Opportunities for Antigen Discovery in Metastatic Breast Cancer.

Immune checkpoint inhibitor-based immunotherapy (ICI) of breast cancer is currently efficacious in a fraction of triple negative breast cancers (TNBC) as these cancers generally carry high tumor mutation burden (TMB) and show increased tumor infiltration by CD8+ T cells. However, most estrogen receptor positive breast cancers (ERBC) have low TMB and/or are infiltrated with immunosuppressive regulatory T cells (Tregs) and thus fail to induce a significant anti-tumor immune response. Our understanding of the immune underpinning of the anti-tumor effects of CDK4/6 inhibitor (CDKi) treatment coupled with new knowledge about the mechanisms of tolerance to self-antigens suggests a way forward, specifically via characterizing and exploiting the repertoire of tumor antigens expressed by metastatic ERBC. These treatment-associated tumor antigens (TATA) may include the conventional tumor neoantigens (TNA) encoded by single nucleotide mutations, TNA encoded by tumor specific aberrant RNA transcription, splicing and DNA replication induced frameshift (FS) events as well as the shared tumor antigens. The latter may include the conventional tumor associated antigens (TAA), cancer-testis antigens (CTA) and antigens encoded by the endogenous retroviral (ERV) like sequences and repetitive DNA sequences induced by ET and CDKi treatment. An approach to identifying these antigens is outlined as this will support the development of a multi-antigen-based immunotherapy strategy for improved targeting of metastatic disease with potential for minimal autoimmune toxicity against normal tissues.

Prostate-derived Ets factor, an oncogenic driver in breast cancer.

Prostate-derived Ets factor (PDEF), a member of the Ets family of transcription factors, differs from other family members in its restricted expression in normal tissues and its unique DNA-binding motif. These interesting attributes coupled with its aberrant expression in cancer have rendered PDEF a focus of increasing interest by tumor biologists. This review provides a current understanding of the characteristics of PDEF expression and its role in breast cancer. The bulk of the evidence is consistent with PDEF overexpression in most breast tumors and an oncogenic role for this transcription factor in breast cancer. In addition, high PDEF expression in estrogen receptor-positive breast tumors showed significant correlation with poor overall survival in several independent cohorts of breast cancer patients. Together, these findings demonstrate PDEF to be an oncogenic driver of breast cancer and a biomarker of poor prognosis in this cancer. Based on this understanding and the limited expression of PDEF in normal human tissues, the development of PDEF-based therapeutics for prevention and treatment of breast cancer is also discussed.

18F-Fluorocholine PET/CT Complementing the Role of Dynamic Contrast-Enhanced MRI for Providing Comprehensive Diagnostic Workup in Prostate Cancer Patients With Suspected Relapse Following Radical Prostatectomy.

PURPOSE: The aim of this study was to compare the diagnostic performance of F-fluorocholine (FCH) PET/CT and dynamic contrast-enhanced MRI (DCE-MRI) of pelvis in restaging prostate cancer (PC) patients with biochemical recurrence (BCR) following radical prostatectomy (RP). METHODS: Twenty PC patients who had undergone RP and had BCR were recruited in this study. All the patients underwent whole-body FCH PET/CT and DCE-MRI of the pelvis. An overall pattern of recurrent disease was analyzed, and diagnostic accuracy for the detection of pelvic disease recurrence by the 2 modalities was evaluated by taking histopathologic analysis as the criterion standard. The whole-body FCH PET/CT images were also analyzed separately for the presence of any extra lesion(s). RESULTS: The initial mean Gleason score was 6.3 ± 1.53 (range, 4-9). The mean prostate-specific antigen levels at the time of relapse were 1.9 ± 2.87 ng/mL (range, 0.24-13.2 ng/mL). MRI findings were positive for primary tumor recurrence in the prostate bed in 6 patients (6/20 [30.0%]), pelvic lymph node metastases in 4 patients (4/20 [20.0%]), and for pelvic skeletal metastases in 2 patients (2/20 [10.0%]), respectively. On the other hand, FCH PET/CT results were positive in the corresponding sites in 7 (7/20 [35.0%]), 9 (9/20 [45.0%]), and 2 patients (2/20 [10.0%]), respectively. F-fluorocholine PET/CT and MRI showed comparable results in terms of sensitivity, specificity, and positive and negative predictive values for PC characterization. The whole-body FCH PET/CT was found to be useful in identifying unknown distant metastases in a significant proportion of patients. CONCLUSIONS: The correlative whole-body FCH PET/CT and pelvic DCE-MRI offer a complementary and comprehensive diagnostic workup for better management of PC patients with BCR following RP.

Prostate derived Ets transcription factor and Carcinoembryonic antigen related cell adhesion molecule 6 constitute a highly active oncogenic axis in breast cancer.

