Increased Antibody Avidity and Cross-Neutralization of Severe Acute Respiratory Syndrome Coronavirus 2 Variants by Hyperimmunized Transchromosomic Bovine-Derived Human Immunoglobulins for Treatment of Coronavirus Disease 2019.

Passive antibody immunotherapeutics directed against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are promising countermeasures for protection and treatment of coronavirus disease 2019 (COVID-19). SARS-CoV-2 variants of concern (VOCs) and variants of interest (VOIs) can impact the clinical efficacy of immunotherapeutics. A fully human polyclonal antibody immunotherapeutic purified from plasma of transchromosomic (Tc) bovines hyperimmunized with SARS-CoV-2 WA-1 spike (SAB-185) is being assessed for efficacy in a phase 2/3 clinical trial when different circulating SARS-CoV-2 variants predominated. We evaluated antibody binding, avidity maturation, and SARS-CoV-2 VOCs/VOIs virus-neutralizing capacity of convalescent plasma compared with different lots of SAB-185 and individual Tc bovine sera sequentially obtained after each vaccination against Alpha, Epsilon, Iota, Gamma, Beta, Kappa, and Delta variants. In contrast to convalescent plasma, sera and SAB-185 derived from hyperimmunized Tc bovines demonstrated higher antibody avidity and more potent cross-neutralizing activity of VOCs/VOIs. Thus, SAB-185 is a potential promising therapeutic candidate for the treatment of patients infected with SARS-CoV-2 variants.

SUSD2 Proteolytic Cleavage Requires the GDPH Sequence and Inter-Fragment Disulfide Bonds for Surface Presentation of Galectin-1 on Breast Cancer Cells.

Galectin-1 (Gal-1) is a 14 kDa protein that has been well characterized for promoting cancer metastasis and tumor immune evasion. By localizing to the cancer cell surface, Gal-1 induces T cell apoptosis through binding T cell surface receptors. The transmembrane protein, Sushi Domain Containing 2 (SUSD2), has been previously shown to be required for Gal-1 surface presentation in breast cancer cells. Western immunoblot analysis revealed that SUSD2 is cleaved into two fragments. However, the significance of this cleavage for Gal-1 surface localization has not been investigated. To define the location of cleavage, a mutagenesis analysis of SUSD2 was performed. Our studies demonstrated that SUSD2 is cleaved at its glycine-aspartic acid-proline-histidine (GDPH) amino acid sequence. Generation of a noncleavable SUSD2 mutant (GDPH∆-SUSD2) showed that SUSD2 cleavage was required for SUSD2 and Gal-1 plasma membrane localization. Noncleavable cysteine mutants were also unable to present Gal-1 at the cell surface, further demonstrating that SUSD2 cleavage is required for Gal-1 surface presentation. Treatment with the serine protease inhibitor, Pefabloc SC, inhibited SUSD2 cleavage in a dose dependent manner, suggesting that SUSD2 is cleaved by a serine protease. Therefore, identification and inhibition of this protease may provide a new therapeutic tool for inhibiting SUSD2 and Gal-1's combined tumorigenic function in breast cancer.

Longitudinal autoantibody responses against tumor-associated antigens decrease in breast cancer patients according to treatment modality.

BACKGROUND: Metastatic breast cancer (BCa) is most often diagnosed months after completion of treatment of the primary tumor when a patient reports physical symptoms. Besides a physical examination, no other alternative recurrence screening method is recommended for routine follow-up care. Detection of autoantibodies against tumor-associated antigens (TAAs) has demonstrated promise for distinguishing healthy women from patients diagnosed with primary BCa. However, it is unknown what changes occur to patient autoantibody levels during and after treatment. METHODS: Three serial blood draws were collected from 200 BCa patients: before treatment, 6 and 12 months after surgery. Patients were categorized according to treatment regimen, including surgery, chemotherapy, radiation, trastuzumab and hormonal therapies. The longitudinal samples were assayed for autoantibody responses against 32 conformation-carrying TAAs using a Luminex multiplex bead assay. RESULTS: The treatment modality groups that had the greatest decrease in autoantibody response levels were radiation + hormonal therapy; radiation + chemotherapy; and radiation + hormonal therapy + chemotherapy. For these three treatment groups, autoantibody responses against 9 TAAs (A1AT, ANGPTL4, CAPC, CST2, DKK1, GFRA1, GRN, LGALS3 and LRP10) were significantly reduced at 12 months after surgery compared to before treatment. One TAA, GRP78, had a significantly increased autoantibody response after 12 months. CONCLUSIONS: Single treatment regimens alone did not significantly alter autoantibodies levels against the studied TAAs. Radiation treatment was the common denominator of the three most affected groups for significant changes in autoantibody response levels.

