Killer cell proteases can target viral immediate-early proteins to control human cytomegalovirus infection in a noncytotoxic manner.

Human cytomegalovirus (HCMV) is the most frequent viral cause of congenital defects and can trigger devastating disease in immune-suppressed patients. Cytotoxic lymphocytes (CD8+ T cells and NK cells) control HCMV infection by releasing interferon-γ and five granzymes (GrA, GrB, GrH, GrK, GrM), which are believed to kill infected host cells through cleavage of intracellular death substrates. However, it has recently been demonstrated that the in vivo killing capacity of cytotoxic T cells is limited and multiple T cell hits are required to kill a single virus-infected cell. This raises the question whether cytotoxic lymphocytes can use granzymes to control HCMV infection in a noncytotoxic manner. Here, we demonstrate that (primary) cytotoxic lymphocytes can block HCMV dissemination independent of host cell death, and interferon-α/ß/γ. Prior to killing, cytotoxic lymphocytes induce the degradation of viral immediate-early (IE) proteins IE1 and IE2 in HCMV-infected cells. Intriguingly, both IE1 and/or IE2 are directly proteolyzed by all human granzymes, with GrB and GrM being most efficient. GrB and GrM cleave IE1 after Asp398 and Leu414, respectively, likely resulting in IE1 aberrant cellular localization, IE1 instability, and functional impairment of IE1 to interfere with the JAK-STAT signaling pathway. Furthermore, GrB and GrM cleave IE2 after Asp184 and Leu173, respectively, resulting in IE2 aberrant cellular localization and functional abolishment of IE2 to transactivate the HCMV UL112 early promoter. Taken together, our data indicate that cytotoxic lymphocytes can also employ noncytotoxic ways to control HCMV infection, which may be explained by granzyme-mediated targeting of indispensable viral proteins during lytic infection.

ARGX-117, a therapeutic complement inhibiting antibody targeting C2.

BACKGROUND: Activation of the classical and lectin pathway of complement may contribute to tissue damage and organ dysfunction of antibody-mediated diseases and ischemia-reperfusion conditions. Complement factors are being considered as targets for therapeutic intervention. OBJECTIVE: We sought to characterize ARGX-117, a humanized inhibitory monoclonal antibody against complement C2. METHODS: The mode-of-action and binding characteristics of ARGX-117 were investigated in detail. Furthermore, its efficacy was analyzed in in vitro complement cytotoxicity assays. Finally, a pharmacokinetic/pharmacodynamic study was conducted in cynomolgus monkeys. RESULTS: Through binding to the Sushi-2 domain of C2, ARGX-117 prevents the formation of the C3 proconvertase and inhibits classical and lectin pathway activation upstream of C3 activation. As ARGX-117 does not inhibit the alternative pathway, it is expected not to affect the antimicrobial activity of this complement pathway. ARGX-117 prevents complement-mediated cytotoxicity in in vitro models for autoimmune hemolytic anemia and antibody-mediated rejection of organ transplants. ARGX-117 exhibits pH- and calcium-dependent target binding and is Fc-engineered to increase affinity at acidic pH to the neonatal Fc receptor, and to reduce effector functions. In cynomolgus monkeys, ARGX-117 dose-dependently reduces free C2 levels and classical pathway activity. A 2-dose regimen of 80 and 20 mg/kg separated by a week, resulted in profound reduction of classical pathway activity lasting for at least 7 weeks. CONCLUSIONS: ARGX-117 is a promising new complement inhibitor that is uniquely positioned to target both the classical and lectin pathways while leaving the alternative pathway intact.

Increased intra-articular granzyme M may trigger local IFN-λ1/IL-29 response in rheumatoid arthritis.

OBJECTIVES: Granzymes are serine proteases involved in eliminating tumour cells and virally infected cells. In addition, extracellular granzyme levels are elevated in inflammatory conditions, including several types of infection and autoimmune diseases, such as rheumatoid arthritis (RA). While GrA and GrB have been associated with RA, a role for the other three granzymes (GrH, GrK, and GrM) in this disease remains unclear. Here, we aimed to investigate the presence and role of GrM and GrK in serum and synovial fluid of patients with RA, psoriatic arthritis, and osteoarthritis. METHODS: Granzyme levels were determined in serum, synovial fluid, peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) of RA patients and relevant control groups. In addition, the link between GrM and inflammatory cytokines in synovial fluid was investigated. RESULTS: Serum GrM and GrK levels were not affected in RA. GrM, but not GrK, levels were elevated in synovial fluid of RA patients. GrM was mainly expressed by cytotoxic lymphocytes in SFMCs with a similar expression pattern as compared with PBMCs. Intra-articular GrM expression correlated with IL-25, IL-29, XCL1, and TNFα levels. Intriguingly, purified GrM triggered the release of IL-29 (IFN-λ1) from human fibroblasts in vitro. CONCLUSIONS: These data indicate that GrM levels are increased in RA synovial fluid and that GrM can stimulate proinflammatory IL-29 release from fibroblasts, suggesting a role of GrM in the pathogenesis of RA.

