Félix Fernández Madrid

Serologic Laboratory Findings in Malignancy

Autoantibodies are extremely promising diagnostic and prognostic biomarkers of cancer, and have the potential to promote early diagnosis and to make a large impact by improving patient outcome and decreasing mortality. Moreover, autoantibodies may be useful reagents in the identification of subjects at risk for cancer, bearing premalignant tissue changes. Great efforts are being made in many laboratories to validate diagnostic panels of autoantibodies with high sensitivity and specificity that could be useful in a clinical setting. It is likely that prospective studies of sufficiently large cohorts of patients and controls using high-throughput technology may allow the identification of biomarkers with diagnostic significance, and perhaps of discrete antigen phenotypes with clinical significance. The identification of TAAs may also be essential for the development of anticancer vaccines, because autoantibodies found in cancer sera target molecules involved in signal transduction, cell-cycle regulation, cell proliferation, and apoptosis, playing important roles in carcinogenesis. On this basis, molecular studies of antigenantibody systems in cancer promise to yield valuable information on the carcinogenic process. TAAs identified by serum antibodies in cancer sera can be natural immunogenic molecules, useful as targets for cancer immunotherapy. An important problem encountered in the practice of medicine is the identification of healthy individuals in the general population who unknowingly are at high risk of developing cancer. For the rheumatologist, a related problem is the identification of those patients with rheumatic diseases who are at high risk for developing a malignant process. These problems encountered in the fields of cancer and the rheumatic diseases can in the future be helped by new diagnostic instruments based on antibodies. The need for promoting the early diagnosis of cancer is a recognized major public health problem in need of significant research support for the validation of multiple promising but inconclusive studies, with the intention of producing diagnostic panels of autoantibodies in various types of cancers. Cancer developing in patients with rheumatic diseases is also an important problem requiring prospective longterm follow-up studies of patients with rheumatic diseases, particularly because some of the new biologic therapies seem to increase the cancer risk. It is possible that a panel of autoantibodies common to patients with cancer and the rheumatic diseases may prove to be of value in the identification of those patients with ADs at high risk for neoplasms.

Autoantibodies in breast cancer sera are not epiphenomena and may participate in carcinogenesis

BackgroundThe objective of this work was to demonstrate that autoantibodies in breast cancer sera are not epiphenomena, and exhibit unique immunologic features resembling the rheumatic autoimmune diseases.MethodsWe performed a comprehensive study of autoantibodies on a collection of sera from women with breast cancer or benign breast disease, undergoing annual screening mammography. All women in this study had suspicious mammography assessment and underwent a breast biopsy. We used indirect immunofluorescence, the crithidia assay for anti-dsDNA antibodies, and multiple ELISAs for extractable nuclear antigens.ResultsAutoantibodies were detected in virtually all patients with breast cancer, predominantly of the IgG1 and IgG3 isotypes. The profile detected in breast cancer sera showed distinctive features, such as antibodies targeting mitochondria, centrosomes, centromeres, nucleoli, cytoskeleton, and multiple nuclear dots. The majority of sera showing anti-mitochondrial antibodies did not react with the M2 component of pyruvate dehydrogenase, characteristic of primary biliary cirrhosis. Anti-centromere antibodies were mainly anti-CENP-B. ELISAs for extractable nuclear antigens and the assays for dsDNA were negative.ConclusionsThe distinctive autoantibody profile detected in BC sera is the expression of tumor immunogenicity. Although some of these features resemble those in the rheumatic autoimmune diseases and primary biliary cirrhosis, the data suggest the involvement of an entirely different set of epithelial antigens in breast cancer. High titer autoantibodies targeting centrosomes, centromeres, and mitochondria were detected in a small group of healthy women with suspicious mammography assessment and no cancer by biopsy; this suggests that the process triggering autoantibody formation starts in the pre-malignant phase and that future studies using validated autoantibody panels may allow detection of breast cancer risk in asymptomatic women.Autoantibodies developing in breast cancer are not epiphenomena, but likely reflect an antigen-driven autoimmune response triggered by epitopes developing in the mammary gland during breast carcinogenesis. Our results support the validity of the multiple studies reporting association of autoantibodies with breast cancer. Results further suggest significant promise for the development of panels of breast cancer-specific, premalignant-phase autoantibodies, as well as studies on the autoantibody response to tumor associated antigens in the pathogenesis of cancer.

Anti-centrosome antibodies in breast cancer are the expression of autoimmunity

Centrosome abnormalities have been observed in nearly all human solid tumors, but their role in tumorigenesis is unclear. We have demonstrated that autoantibodies reacting with antigens in centrosomes are frequently found in BC sera. In this work, we attempted to characterize the centrosome antigens associated with BC. We immunoscreened a T7 cDNA library of BC proteins with BC sera, and the autoantigens identified were printed as a microarray and hybridized with BC and control sera. We used immunohistochemistry (IHC) to investigate the expression of the cloned autoantigens in BC tissue. Immunoscreening with BC sera led to the cloning of autoantibodies recognizing epitopes developing in a family of proteins located on centrosomes such as peri-centriolar material-1, isomorph CRA, stathmin1, HS actin gamma1, SUMO/sentrin peptidase 2, and ubiquitin-conjugating enzyme E2 variant 1. Antibody reactivity to these proteins that are associated with centrosome assembly and/or microtubule function was highly associated with the diagnosis of BC. IHC staining of formalin-fixed paraffin-embedded sections with specific antibodies showed that aurora and stathmin are expressed in BC. The discovery of autoantibodies to important centrosome antigens associated with BC suggests that this immune reactivity could be related to autoimmunity developing in BC. Our finding that some of these antibodies are also present in a group of healthy women suggests that breakdown of tolerance to centrosome proteins may occur early in breast carcinogenesis and that autoantibodies to centrosome antigens might be biomarkers of early BC.