Sushi Domain Containing 2 (SUSD2) inhibits platelet activation and binding to high-grade serous ovarian carcinoma cells.

Platelets play a central role in primary hemostasis affecting tumor survival and metastases. Tumors induce platelets to aggregate and bind to the cancer cells, resulting in protection from immune surveillance and often leading to thrombocytosis. In ovarian cancer (OvCa), one-third of patients present with thrombocytosis, a diagnosis that correlates with shorter survival. SUSD2 (SUShi Domain containing 2), a type I transmembrane protein, shown to inhibit metastatic processes in high-grade serous ovarian carcinoma (HGSOC), is expressed on endothelial cells and thus may influence platelet reactivity. As such, we hypothesized that SUSD2 levels in ovarian cancer-derived cell lines influence platelet activation. We incubated OvCa non-targeting (NT) and SUSD2 knockdown (KD) cell lines with labeled platelets and quantified platelet binding, as well as GPIIb/IIIa integrin activation. The role of GPIIb/IIIa in tumor cell/platelet interaction was also examined by measuring cell-cell adhesion in the presence of eptifibatide. We found that platelets exposed to OvCa cells with low SUSD2 expression display increased tumor cell-platelet binding along with an increase in GPIIb/IIIa receptor activation. As such, platelet activation and binding to HGSOC cells was inversely correlated with the presence of SUSD2. This represents one of the first tumor proteins known to provide differential platelet interaction based on protein status.

The Impact of Medicaid Expansion Under the Affordable Care Act on the Gap Between American Indians and Whites in Breast Cancer Management and Prognosis.

BACKGROUND: Breast cancer (BC) death rates in the USA have not significantly declined for American Indians (AIs) in comparison to Whites. Our objective was to determine whether Medicaid Expansion as part of the Affordable Care Act led to improved BC outcomes for AIs relative to Whites. PATIENTS AND METHODS: Using the National Cancer Database, we conducted a retrospective cohort study. Included were BC patients who were AI and White; 40 to 64 years of age; diagnosed in 2009 to 2016; lived in states that expanded Medicaid in January 2014, and states that did not expand Medicaid. Our outcomes were stage at diagnosis, insurance status, timely treatment, and 3-year mortality. RESULTS: There were 359,484 newly diagnosed BC patients, 99.49% White, 0.51% AI. Uninsured rates declined more in the expansion states than in the nonexpansion states (OR = 0.44, 95% CI: 0.15-0.97, P < 0.001). Lower rates of Stage I BC diagnosis was found in AIs compared to Whites (46.58% vs. 55.33%, P < .001); these differential rates did not change after Medicaid expansion. Rates of definitive treatment initiation within 30 days of diagnosis declined after Medicaid expansion (P < .001); there was a smaller decline in the expansion states (OR 1.118, 95% CI: 1.09, 1.15, P < .001). Three year mortality was not different between expansion and nonexpansion states post Medicaid expansion. CONCLUSIONS: In newly diagnosed BCs, uninsured rates declined more in the states that expanded Medicaid in January 2014. Timely treatment post Medicaid expansion declined less in states that expanded Medicaid. There was no differential benefit of Medicaid expansion in the 2 races.

Differences in Breast Cancers Among American Indian/Alaska Native and non-Hispanic Whites in the USA.