We previously reported overexpression of Prostate derived Ets transcription factor (PDEF) in breast cancer and its role in breast cancer progression, supporting PDEF as an attractive target in this cancer. The goal of this research was to identify specific PDEF induced molecules that, like PDEF, show overexpression in breast tumors and a role in breast tumor progression. PDEF expression was down regulated by shRNA in MCF-7 human breast tumor cell line, and probes from PDEF down-regulated and control MCF-7 cells were used to screen the HG-U133A human gene chips. These analyses identified 1318 genes that were induced two-fold or higher by PDEF in MCF-7 cells. Further analysis of three of these genes, namely CEACAM6, S100A7 and B7-H4, in relation to PDEF in primary breast tumors showed that in 82% of ER+, 67% of Her2 overexpressing and 24% of triple-negative breast tumors both PDEF and CEACAM6 expression was elevated 10-fold or higher in comparison to normal breast tissue. Overall, 72% (94 of 131) of the primary breast tumors showed 10-fold or higher expression of both PDEF and CEACAM6. In contrast, S100A7 and B7-H4 failed to show concordant elevated expression with PDEF in primary tumors. To determine the significance of elevated PDEF and CEACAM6 expression to tumor phenotype, their expression was down regulated by specific siRNAs in human breast tumor cell lines. This resulted in the loss of viability of tumor cells in vitro, supporting an oncogenic role for both PDEF and CEACAM6 in breast cancer. Together, these findings show that PDEF-CEACAM6 is a highly active oncogenic axis in breast cancer and suggest that targeting of these molecules should provide novel treatments for most breast cancer patients.

PDEF in prostate cancer.

Prostate-derived Ets factor (PDEF) is a relatively recently described member of the Ets family of transcription factors. It differs from other family members in its restricted and epithelial-specific expression in normal tissues and its unique DNA-binding motif that together may impart interesting specificity to its function. This communication reviews our current understanding of the expression characteristics of PDEF in normal prostate and in prostate cancer. Also, the biochemical and genetic evidence relating to the role of this transcription factor in prostate cancer is reviewed. Most evidence is consistent with an oncogenic role for PDEF in prostate cancer. Specific observations about the loss of PDEF expression in prostate tumors and its apparent role as a prostate tumor suppressor are also discussed. PDEF is one of the few transcription factors with potential to have a significant impact on the management of prostate cancer. A better understanding of its biology and its role in prostate cancer is urgently needed.

PDEF and PDEF-induced proteins as candidate tumor antigens for T cell and antibody-mediated immunotherapy of breast cancer.

Novel breast tumor antigens are needed to develop T cell and antibody-based vaccine immunotherapy approach against breast cancer. To this purpose, we have previously shown that PDEF is frequently over expressed in human breast tumors and exhibits highly restricted expression in normal human tissues that is primarily limited to normal prostate. Moreover, PDEF expression correlates with poor overall survival for breast cancer patients. Additionally, Pse (prostate-specific Ets, mouse homologue of PDEF) is immunogenic in female mice and PDEF sequence contains HLA-A2 binding potentially immunogenic peptides. Together, these observations support PDEF as a novel candidate breast tumor antigen. Further, we have identified certain PDEF-induced proteins including CEACAM6, B7-H4, and S100A7 as additional candidate breast tumor antigens.

Sam-pointed domain containing Ets transcription factor in luminal breast cancer pathogenesis.

We previously described frequent overexpression of Sam-pointed domain containing Ets transcription factor (SPDEF), also known as PDEF, in human breast cancer, and suggested a role for this transcription factor in breast tumor progression. To seek evidence in support of this hypothesis, the MCF-12A breast epithelial cell line was transfected with an SPDEF expression plasmid or with control vector plasmid and the transfected cells tested for their tumorigenic growth in vivo. The data showed that SPDEF expression in MCF-12A cells induced accelerated tumor growth in severe combined immune deficient mice compared with vector-transfected MCF-12A cells. Furthermore, Gene Expression Omnibus and Oncomine databases were mined to determine any correlation between SPDEF expression levels and clinical outcome. High SPDEF expression correlated with poor overall survival of patients with estrogen receptor+ breast cancer, in three independent data sets. In contrast, little correlation was observed between SPDEF expression and cancer relapse or remote metastases. SPDEF expression was further found to be restricted to tumors arising in the luminal epithelial lineage including estrogen receptor+ luminal subtype breast tumors, Her2/neu-positive tumors, and apocrine carcinomas. In contrast, little SPDEF expression was found in the basal subtype of breast tumors. Based on these results, we hypothesize that SPDEF has a function in the specification of the progenitor cells of the luminal epithelial lineage that become targets of oncogenesis in luminal breast cancer.

Expression characteristics of prostate-derived Ets factor support a role in breast and prostate cancer progression.

