Proteogenomic characterization of difficult-to-treat breast cancer with tumor cells enriched through laser microdissection.

BACKGROUND: Breast cancer (BC) is the most commonly diagnosed cancer and the leading cause of cancer death among women globally. Despite advances, there is considerable variation in clinical outcomes for patients with non-luminal A tumors, classified as difficult-to-treat breast cancers (DTBC). This study aims to delineate the proteogenomic landscape of DTBC tumors compared to luminal A (LumA) tumors. METHODS: We retrospectively collected a total of 117 untreated primary breast tumor specimens, focusing on DTBC subtypes. Breast tumors were processed by laser microdissection (LMD) to enrich tumor cells. DNA, RNA, and protein were simultaneously extracted from each tumor preparation, followed by whole genome sequencing, paired-end RNA sequencing, global proteomics and phosphoproteomics. Differential feature analysis, pathway analysis and survival analysis were performed to better understand DTBC and investigate biomarkers. RESULTS: We observed distinct variations in gene mutations, structural variations, and chromosomal alterations between DTBC and LumA breast tumors. DTBC tumors predominantly had more mutations in TP53, PLXNB3, Zinc finger genes, and fewer mutations in SDC2, CDH1, PIK3CA, SVIL, and PTEN. Notably, Cytoband 1q21, which contains numerous cell proliferation-related genes, was significantly amplified in the DTBC tumors. LMD successfully minimized stromal components and increased RNA-protein concordance, as evidenced by stromal score comparisons and proteomic analysis. Distinct DTBC and LumA-enriched clusters were observed by proteomic and phosphoproteomic clustering analysis, some with survival differences. Phosphoproteomics identified two distinct phosphoproteomic profiles for high relapse-risk and low relapse-risk basal-like tumors, involving several genes known to be associated with breast cancer oncogenesis and progression, including KIAA1522, DCK, FOXO3, MYO9B, ARID1A, EPRS, ZC3HAV1, and RBM14. Lastly, an integrated pathway analysis of multi-omics data highlighted a robust enrichment of proliferation pathways in DTBC tumors. CONCLUSIONS: This study provides an integrated proteogenomic characterization of DTBC vs LumA with tumor cells enriched through laser microdissection. We identified many common features of DTBC tumors and the phosphopeptides that could serve as potential biomarkers for high/low relapse-risk basal-like BC and possibly guide treatment selections.

Comparative Survival Analysis of Invasive Breast Cancer Patients Treated by a U.S. Military Medical Center and Matched Patients From the U.S. General Population.

OBJECTIVE: Many differences between U.S. military beneficiaries and the U.S. general population, including differences in health care access, are known factors affecting invasive breast cancer outcomes. Thus, comparing the two populations for any outcome differences and their contributing factors may provide insights to breast cancer prognosis. METHODS: Using a marginal Cox proportional hazards regression model, we compared disease-specific survival (DSS) and 5-year DSS rates between 418 patients from the Clinical Breast Care Project at the Walter Reed National Military Medical Center (CBCP-WR) and a set of 1:5 randomly matched patients from the Surveillance, Epidemiology, and End Results program. Patients were compared in the "demographic model" (adjusted by diagnosis year, age, and race) and the "overall model" (further adjusted by estrogen receptor, progesterone receptor, stage, and grade). RESULTS: In the "overall model," CBCP-WR patients were less likely overall to die from breast cancer (hazard ratio [HR] = 0.631, 95% confidence interval [CI] = 0.437-0.911; p = 0.014). This increase in survival was also significant in African American patients (HR = 0.524, 95% CI = 0.277-0.992; p = 0.047) and patients older than 50 (HR = 0.511, 95% CI = 0.306-0.854; p = 0.010). The advantage in 5-year DSS rate for CBCP-WR patients was 5.3% (93.1% vs. 87.8%; p < 0.001) in the "demographic model" and 3.4% (91.3% vs. 87.9%; p = 0.018) in the "overall model." CONCLUSION: CBCP-WR patients demonstrated significantly better DSS over matched SEER patients. Although a portion of the outcome disparity, i.e., 36% of the 5.3% DSS rate difference, could be explained by differences in tumor characteristics, the cause(s) behind the majority of the disparity has yet to be identified. Identification and further analysis of contributing factors to survival differences have the potential to improve clinical practice and outcomes for invasive breast cancer patients.

