risk for non-Hodgkin lymphoma (AIDS-NHL). However, the mechanisms that promote the development of AIDS-NHL are not fully understood. METHODS: In this study, serum levels of several cytokines and other molecules associated with immune activation were measured in specimens collected longitudinally during 1 to 5 years preceding AIDS-NHL diagnosis, in 176 AIDS-NHL cases and 176 HIV(+) controls from the Multicenter AIDS Cohort Study (MACS). RESULTS: Multivariate analyses revealed that serum levels of immunoglobulin free light chains (FLC), interleukin (IL)-6, IL-10, IP-10/CXCL10, neopterin, and TNF-α were elevated in those HIV(+) individuals who went on to develop AIDS-NHL. In addition, the fraction of specimens with detectable IL-2 was increased and the fraction with detectable IL-4 was decreased in these subjects. CONCLUSIONS: These results suggest that long-term, chronic immune activation, possibly driven by macrophage-produced cytokines, precedes development of NHL in HIV(+) individuals. IMPACT: FLC, IL-6, IL-10, IP-10/CXCL10, neopterin, and TNF-α may serve as biomarkers for AIDS-NHL. .

Levels of murine, but not human, CXCL13 are greatly elevated in NOD-SCID mice bearing the AIDS-associated Burkitt lymphoma cell line, 2F7.

Currently, few rodent models of AIDS-associated non-Hodgkin's lymphoma (AIDS-NHL) exist. In these studies, a novel mouse/human xenograft model of AIDS-associated Burkitt lymphoma (AIDS-BL) was created by injecting cells of the human AIDS-BL cell line, 2F7, intraperitoneally into NOD-SCID mice. Mice developed tumors in the peritoneal cavity, with metastases to the spleen, thymus, and mesenteric lymph nodes. Expression of the chemokine receptor, CXCR5, was greatly elevated in vivo on BL tumor cells in this model, as shown by flow cytometry. CXCL13 is the ligand for CXCR5, and serum and ascites levels of murine, but not human, CXCL13 showed a striking elevation in tumor-bearing mice, with levels as high as 200,000 pg/ml in ascites, as measured by ELISA. As shown by immunohistochemistry, murine CXCL13 was associated with macrophage-like tumor-infiltrating cells that appeared to be histiocytes. Blocking CXCR5 on 2F7 cells with neutralizing antibodies prior to injection into the mice substantially delayed tumor formation. The marked elevations in tumor cell CXCR5 expression and in murine CXCL13 levels seen in the model may potentially identify an important link between tumor-interacting histiocytes and tumor cells in AIDS-BL. These results also identify CXCL13 as a potential biomarker for this disease, which is consistent with previous studies showing that serum levels of CXCL13 were elevated in human subjects who developed AIDS-lymphoma. This mouse model may be useful for future studies on the interactions of the innate immune system and AIDS-BL tumor cells, as well as for the assessment of potential tumor biomarkers for this disease.