Sialic Acid-Engineered IL4-10 Fusion Protein is Bioactive and Rapidly Cleared from the Circulation.

PURPOSE: Modulating sialylation of therapeutic glycoproteins may be used to influence their clearance and systemic exposure. We studied the effect of low and high sialylated IL4-10 fusion protein (IL4-10 FP) on in vitro and in vivo bioactivity and evaluated the effect of differential sialylation on pharmacokinetic parameters. METHODS: CHO cell lines producing low (IL4-10 FP lowSA) and high sialylated (IL4-10 FP highSA) fusion protein were generated. Bioactivity of the proteins was evaluated in an LPS-stimulated whole blood assay. Pharmacokinetics were studied in rats, analyzing plasma levels of IL4-10 FP upon intravenous injection. In vivo activity was assessed in an inflammatory pain mice model upon intrathecal injection. RESULTS: IL4-10 FP lowSA and IL4-10 FP highSA had similar potency in vitro. The pharmacokinetics study showed a 4-fold higher initial systemic clearance of IL4-10 FP lowSA, whereas the calculated half-life of both IL4-10 FP lowSA and IL4-10 FP highSA was 20.7 min. Finally, both IL4-10 FP glycoforms inhibited persistent inflammatory pain in mice to the same extent. CONCLUSIONS: Differential sialylation of IL4-10 fusion protein does not affect the in vitro and in vivo activity, but clearly results in a difference in systemic exposure. The rapid systemic clearance of low sialylated IL4-10 FP could be a favorable characteristic to minimize systemic exposure after administration in a local compartment.

FcRγ-chain ITAM signaling is critically required for cross-presentation of soluble antibody-antigen complexes by dendritic cells.

The uptake of Ag-Ab immune complexes (IC) after the ligation of activating FcγR on dendritic cells (DC) leads to 100 times more efficient Ag presentation than the uptake of free Ags. FcγRs were reported to facilitate IC uptake and simultaneously induce cellular activation that drives DC maturation and mediates efficient T cell activation. Activating FcγRs elicit intracellular signaling via the ITAM domain of the associated FcRγ-chain. Studies with FcRγ-chain knockout (FcRγ(-/-)) mice reported FcRγ-chain ITAM signaling to be responsible for enhancing both IC uptake and DC maturation. However, FcRγ-chain is also required for surface expression of activating FcγRs, hampering the dissection of ITAM-dependent and independent FcγR functions in FcRγ(-/-) DCs. In this work, we studied the role of FcRγ-chain ITAM signaling using DCs from NOTAM mice that express normal surface levels of activating FcγR, but lack functional ITAM signaling. IC uptake by bone marrow-derived NOTAM DCs was reduced compared with wild-type DCs, but was not completely absent as in FcRγ(-/-) DCs. In NOTAM DCs, despite the uptake of ICs, both MHC class I and MHC class II Ag presentation was completely abrogated similar to FcRγ(-/-) DCs. Secretion of cytokines, upregulation of costimulatory molecules, and Ag degradation were abrogated in NOTAM DCs in response to FcγR ligation. Cross-presentation using splenic NOTAM DCs and prolonged incubation with OVA-IC was also abrogated. Interestingly, in this setup, proliferation of CD4(+) OT-II cells was induced by NOTAM DCs. We conclude that FcRγ-chain ITAM signaling facilitates IC uptake and is essentially required for cross-presentation, but not for MHC class II Ag presentation.

Cellular immunotherapy for medulloblastoma.