Positive Association of Fibroadenomatoid Change with HER2-Negative Invasive Breast Cancer: A Co-Occurrence Study.

BACKGROUND: Risk assessment of a benign breast disease/lesion (BBD) for invasive breast cancer (IBC) is typically done through a longitudinal study. For an infrequently-reported BBD, the shortage of occurrence data alone is a limiting factor to conducting such a study. Here we present an approach based on co-occurrence analysis, to help address this issue. We focus on fibroadenomatoid change (FAC), an under-studied BBD, as our preliminary analysis has suggested its previously unknown significant co-occurrence with IBC. METHODS: A cohort of 1667 female patients enrolled in the Clinical Breast Care Project was identified. A single experienced breast pathologist reviewed all pathology slides for each case and recorded all observed lesions, including FAC. Fibroadenoma (FA) was studied for comparison since FAC had been speculated to be an immature FA. FA and Fibrocystic Changes (FCC) were used for method validation since they have been comprehensively studied. Six common IBC and BBD risk/protective factors were also studied. Co-occurrence analyses were performed using logistic regression models. RESULTS: Common risk/protective factors were associated with FA, FCC, and IBC in ways consistent with the literature in general, and they were associated with FAC, FA, and FCC in distinct patterns. Age was associated with FAC in a bell-shape curve so that middle-aged women were more likely to have FAC. We report for the first time that FAC is positively associated with IBC with odds ratio (OR) depending on BMI (OR = 6.78, 95%CI = 3.43-13.42 at BMI<25 kg/m2; OR = 2.13, 95%CI = 1.20-3.80 at BMI>25 kg/m2). This association is only significant with HER2-negative IBC subtypes. CONCLUSIONS: We conclude that FAC is a candidate risk factor for HER2-negative IBCs, and it is a distinct disease from FA. Co-occurrence analysis can be used for initial assessment of the risk for IBC from a BBD, which is vital to the study of infrequently-reported BBDs.

Role for chondroitin sulfate glycosaminoglycan in NEDD9-mediated breast cancer cell growth.

There are lines of evidence demonstrating that NEDD9 (Cas-L, HEF-1) plays a key role in the development, progression, and metastasis of breast cancer cells. We previously reported that NEDD9 plays a critical role for promoting migration and growth of MDA-MB-231. In order to further characterize the mechanisms of NEDD9-mediated cancer migration and growth, stable cells overexpressing NEDD9 were generated using HCC38 as a parental cell line which expresses low level of endogenous NEDD9. Microarray studies demonstrated that core proteins of CD44 and Serglycin were markedly upregulated in HCC38(NEDD9) cells compared to HCC38(Vector) cells, while those of Syndecan-1, Syndecan-2, and Versican were downregulated in HCC38(NEDD9). Importantly, enzymes generating chondroitin sulfate glycosaminoglycans (CS) such as CHST11, CHST15, and CSGALNACT1 were upregulated in HCC38(NEDD9) compared to HCC38(Vector). Immunofluorescence studies using specific antibody, GD3G7, confirmed the enhanced expression of CS-E subunit in HCC38(NEDD9). Immunoprecipitation and western blotting analysis demonstrated that CS-E was attached to CD44 core protein. We demonstrated that removing CS by chondroitinase ABC significantly inhibited anchorage-independent colony formation of HCC38(NEDD9) in methylcellulose. Importantly, the fact that GD3G7 significantly inhibited colony formation of HCC38(NEDD9) cells suggests that CS-E subunit plays a key role in this process. Furthermore, treatment of HCC38(NEDD9) cells with chondroitinase ABC or GD3G7 significantly inhibited mammosphere formation. Exogenous addition of CS-E enhanced colony formation and mammosphere formation of HCC38 parental and HCC38(Vector) cells. These results suggest that NEDD9 regulates the synthesis and expression of tumor associated glycocalyx structures including CS-E, which plays a key role in promoting and regulating breast cancer progression and metastasis and possibly stem cell phenotypes.