The purpose of this study was to understand the characteristics of prostate-derived Ets factor (PDEF) protein expression in breast and prostate cancer progression. A polyclonal antibody specific to PDEF was raised and reacted with tissue microarrays consisting of benign breast, in situ ductal, invasive ductal, and invasive lobular breast carcinomas. The antibody was also reacted with tissue microarrays, including benign prostate, prostate intraepithelial neoplasias (PINs), and prostate carcinomas. Increased expression of PDEF was identified in 18%, 50%, 46%, and 51% of benign breast tissues, intraductal, invasive ductal, and invasive lobular carcinomas, respectively. Importantly, in matched samples of benign breast vs tumor, 90% showed higher expression of PDEF in the tumor tissue. Moreover, in invasive breast carcinomas, increased PDEF expression tended to correlate with Her2/neu overexpression. Increased expression of PDEF was also found in 27%, 33%, and 40% of benign prostate tissues, PIN samples, and prostate adenocarcinomas, respectively. Again, in matching samples of cancer vs benign and cancer vs PIN, 68% and 70%, respectively, showed increased expression in the malignant tissue. Moreover, PDEF was found to be more highly expressed in tumors with intermediate or high Gleason score compared with low-grade tumors (P < .01). In addition, R1881 treatment induced PDEF expression in the LNCaP prostate tumor cell line, suggesting regulation of PDEF by androgens in vivo. Together, these results for the first time show frequent increased expression of PDEF protein in breast and prostate tumors and support a role for PDEF in breast and prostate cancer progression.

Prostate-derived Ets factor is overexpressed in serous epithelial ovarian tumors.

The frequent overexpression of prostate-derived Ets factor (PDEF) mRNA in ovarian cancer has been previously reported. The aim of this study was to evaluate PDEF protein expression in ovarian cancer and how this expression might vary at different stages of epithelial ovarian tumors in comparison to normal ovary. A new rabbit polyclonal antibody to PDEF was prepared, and immunohistochemistry was performed on tissue sections from 12 normal ovaries, 10 cases of benign serous cystadenoma, 17 cases of low malignant potential tumor, 19 cases of stage 1, and 15 cases of advanced stage primary epithelial (serous) ovarian carcinomas and their peritoneal metastases. Expression levels were assessed based on the percentage of positively staining cells and the intensity of staining. All 12 normal ovary and 10 benign serous cystadenoma cases were negative for PDEF expression. In contrast, 6 of 17 (35%) low malignant potential tumors, 5 of 19 (27%) stage 1, and 5 of 15 (33%) advanced stage ovarian tumors stained positive for PDEF expression. Together, these results show frequent overexpression of PDEF protein in epithelial ovarian tumors and its lack of expression in normal ovary and cystadenomas, and this supports a role for PDEF in ovarian tumorigenesis. Furthermore, these results suggest that PDEF is a potential marker and target in ovarian cancer.

Prostate derived Ets transcription factor shows better tumor-association than other cancer-associated molecules.

We previously reported that prostate derived Ets transcription factor (PDEF) is a breast tumor-associated molecule. To obtain further insights into PDEF expression in other human tumor types, a cDNA library database from human adult normal and tumor tissues was compiled and searched for PDEF distribution. The results showed that PDEF is present at relative higher frequencies in the cDNA libraries from brain, breast, lung and ovarian tumors in comparison to those from the corresponding normal tissues. RT/PCR analysis of PDEF expression in ovarian tumors confirmed that PDEF is expressed in 36 out of 51 (71%) ovarian tumors. Further comparison of the distribution of PDEF with other widely recognized cancer-associated molecules showed that PDEF has more restricted distributions than Her-2/neu, Bcl-2, survivin or telomerase in cDNA libraries from normal human tissues and more increased distribution than Her-2/neu, CA-125, Bcl-2, survivin and telomerase in cDNA libraries from brain (except survivin), breast, lung and ovarian tumors. These data together show a better tumor-association for PDEF and suggest that PDEF is a more suitable target for developing specific cancer therapies.

Tumor antigens and markers for breast and ovarian cancers.

Use of vaccines to prevent and treat breast and ovarian cancer is a highly attractive approach because of the expected minimal side effects and the potential to predict individuals likely to benefit from vaccination. To fully harness the capacity of the immune system for this purpose, it is necessary to characterize tumor antigens for these cancers so that purified antigens can be tested for their immunogenicity in individual patients and for their suitability as targets of vaccine-induced immunity. Discovery of novel breast and ovarian tumor antigens is also necessary for developing multi-antigen vaccines composed of multiple tumor antigens. Such vaccines are expected to induce diverse immune responses and minimize emergence of antigen-loss variant tumors that are resistant to vaccine-induced immunity. With the exception of melanomas, for most human cancers including breast and ovarian cancers the repertoire of known tumor antigens remains relatively small. In this review we will discuss the importance of characterizing tumor antigens for use in vaccination against cancer and then summarize antigens that have been characterized for human breast and ovarian cancers. We will also emphasize that identification of a novel tumor antigen, while an important first step, needs to be followed by a multi-step process of validation of that antigen. The steps in this validation process are i) to demonstrate that a tumor antigen is over-expressed at a reasonable frequency in primary tumors and in metastases; ii) to demonstrate the immunogenicity of a tumor antigen in an appropriate animal model; iii) to demonstrate its immunogenicity and safety in humans. Additional considerations in this review include: i) discussion of the potential of breast and ovarian tumor antigens as markers for early detection and for monitoring tumor burden in cancer patients; ii) discussion of their potential as prognostic markers of breast and ovarian cancers; and iii) discussion of a unique class of tumor antigens and markers that induce expression of multiple other tumor antigens and markers. Finally, we will discuss the present evidence for potential for autoimmunity that might accompany antitumor vaccination.