Medulloblastoma (MB) is the most common malignant brain tumor in children, making up ~20% of all primary pediatric brain tumors. Current therapies consist of maximal surgical resection and aggressive radio- and chemotherapy. A third of the treated patients cannot be cured and survivors are often left with devastating long-term side effects. Novel efficient and targeted treatment is desperately needed for this patient population. Cellular immunotherapy aims to enhance and utilize immune cells to target tumors, and has been proven successful in various cancers. However, for MB, the knowledge and possibilities of cellular immunotherapy are limited. In this review, we provide a comprehensive overview of the current status of cellular immunotherapy for MB, from fundamental in vitro research to in vivo models and (ongoing) clinical trials. In addition, we compare our findings to cellular immunotherapy in glioma, an MB-like intracranial tumor. Finally, future possibilities for MB are discussed to improve efficacy and safety.

Serpin B9 controls tumor cell killing by CAR T cells.

BACKGROUND: Initial clinical responses with gene engineered chimeric antigen receptor (CAR) T cells in cancer patients are highly encouraging; however, primary resistance and also relapse may prevent durable remission in a substantial part of the patients. One of the underlying causes is the resistance mechanisms in cancer cells that limit effective killing by CAR T cells. CAR T cells exert their cytotoxic function through secretion of granzymes and perforin. Inhibition of granzyme B (GrB) can underlie resistance to T cell-mediated killing, and it has been shown that serine proteinase inhibitor serpin B9 can effectively inhibit GrB. We aimed to determine whether expression of serpin B9 by cancer cells can lead to resistance toward CAR T cells. METHODS: Serpin B9 gene and protein expression were examined by R2 or DepMap database mining and by western blot or flow cytometric analysis, respectively. Coculture killing experiments were performed with melanoma cell line MeWo, diffuse large B cell lymphoma (DLBCL) cell line OCI-Ly7 or primary chronic lymphocytic leukemia (CLL) cells as target cells and natural killer cell line YT-Indy, CD20 CAR T cells or CD19 CAR T cells as effector cells and analyzed by flow cytometry. RESULTS: Serpin B9 protein expression was previously shown to be associated with clinical outcome in melanoma patients and in line with these observations we demonstrate that enforced serpin B9 expression in melanoma cells reduces sensitivity to GrB-mediated killing. Next, we examined serpin B9 expression in a wide array of primary tumor tissues and human cell lines to find that serpin B9 is uniformly expressed in B-cell lymphomas and most prominently in DLBCL and CLL. Subsequently, using small interfering RNA, we silenced serpin B9 expression in DLBCL cells, which increased their sensitivity to CD20 CAR T cell-mediated killing. In addition, we showed that co-ulture of primary CLL cells with CD20 CAR T cells results in selection of serpin B9-high CLL cells, suggesting these cells resist CAR T-cell killing. CONCLUSIONS: Overall, the data indicate that serpin B9 is a resistance mediator for CAR T cell-mediated tumor cell killing that should be inhibited or bypassed to improve CAR T-cell responses.

Cleavage of Notch1 by granzyme B disables its transcriptional activity.

Granzyme-mediated cell death is the main pathway for cytotoxic lymphocytes to kill virus-infected and tumour cells. A major player in this process is GrB (granzyme B), which triggers apoptosis in both caspase-dependent and caspase-independent pathways. A caspase-independent substrate of GrB is the highly conserved transmembrane receptor Notch1. The GrB cleavage sites in Notch1 and functional consequences of Notch1 cleavage by GrB were unknown. In the present study, we confirmed that Notch1 is a direct and caspase-independent substrate of GrB. We demonstrate that GrB cleaved the intracellular Notch1 domain at least twice at two distinct aspartic acids, Asp1860 and Asp1961. GrB cleavage of Notch1 can occur in all subcellular compartments, during maturation of the receptor, at the membrane, and in the nucleus. GrB also displayed perforin-independent functions by cleaving the extracellular domain of Notch1. Overall, cleavage of Notch1 by GrB resulted in a loss of transcriptional activity, independent of Notch1 activation. We conclude that GrB disables Notch1 function, probably resulting in anti-cellular proliferation and cell death signals.

Collagen Fragments Produced in Cancer Mediate T Cell Suppression Through Leukocyte-Associated Immunoglobulin-Like Receptor 1.