DNA aptamers against exon v10 of CD44 inhibit breast cancer cell migration.

CD44 adhesion molecules are expressed in many breast cancer cells and have been demonstrated to play a key role in regulating malignant phenotypes such as growth, migration, and invasion. CD44 is an integral transmembrane protein encoded by a single 20-exon gene. The diversity of the biological functions of CD44 is the result of the various splicing variants of these exons. Previous studies suggest that exon v10 of CD44 plays a key role in promoting cancer invasion and metastasis, however, the molecular mechanisms are not clear. Given the fact that exon v10 is in the ectodomain of CD44, we hypothesized that CD44 forms a molecular complex with other cell surface molecules through exon v10 in order to promote migration of breast cancer cells. In order to test this hypothesis, we selected DNA aptamers that specifically bound to CD44 exon v10 using Systematic Evolution of Ligands by Exponential Enrichment (SELEX). We selected aptamers that inhibited migration of breast cancer cells. Co-immunoprecipitation studies demonstrated that EphA2 was co-precipitated with CD44. Pull-down studies demonstrated that recombinant CD44 exon v10 bound to EphA2 and more importantly aptamers that inhibited migration also prevented the binding of EphA2 to exon v10. These results suggest that CD44 forms a molecular complex with EphA2 on the breast cancer cell surface and this complex plays a key role in enhancing breast cancer migration. These results provide insight not only for characterizing mechanisms of breast cancer migration but also for developing target-specific therapy for breast cancers and possibly other cancer types expressing CD44 exon v10.

QAIT: a quality assurance issue tracking tool to facilitate the improvement of clinical data quality.

In clinical and translational research as well as clinical trial projects, clinical data collection is prone to errors such as missing data, and misinterpretation or inconsistency of the data. A good quality assurance (QA) program can resolve many such errors though this requires efficient communications between the QA staff and data collectors. Managing such communications is critical to resolving QA problems but imposes a major challenge for a project involving multiple clinical and data processing sites. We have developed a QA issue tracking (QAIT) system to support clinical data QA in the Clinical Breast Care Project (CBCP). This web-based application provides centralized management of QA issues with role-based access privileges. It has greatly facilitated the QA process and enhanced the overall quality of the CBCP clinical data. As a stand-alone system, QAIT can supplement any other clinical data management systems and can be adapted to support other projects.

Tissue imprints: assessing their potential for routine biobanking collection.

Biomedical research depends on the availability of good quality biospecimens. Unfortunately, certain specimens are scarce due to disease rarity or size restrictions of surgical materials. To increase access to limited surgical specimens, Biobanks need to reassess and adjust their collection programs. We evaluated the feasibility of adapting "touch imprints" to gain access to limited surgical specimens as well as to maximize the use of "precious" specimens. We utilized 12 kidney samples for touch imprints on defined areas of microscope glass slides and FTA paper. DNA was isolated from glass slides on the day of preparation, Day 0, and from glass slide and FTA paper preparations after two weeks of storage at room temperature and -80°C. Yield and purity of DNA from reference kidney samples were compared to DNA from the touch imprints and the quality determined by real-time PCR using the amplification of Cyclophilin A (Cyc A) as an index. DNA quality for glass slides at Day 0 was not significantly different from DNA after two weeks at room temperature (glass at room temperature; p=0.111 and 0.097, yield and purity, respectively) and after two weeks at -80°C (glass -80°C; p=0.358 and 0.281, yield and purity, respectively). Glass slide DNA at room temperature and -80°C were not significantly different (p=0.795 and 0.146 for yield and purity, respectively). DNA from FTA paper at room temperature and from FTA paper at -80°C were significantly different from glass at room temperature and glass at -80°C (p=0.002, respectively). Threshold values for Cyc A were ≤28 for the reference DNA and ≤32 for DNA from glass and FTA paper. This study demonstrates that touch preparations on microscope glass slides and FTA paper can provide sufficient and good quality DNA suitable for PCR. Touch imprints could therefore be adopted by biobanks to collect and bank biological materials from limited surgical specimens.