IMPORTANCE: Breast cancer (BC) death rates have not improved for American Indian/Alaska Native (AI/AN) women, whereas, it has significantly decreased for non-Hispanic White (White) women. OBJECTIVE: Delineate the differences in patient and tumor characteristics among AI/AN and Whites with BC, and its impact on age and stage at diagnosis as well as overall survival (OS). METHODS: Hospital-based, cohort study using the National Cancer Database to identify female AI/AN and Whites diagnosed with BC between the years 2004 and 2016. RESULTS: BC in 6866 AI/AN (0.3%) and 1,987,324 Whites (99.7%) were studied. The median age at diagnosis was 58 for AI/AN and 62 for Whites. AI BC patients traveled double the distance for treatment, lived in lower median income zip codes, had a higher percentage of uninsured, higher comorbidities, lower percentage of Stage 0/I, larger tumor size, greater number of positive lymph nodes, higher proportion of triple negative and HER2-positive BC than Whites. All the above comparisons were significant, p<0.001. Association between patient/tumor characteristics with age and stage at diagnosis was not significantly different between AI/AN and Whites. Unadjusted OS was worse for AI/AN as compared to Whites (HR=1.07, 95% CI=1.01-1.14, p=0.023). After adjustment of all covariates, OS was not different (HR=1.038, 95%CI=0.902-1.195, p=0.601). CONCLUSION: There were significant differences in patient/tumor characteristics among AI/AN and White BC which adversely impacted OS in AI/AN. However, when adjusted for various covariates, the survival was similar, suggesting that the worse survival in AI/AN is mostly the impact of known biological, socio-economic, and environmental determinants of health.

Polyclonal Antibodies Derived from Transchromosomic Bovines Vaccinated with the Recombinant F1-V Vaccine Increase Bacterial Opsonization In Vitro and Protect Mice from Pneumonic Plague.

Plague is an ancient disease that continues to be of concern to both the public health and biodefense research communities. Pneumonic plague is caused by hematogenous spread of Yersinia pestis bacteria from a ruptured bubo to the lungs or by directly inhaling aerosolized bacteria. The fatality rate associated with pneumonic plague is significant unless effective antibiotic therapy is initiated soon after an early and accurate diagnosis is made. As with all bacterial pathogens, drug resistance is a primary concern when developing strategies to combat these Yersinia pestis infections in the future. While there has been significant progress in vaccine development, no FDA-approved vaccine strategy exists; thus, other medical countermeasures are needed. Antibody treatment has been shown to be effective in animal models of plague. We produced fully human polyclonal antibodies in transchromosomic bovines vaccinated with the recombinant F1-V plague vaccine. The resulting human antibodies opsonized Y. pestis bacteria in the presence of RAW264.7 cells and afforded significant protection to BALB/c mice after exposure to aerosolized Y. pestis. These data demonstrate the utility of this technology to produce large quantities of non-immunogenic anti-plague human antibodies to prevent or possibly treat pneumonic plague in human.

Human immunoglobulin from transchromosomic bovines hyperimmunized with SARS-CoV-2 spike antigen efficiently neutralizes viral variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with amino-acid substitutions and deletions in spike protein (S) can reduce the effectiveness of monoclonal antibodies (mAbs) and may compromise immunity induced by vaccines. We report a polyclonal, fully human, anti-SARS-CoV-2 immunoglobulin produced in transchromosomic bovines (Tc-hIgG-SARS-CoV-2) hyperimmunized with two doses of plasmid DNA encoding the SARS-CoV-2 Wuhan strain S gene, followed by repeated immunization with S protein purified from insect cells. The resulting Tc-hIgG-SARS-CoV-2, termed SAB-185, efficiently neutralizes SARS-CoV-2, and vesicular stomatitis virus (VSV) SARS-CoV-2 chimeras in vitro. Neutralization potency was retained for S variants including S477N, E484K, and N501Y, substitutions present in recent variants of concern. In contrast to the ease of selection of escape variants with mAbs and convalescent human plasma, we were unable to isolate VSV-SARS-CoV-2 mutants resistant to Tc-hIgG-SARS-CoV-2 neutralization. This fully human immunoglobulin that potently inhibits SARS-CoV-2 infection may provide an effective therapeutic to combat COVID-19.

Pathways to personalized medicine for breast and prostate cancers: emerging diagnostic methods and prognostic biomarkers.

Early and personal diagnosis to breast a prostate cancer is crucial for optimizing treatments leading to long-term patient survival. Once cancer metastasizes from the breast or prostate to other tissues of the body, therapies are limited, and there is no cure for the diseases. Currently used screening modalities for breast and prostate cancers have limitations. Routine screening for breast cancer includes clinical breast exams and mammograms. Improvements in imaging techniques, such as magnetic resonance, ultrasound, digital breast tomosynthesis and ductography are being explored as adjuncts to mammography. A new approach to breast cancer screening involves the detection of abnormalities at the cellular level and uses various means to collect cellular material from the ductal system, including nipple aspirate fluid, breast ductal lavage, fiberoptic ductoscopy and random periareolar fine needle aspiration. Current screening methods for prostate cancer include digital rectal exam and serum PSA levels. However, these methods offer low sensitivity and specificity and do not allow differentiation between significant- and minimal-risk cancers. New approaches to prostate cancer screening involve different calculations using PSA, as well as molecular urine tests. With the recent advances in microarray technologies and whole-genome sequencing of tumors, the identification of specific biomarkers for diagnosis and prognosis, as well as new therapeutic targets, is quickly paving the way for personalized medicine. In the future, routine patient care will include using the molecular signature of a patient's disease to guide treatment.