The tumor microenvironment (TME) is a complex structure comprised of tumor, immune and stromal cells, vasculature, and extracellular matrix (ECM). During tumor development, ECM homeostasis is dysregulated. Collagen remodeling by matrix metalloproteinases (MMPs) generates specific collagen fragments, that can be detected in the circulation of cancer patients and correlate with poor disease outcome. Leukocyte-Associated Immunoglobulin-like Receptor-1 (LAIR-1) is an inhibitory collagen receptor expressed on immune cells in the TME and in the circulation. We hypothesized that in addition to ECM collagen, collagen fragments produced in cancer can mediate T cell immunosuppression through LAIR-1. Our analyses of TCGA datasets show that cancer patients with high tumor mRNA expression of MMPs, collagen I and LAIR-1 have worse overall survival. We show that in vitro generated MMP1 or MMP9 collagen I fragments bind to and trigger LAIR-1. Importantly, LAIR-1 triggering by collagen I fragments inhibits CD3 signaling and IFN-γ secretion in a T cell line. LAIR-2 is a soluble homologue of LAIR-1 with higher affinity for collagen and thereby acts as a decoy receptor. Fc fusion proteins of LAIR-2 have potential as cancer immunotherapeutic agents and are currently being tested in clinical trials. We demonstrate that collagen fragment-induced inhibition of T cell function could be reversed by LAIR-2 fusion proteins. Overall, we show that collagen fragments produced in cancer can mediate T cell suppression through LAIR-1, potentially contributing to systemic immune suppression. Blocking the interaction of LAIR-1 with collagen fragments could be an added benefit of LAIR-1-directed immunotherapy.

Extracellular Granzyme K Modulates Angiogenesis by Regulating Soluble VEGFR1 Release From Endothelial Cells.

Angiogenesis is crucial for normal development and homeostasis, but also plays a role in many diseases including cardiovascular diseases, autoimmune diseases, and cancer. Granzymes are serine proteases stored in the granules of cytotoxic cells, and have predominantly been studied for their pro-apoptotic role upon delivery in target cells. A growing body of evidence is emerging that granzymes also display extracellular functions, which largely remain unknown. In the present study, we show that extracellular granzyme K (GrK) inhibits angiogenesis and triggers endothelial cells to release soluble VEGFR1 (sVEGFR1), a decoy receptor that inhibits angiogenesis by sequestering VEGF-A. GrK does not cleave off membrane-bound VEGFR1 from the cell surface, does not release potential sVEGFR1 storage pools from endothelial cells, and does not trigger sVEGFR1 release via protease activating receptor-1 (PAR-1) activation. GrK induces de novo sVEGFR1 mRNA and protein expression and subsequent release of sVEGFR1 from endothelial cells. GrK protein is detectable in human colorectal tumor tissue and its levels positively correlate with sVEGFR1 protein levels and negatively correlate with T4 intratumoral angiogenesis and tumor size. In conclusion, extracellular GrK can inhibit angiogenesis via secretion of sVEGFR1 from endothelial cells, thereby sequestering VEGF-A and impairing VEGFR signaling. Our observation that GrK positively correlates with sVEGFR1 and negatively correlates with angiogenesis in colorectal cancer, suggest that the GrK-sVEGFR1-angiogenesis axis may be a valid target for development of novel anti-angiogenic therapies in cancer.

Reformatting palivizumab and motavizumab from IgG to human IgA impairs their efficacy against RSV infection in vitro and in vivo.

Respiratory syncytial virus (RSV) infection is a leading cause of hospitalization and mortality in young children. Protective therapy options are limited. Currently, palivizumab, a monoclonal IgG1 antibody, is the only licensed drug for RSV prophylaxis, although other IgG antibody candidates are being evaluated. However, at the respiratory mucosa, IgA antibodies are most abundant and act as the first line of defense against invading pathogens. Therefore, it would be logical to explore the potential of recombinant human IgA antibodies to protect against viral respiratory infection, but very little research on the topic has been published. Moreover, it is unknown whether human antibodies of the IgA isotype are better suited than those of the IgG isotype as antiviral drugs to combat respiratory infections. To address this, we generated various human IgA antibody formats of palivizumab and motavizumab, two well-characterized human IgG1 anti-RSV antibodies. We evaluated their efficacy to prevent RSV infection in vitro and in vivo and found similar, but somewhat decreased efficacy for different IgA subclasses and formats. Thus, reformatting palivizumab or motavizumab into IgA reduces the antiviral potency of either antibody. Moreover, our results indicate that the efficacy of intranasal IgA prophylaxis against RSV infection in human FcαRI transgenic mice is independent of Fc receptor expression.

Development and Validation of a Multiplex Non-HLA Antibody Assay for the Screening of Kidney Transplant Recipients.