Autoantibodies in Breast Cancer Identify Proteins Involved in Self- Renewal and Epigenetic Chromatin Remodeling

Recent studies have shown that autoantibodies developing in cancer patients are tumor-associated and are promising biomarkers for the diagnosis and prognosis of cancer. Here we report a panel of signal transduction molecules with partial sequences identical to the oncogene Bmi-1, NIFK, a nucleolar protein interacting with the FHA domain of Ki- 67, TAB182, a protein interacting with tankyrase-1, CNOT6L, a subunit of the CCR4-NOT complex and to Elp3, a subunit of the elongator complex that are recognized as autoantigens by breast cancer sera with the ability to discriminate between invasive breast cancer and normal sera with high sensitivity and specificity. The proteins bearing the epitopes recognized by these antibodies have conserved regions involved in protein-protein interactions participating in regulatory processes such as self renewal, cell proliferation and survival, chromatin modulation, transcriptional silencing and organ patterning, usually ascribed to stem cell function. In this work we demonstrate by autoantigen microarray analysis that autoantibodies in breast cancer sera have the potential to delineate pathway connectivity. Our data indicate that several pathways involved in maintaining telomere stability are the target of an autoimmune reaction in breast cancer. These find- ings suggest that autoantibodies with the ability to recognize autoantigens involved in self-renewal and epigenetic chro- matin remodeling have the potential to predict an invasive tendency of breast cancer.

Abstract P2-08-01: Autoimmunity in breast carcinogenesis. A new paradigm

The B cell response in breast cancer [BC] may have a tumor-promoting effect. The objective of this work was to demonstrate that TAAs and cytokines inflict autoimmune damage to the breast creating a chronic inflammatory milieu that promotes BC progression Methods: The DCIS.com nude mouse xenograft model of BC was treated with rituximab and dexamethasone. Immunoscreening of a cDNA library, PCR, and sequence determination. Immunohistochemistry [IHC] for inflammatory cytokines, immunoglobulins and complement and the TUNEL assay. Results: Deposition of immunoglobulins and complement was shown on breast epithelial and stromal cells and the involvement of IL-4, IL-6, IL-17, TNF-alpha and NFkB was demonstrated by IHC staining. We identified autoantibodies recognizing multiple human breast epithelial and stromal antigens and the Fc and V regions of immunoglobulins. Rituximab and dexamethasone treatment markedly modified the macroscopic and microscopic appearance of BC and the pattern of cytokine deposition, induced increase in vascularity, marked stromal hypertrophy and massive debulking of the tumor by drastically reducing the tumor mass through apoptosis. Conclusions : These results suggest that autoantibody- and cytokine-mediated autoimmune damage, triggered by TAAs creates a chronic inflammatory milieu with generation of pro-inflammatory and tumor promoting signals supporting BC progression. The dramatic results of the treatment of the xenograft model with rituximab and dexamethasone illustrate the potential of treating BC as a chronic rheumatic autoimmune disease. The demonstration of a major role of autoimmunity in BC progression may have a transformative impact on prevention, diagnosis and treatment of BC. Citation Format: Felix Fernandez Madrid, Marie-Claire Maroun, Leonard Lipovich, Larry Tait, Azadeh Stark, Richard Zarbo, Lisa Polin, Dhananjay Chitale. Autoimmunity in breast carcinogenesis. A new paradigm [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-08-01.

Abstract P2-08-02: Autoimmunity to centrosomal and proteasome proteins in breast cancer. A link to chromosome instability?

BACKGROUND. Perturbation of centrosomal or centrosome-associated proteins has been observed in nearly all human solid tumors and has been implicated in the origin of chromosomal instability. Ubiquitin-proteasome degradation is highly dependent on the organizing capabilities of the centrosomes. Here we report that autoantibodies in breast cancer [BC] sera target centrosomal proteins as well as important proteins involved in proteasome protein degradation. METHODS. We immunoscreened a T7 cDNA library of BC proteins and the association of the cloned autoantigens with BC was studied by autoantigen microarray analysis. We used immunohistochemistry [IHC] to investigate the expression of the centrosome and centrosome-associated autoantigens identified. RESULTS. Immunoscreening with BC sera led to the identification of autoantibodies recognizing epitopes developing in a family of proteins located on the centrosomes such as NIMA-related kinase 7, dynein heavy chain domain 3, peri-centriolar material-1, isomorph CRA, and stathmin-1, the ubiquitin-conjugating enzyme E2, the proteasome 26S subunit and the SUMO/sentrin peptidase. Antibody reactivity to these proteins which are associated with centrosome assembly, microtubule function and protein degradation were highly associated with the diagnosis of BC. IHC staining of paraffin-embedded BC sections with specific antibodies showed that aurora and stathmin-1 and other centrosome antigens are expressed in BC. CONCLUSIONS. The discovery of autoantibodies targeting important centrosome and proteasome proteins associated with BC suggests that this immune reactivity could be related to autoimmunity developing in BC. Our findings indicate that these autoantibodies might be biomarkers of early BC and suggest the possibility of a link with chromosomal instability in BC. Citation Format: Marie-Claire Maroun, Ofelia Olivero, Judith Abrams, Wei Chen, Azadeh Stark, Carol Peebles, Larry Tait, Dhanajay Chitale, Felix Fernandez Madrid. Autoimmunity to centrosomal and proteasome proteins in breast cancer. A link to chromosome instability? [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-08-02.