SUSD2 expression correlates with decreased metastasis and increased survival in a high-grade serous ovarian cancer xenograft murine model.

The cause of death among high-grade serous ovarian cancer (HGSOC) patients involves passive dissemination of cancer cells within the peritoneal cavity and subsequent implantation of cancer spheroids into adjacent organs. Sushi Domain Containing 2 (SUSD2) encodes a type I transmembrane protein containing several functional domains inherent to adhesion molecules. Previous studies using in vitro methods have indicated that SUSD2 functions as a tumor suppressor in several cancers, including HGSOC. In this study, we generated a HGSOC xenograft mouse model to investigate SUSD2 expression in the context of HGSOC late-stage metastasis and overall survival. OVCAR3 cells with knock-down expression of SUSD2 (OVCAR3 SUSD2-KD) or endogenous expression of SUSD2 (OVCAR3-Non-Targeting (NT)) were injected into the peritoneal cavity of athymic nude mice. Immunohistochemistry analysis was utilized to identify infiltrating cancer cells and metastatic tumors in mouse ovaries, pancreas, spleen, omentum and liver. OVCAR3-NT mice developed significantly less cancer cell infiltrate and tumors in their pancreas and omentum compared to OVCAR3 SUSD2-KD mice. Furthermore, OVCAR3-NT mice displayed a longer median survival when compared to OVCAR3 SUSD2-KD mice (175 days and 185.5 days, respectively; p-value 0.0159). Altogether, the findings generated through the preclinical mouse model suggest that increased SUSD2 expression in HGSOC impedes in vivo metastasis to pancreas and omentum. These results correlate to longer median survival and prove to be consistent with previous findings showing prolonged survival of HGSOC patients with high SUSD2-expressing primary tumors.

Interspecies signaling between Veillonella atypica and Streptococcus gordonii requires the transcription factor CcpA.

Streptococcus gordonii and Veillonella atypica, two early-colonizing members of the dental plaque biofilm, participate in a relationship that results in increased transcription of the S. gordonii gene amyB, encoding an alpha-amylase. We show that the transcription factor CcpA is required for this interspecies interaction.

SUSD2 promotes tumor-associated macrophage recruitment by increasing levels of MCP-1 in breast cancer.

Tumor-associated macrophages (TAMs) play a role in tumor angiogenesis and are recruited into the tumor microenvironment (TME) by secreted chemokines, including Monocyte Chemoattractant Protein-1 (MCP-1/CCL2). Angiogenesis is required to sustain proliferation and enable metastasis of breast cancer (BCa) cells. Understanding the underlying mechanisms of TAM recruitment would allow for the identification of desperately needed novel drug targets. Sushi Domain Containing 2 (SUSD2), a transmembrane protein on BCa cells, was previously shown to promote tumor angiogenesis in a murine model. To identify the role of SUSD2 in angiogenesis, 175 human breast tumors were surveyed by immunohistochemical analysis for the presence of SUSD2 and macrophages. Tumors with high levels of SUSD2 staining contained 2-fold more TAMs, mainly of the M2 pro-angiogenic phenotype. An in vitro co-culture model system was developed by differentiating SC monocytes into SC M0 macrophages. A 2-fold increase in polarized M2 macrophages was observed when M0 macrophages were incubated with SUSD2-expressing BCa cells compared to cancer cells that do not contain SUSD2. Since MCP-1 is known to recruit macrophages, levels of MCP-1 were compared between SUSD2-expressing MDA-MB-231 and MBA-MB-231-vector control cell lines. MCP-1 RNA, intracellular protein and secreted MCP-1 were all significantly increased compared to the vector control. Knockdown of SUSD2 in SKBR3 resulted in significantly decreased levels of secreted MCP-1. Consistently, increased levels of MCP-1 were observed in Susd2-expressing tumors generated from an in vivo isogeneic mouse model compared to the vector control tumors. Because SUSD2 recruits macrophages into the TME and promotes M2 polarization, inhibiting the function of SUSD2 may be an effective therapy for breast cancer patients.