The best treatment for patients with end-stage renal disease is kidney transplantation. Although graft survival rates have improved in the last decades, patients still may lose their grafts partly due to the detrimental effects of donor-specific antibodies (DSA) against human leukocyte antigens (HLA) and to a lesser extent also by antibodies directed against non-HLA antigens expressed on the donor endothelium. Assays to detect anti-HLA antibodies are already in use for many years and have been proven useful for transplant risk stratification. Currently, there is a need for assays to additionally detect multiple non-HLA antibodies simultaneously in order to study their clinical relevance in solid organ transplantation. This study describes the development, technical details and validation of a high-throughput multiplex assay for the detection of antibodies against 14 non-HLA antigens coupled directly to MagPlex microspheres or indirectly via a HaloTag. The non-HLA antigens have been selected based on a literature search in patients with kidney disease or following transplantation. Due to the flexibility of the assay, this approach can be used to include alternative antigens and can also be used for screening of other organ transplant recipients, such as heart and lung.

Nuclear DDX3 expression predicts poor outcome in colorectal and breast cancer.

PURPOSE: DEAD box protein 3 (DDX3) is an RNA helicase with oncogenic properties that shuttles between the cytoplasm and nucleus. The majority of DDX3 is found in the cytoplasm, but a subset of tumors has distinct nuclear DDX3 localization of yet unknown biological significance. This study aimed to evaluate the significance of and mechanisms behind nuclear DDX3 expression in colorectal and breast cancer. METHODS: Expression of nuclear DDX3 and the nuclear exporter chromosome region maintenance 1 (CRM1) was evaluated by immunohistochemistry in 304 colorectal and 292 breast cancer patient samples. Correlations between the subcellular localization of DDX3 and CRM1 and the difference in overall survival between patients with and without nuclear DDX3 were studied. In addition, DDX3 mutants were created for in vitro evaluation of the mechanism behind nuclear retention of DDX3. RESULTS: DDX3 was present in the nucleus of 35% of colorectal and 48% of breast cancer patient samples and was particularly strong in the nucleolus. Nuclear DDX3 correlated with worse overall survival in both colorectal (hazard ratio [HR] 2.34, P<0.001) and breast cancer (HR 2.39, P=0.004) patients. Colorectal cancers with nuclear DDX3 expression more often had cytoplasmic expression of the nuclear exporter CRM1 (relative risk 1.67, P=0.04). In vitro analysis of DDX3 deletion mutants demonstrated that CRM1-mediated export was most dependent on the N-terminal nuclear export signal. CONCLUSION: Overall, we conclude that nuclear DDX3 is partially CRM1-mediated and predicts worse survival in colorectal and breast cancer patients, putting it forward as a target for therapeutic intervention with DDX3 inhibitors under development in these cancer types.

Reduced serpinB9-mediated caspase-1 inhibition can contribute to autoinflammatory disease.

Patients who suffer from autoinflammatory disease (AID) exhibit seemingly uncontrolled release of interleukin (IL)-1ß. The presence of this inflammatory cytokine triggers immune activation in absence of pathogens and foreign material. The mechanisms that contribute to 'sterile inflammation' episodes in AID patients are not fully understood, although for some AIDs underlying genetic causes have been identified. We show that the serine protease inhibitor B9 (serpinB9) regulates IL-1ß release in human monocytes. SerpinB9 function is more commonly known for its role in control of granzyme B. SerpinB9 however also serves to restrain IL-1ß maturation through caspase-1 inhibition. We here describe an autoinflammatory disease-associated serpinB9 (c.985G>T, A329S) variant, which we discovered in a patient with unknown AID. Using patient cells and serpinB9 overexpressing monocytic cells, we show the A329S variant of serpinB9 exhibits unobstructed granzyme B inhibition, but compromised caspase-1 inhibition. SerpinB9 gene variants might contribute to AID development.

Granzymes A and K differentially potentiate LPS-induced cytokine response.

Granzymes are serine proteases that, upon release from cytotoxic cells, induce apoptosis in tumor cells and virally infected cells. In addition, a role of granzymes in inflammation is emerging. Recently, we have demonstrated that extracellular granzyme K (GrK) potentiates lipopolysaccharide (LPS)-induced cytokine response from monocytes. GrK interacts with LPS, disaggregates LPS micelles, and stimulates LPS-CD14 binding and Toll-like receptor signaling. Here we show that human GrA also potentiates cytokine responses in human monocytes initiated by LPS or Gram-negative bacteria. Similar to GrK, this effect is independent of GrA catalytic activity. Unlike GrK, however, GrA does not bind to LPS, has little influence on LPS micelle disaggregation, and does not augment LPS-CD14 complex formation. We conclude that GrA and GrK differentially modulate LPS-Toll-like receptor signaling in monocytes, suggesting functional redundancy among cytotoxic lymphocyte proteases in the anti-bacterial innate immune response.