Characterization of overlapping XAGE-1 transcripts encoding a cancer testis antigen expressed in lung, breast, and other types of cancers.

Cancer testis (CT) antigens have an expression pattern that is predominantly restricted to testis in normal tissues, yet they are expressed in many different histological types of cancers. One previously described member of the CT antigen family, XAGE-1, was shown to be expressed in Ewing's sarcomas and rhabdomyosarcomas. Here we show that XAGE-1 is also expressed in breast cancer, prostate cancer, and different types of lung cancers, including lung squamous cell carcinoma, adenocarcinoma, small cell lung carcinoma, and non-small cell lung carcinoma. In addition, XAGE-1 mRNA was present in ovarian cancer, melanoma, glioblastoma, T-cell lymphoma, chronic myelogenous leukemia, and histiocytic lymphoma cell lines. We also characterized the XAGE-1 transcript by primer extension analysis and found that transcription of the XAGE-1 gene is initiated from two distinct start sites, resulting in two overlapping transcripts, XAGE-1a and XAGE-1b. XAGE-1a contains two in-frame ATG translational start codons; whereas XAGE-1b initiates downstream of the first ATG start codon. Our results suggest that XAGE-1b is the dominant transcript, and that translation begins with the second ATG start codon, producing a 9 kDa protein. Because XAGE-1 is expressed in such a diverse range of cancers, it has potential to be used as a target for many cancer immunotherapies.

Classifying patients for breast cancer by detection of autoantibodies against a panel of conformation-carrying antigens.

Patients with breast cancer elicit an autoantibody response against cancer proteins, which reflects and amplifies the cellular changes associated with tumorigenesis. Detection of autoantibodies in plasma may provide a minimally invasive mechanism for early detection of breast cancer. To identify cancer proteins that elicit a humoral response, we generated a cDNA library enriched for breast cancer genes that encode membrane and secreted proteins, which are more likely to induce an antibody response compared with intracellular proteins. To generate conformation-carrying antigens that are efficiently recognized by patients' antibodies, a eukaryotic expression strategy was established. Plasma from 200 patients with breast cancer and 200 age-matched healthy controls were measured for autoantibody activity against 20 different antigens designed to have conformational epitopes using ELISA. A conditional logistic regression model was used to select a combination of autoantibody responses against the 20 different antigens to classify patients with breast cancer from healthy controls. The best combination included ANGPTL4, DKK1, GAL1, MUC1, GFRA1, GRN, and LRRC15; however, autoantibody responses against GFRA1, GRN, and LRRC15 were inversely correlated with breast cancer. When the autoantibody responses against the 7 antigens were added to the base model, including age, BMI, race and current smoking status, the assay had the following diagnostic capabilities: c-stat (95% CI), 0.82 (0.78-0.86); sensitivity, 73%; specificity, 76%; and positive likelihood ratio (95% CI), 3.04 (2.34-3.94). The model was calibrated across risk deciles (Hosmer-Lemeshow, P = 0.13) and performed well in specific subtypes of breast cancer including estrogen receptor positive, HER-2 positive, invasive, in situ and tumor sizes >1 cm.

Multiple functions of sushi domain containing 2 (SUSD2) in breast tumorigenesis.

Routinely used therapies are not adequate to treat the heterogeneity of breast cancer, and consequently, more therapeutic targets are desperately needed. To identify novel targets, we generated a breast cancer cDNA library enriched for genes that encode membrane and secreted proteins. From this library we identified SUSD2 (Sushi Domain Containing 2), which encodes an 822-amino acid protein containing a transmembrane domain and functional domains inherent to adhesion molecules. Previous studies describe the mouse homolog, Susd2, but there are no studies on the human gene associated with breast cancer. Immunohistochemical analysis of human breast tissues showed weak or no expression of SUSD2 in normal epithelial cells, with the endothelial lining of vessels staining positive for SUSD2. However, staining was observed in pathologic breast lesions and in lobular and ductal carcinomas. SUSD2 interacts with galectin-1 (Gal-1), a 14-kDa secreted protein that is synthesized by carcinoma cells and promotes tumor immune evasion, angiogenesis, and metastasis. Interestingly, we found that localization of Gal-1 on the surface of cells is dependent on the presence of SUSD2. Various phenotype assays indicate that SUSD2 increases the invasion of breast cancer cells and contributes to a potential immune evasion mechanism through induction of apoptosis of Jurkat T cells. Using a syngeneic mouse model, we observed accelerated tumor formation and decreased survival in mice with tumors expressing Susd2. We found significantly fewer CD4 tumor infiltrating lymphocytes in mice with tumors expressing Susd2. Together, our findings provide evidence that SUSD2 may represent a promising therapeutic target for breast cancer.