Improved in vivo anti-tumor effects of IgA-Her2 antibodies through half-life extension and serum exposure enhancement by FcRn targeting.

Antibody therapy is a validated treatment approach for several malignancies. All currently clinically applied therapeutic antibodies (Abs) are of the IgG isotype. However, not all patients respond to this therapy and relapses can occur. IgA represents an alternative isotype for antibody therapy that engages FcαRI expressing myeloid effector cells, such as neutrophils and monocytes. IgA Abs have been shown to effectively kill tumor cells both in vitro and in vivo. However, due to the short half-life of IgA Abs in mice, daily injections are required to reach an effect comparable to IgG Abs. The relatively long half-life of IgG Abs and serum albumin arises from their capability of interacting with the neonatal Fc receptor (FcRn). As IgA Abs lack a binding site for FcRn, we generated IgA Abs with the variable regions of the Her2-specific Ab trastuzumab and attached an albumin-binding domain (ABD) to the heavy or light chain (HCABD/LCABD) to extend their serum half-life. These modified Abs were able to bind albumin from different species in vitro. Furthermore, tumor cell lysis of IgA-Her2-LCABD Abs in vitro was similar to unmodified IgA-Her2 Abs. Pharmacokinetic studies in mice revealed that the serum exposure and half-life of the modified IgA-Her2 Abs was extended. In a xenograft mouse model, the modified IgA1 Abs exhibited a slightly, but significantly, improved anti-tumor response compared to the unmodified Ab. In conclusion, empowering IgA Abs with albumin-binding capacity results in in vitro and in vivo functional Abs with an enhanced exposure and prolonged half-life.

TCF and Groucho-related genes influence pituitary growth and development.

Mutations in the prophet of PIT1 gene (PROP1) are the most common cause of multiple pituitary hormone deficiency in humans; however, the mechanism of PROP1 action is not well understood. We report that Prop1 is essential for dorsally restricted expression of a Groucho-related gene, transducin-like enhancer of split 3 (Tle3), which encodes a transcriptional corepressor. Deficiency of a related gene, amino terminal enhancer of split (Aes), causes pituitary anomalies and growth insufficiency. TLE3 and AES have been shown to interact with TCF/LEF (transcripiton factors of the T cell-specific and lymphoid enhancer specific group) family members in cell culture systems. In the absence of TCF4 (Tcf7L2), Prop1 levels are elevated, pituitary hyperplasia ensues and palate closure is abnormal. Thus, we demonstrate that Tcf4 and Aes influence pituitary growth and development, and place Tcf4 and Tle3 in the genetic hierarchy with Prop1.

Validation of DNA promoter hypermethylation biomarkers in breast cancer--a short report.

PURPOSE: DNA promoter hypermethylation of tumor suppressor genes is known to occur early in cancer development, including breast cancer. To improve early breast cancer detection, we aimed to investigate whether the identification of DNA promoter hypermethylation might be of added value. METHODS: The methylation status of a panel of 19 candidate genes (AKR1B1, ALX1, ARHGEF7, FZD10, GHSR, GPX7, GREM1, GSTP1, HOXD1, KL, LHX2, MAL, MGMT, NDRG2, RASGRF2, SFRP1, SFRP2, TM6SF1 and TMEFF2) was determined in formalin-fixed paraffin-embedded normal breast and breast cancer tissue samples using gel-based methylation-specific PCR (MSP). RESULTS: The promoters of the AKR1B1, ALX1, GHSR, GREM1, RASGRF2, SFRP2, TM6SF1 and TMEFF2 genes were found to be significantly differentially methylated in normal versus malignant breast tissues. Based on sensitivity, specificity and logistic regression analyses the best performing genes for detecting breast cancer were identified. Through multivariate analyses, we found that AKR1B1 and TM6SF1 could detect breast cancer with an area under the curve (AUC) of 0.986 in a receiver operating characteristic (ROC) assessment. CONCLUSIONS: Based on our data, we conclude that AKR1B1 and TM6SF1 may serve as candidate methylation biomarkers for early breast cancer detection. Further studies are underway to evaluate the methylation status of these genes in body fluids, including nipple aspirates and blood.