Identification of proangiogenic genes and pathways by high-throughput functional genomics: TBK1 and the IRF3 pathway.

A genome-wide phenotype screen was used to identify factors and pathways that induce proliferation of human umbilical vein endothelial cells (HUVEC). HUVEC proliferation is a recognized marker for factors that modulate vascularization. Screening "hits" included known proangiogenic factors, such as VEGF, FGF1, and FGF2 and additional factors for which a direct association with angiogenesis was not previously described. These include the kinase TBK1 as well as Toll-like receptor adaptor molecule and IFN regulatory factor 3. All three proteins belong to one signaling pathway that mediates induction of gene expression, including a mixture of secreted factors, which, in concert, mediate proliferative activity toward endothelial cells. TBK1 as the "trigger" of this pathway is induced under hypoxic conditions and expressed at significant levels in many solid tumors. This pattern of expression and the decreased expression of angiogenic factors in cultured cells upon RNA-interference-mediated ablation suggests that TBK1 is important for vascularization and subsequent tumor growth and a target for cancer therapy.

High expression of a cytokeratin-associated protein in many cancers.

We have described previously a cDNA library made from membrane-bound polysomal mRNA prepared from breast and prostate cancer cell lines. The library is highly enriched for cDNAs encoding membrane proteins, secreted proteins, and cytokeratins. To characterize this library, 25,277 cDNA clones were sequenced and aligned with various databases; 1,439 clones did not align with known genes. From this set of clones we identified a previously uncharacterized gene encoding a 334-aa protein. Although protein structural motif prediction programs indicate that the gene encodes a membrane protein comprising a signal sequence, a series of leucine-rich repeats, and a single transmembrane domain with a cytoplasmic tail, confocal microscopy of MCF7 breast cancer cells demonstrates that the protein is not directly associated with the plasma membrane or intracellular membranes but instead colocalizes with intermediate filaments and cytokeratins within the cell. Immunofluorescence studies also show that protein expression is increased greatly in mitotic MCF7 cells, and immunohistochemistry demonstrates its expression in human breast cancer cells. Analysis of mRNA levels in 25 different normal tissues by RT-PCR shows that this gene is expressed highly in normal prostate and salivary gland, very weakly in colon, pancreas, and intestine, and not at all in other tissues. RT-PCR studies on human cancer samples show that the RNA is expressed highly in many cancer cell lines and cancer specimens, including 26 of 33 human breast cancers, 3 of 3 prostate cancers, 3 of 3 colon cancers, and 3 of 3 pancreatic cancers. We name the protein CAPC, cytokeratin-associated protein in cancer.

Discovery of the breast cancer gene BASE using a molecular approach to enrich for genes encoding membrane and secreted proteins.

To identify unknown membrane proteins that could be used as targets for breast and prostate cancer immunotherapies and secreted proteins to be used as diagnostic markers, a cDNA library was generated from membrane-associated polyribosomal RNA derived from four breast cancer cell lines, one normal breast cell line, and a prostate cancer cell line. The membrane-associated polyribosomal cDNA library was subtracted with RNA from normal brain, liver, lung, kidney, and muscle. Of the 15,581 clones sequenced from the subtracted cDNA library, sequences from 10,506 clones map to known genes, but 5,075 sequences, representing 3,181 unique transcripts, are not associated with known genes. As one example, we experimentally investigated expression of a previously uncharacterized breast cancer gene that encodes a secreted protein designated BASE (breast cancer and salivary gland expression). BASE is expressed in many breast cancers but not in essential normal tissues including the five organs used for subtraction. Further analysis of this library should yield additional gene products of use in the diagnosis or treatment of breast or prostate cancer.