FH535 inhibited migration and growth of breast cancer cells.

There is substantial evidence indicating that the WNT signaling pathway is activated in various cancer cell types including breast cancer. Previous studies reported that FH535, a small molecule inhibitor of the WNT signaling pathway, decreased growth of cancer cells but not normal fibroblasts, suggesting this pathway plays a role in tumor progression and metastasis. In this study, we tested FH535 as a potential inhibitor for malignant phenotypes of breast cancer cells including migration, invasion, and growth. FH535 significantly inhibited growth, migration, and invasion of triple negative (TN) breast cancer cell lines (MDA-MB231 and HCC38) in vitro. We demonstrate that FH535 was a potent growth inhibitor for TN breast cancer cell lines (HCC38 and MDA-MB-231) but not for other, non-TN breast cancer cell lines (MCF-7, T47D or SK-Br3) when cultured in three dimensional (3D) type I collagen gels. Western blotting analyses suggest that treatment of MDA-MB-231 cells with FH535 markedly inhibited the expression of NEDD9 but not activations of FAK, Src, or downstream targets such as p38 and Erk1/2. We demonstrated that NEDD9 was specifically associated with CSPG4 but not with ß1 integrin or CD44 in MDA-MB-231 cells. Analyses of gene expression profiles in breast cancer tissues suggest that CSPG4 expression is higher in Basal-type breast cancers, many of which are TN, than any other subtypes. These results suggest not only a mechanism for migration and invasion involving the canonical WNT-signaling pathways but also novel strategies for treating patients who develop TN breast cancer.

Analysis of matrix metalloproteinase-1 gene polymorphisms and expression in benign and malignant breast tumors.

A guanine insertion polymorphism in matrix metalloproteinase-1 promoter (MMP-1 2G) is linked to early onset and aggressiveness in cancer. We determined the role of MMP-1 2G on MMP-1 expression and breast cancer severity in patients with breast diseases. We observed no significant difference in genotype distribution among different disease groups. However, MMP-1 expression was significantly higher in atypical ductal hyperplasia than in benign breast disease and in invasive breast cancer compared to in situ breast cancer. MMP-1 2G insertion polymorphism in the invasive group also correlated significantly with the expression of MMP-1 and breast cancer prognostic markers HER2 and P53.

DW4TR: A Data Warehouse for Translational Research.

The linkage between the clinical and laboratory research domains is a key issue in translational research. Integration of clinicopathologic data alone is a major task given the number of data elements involved. For a translational research environment, it is critical to make these data usable at the point-of-need. Individual systems have been developed to meet the needs of particular projects though the need for a generalizable system has been recognized. Increased use of Electronic Medical Record data in translational research will demand generalizing the system for integrating clinical data to support the study of a broad range of human diseases. To ultimately satisfy these needs, we have developed a system to support multiple translational research projects. This system, the Data Warehouse for Translational Research (DW4TR), is based on a light-weight, patient-centric modularly-structured clinical data model and a specimen-centric molecular data model. The temporal relationships of the data are also part of the model. The data are accessed through an interface composed of an Aggregated Biomedical-Information Browser (ABB) and an Individual Subject Information Viewer (ISIV) which target general users. The system was developed to support a breast cancer translational research program and has been extended to support a gynecological disease program. Further extensions of the DW4TR are underway. We believe that the DW4TR will play an important role in translational research across multiple disease types.

An issue tracking system to facilitate the enhancement of clinical data quality in the clinical breast care project.

An online issue tracking (QAIT) system was developed to support the QA of questionnaire-based clinical data and tissue banking in the Clinical Breast Care Project (CBCP). The web-based system provides a centralized storage and management of QA issues and role-based access to related information and functions via internet. The QAIT system greatly improved the QA process for the CBCP clinical data and tissue banking and can be easily adapted to other applications.

Genome assembly comparison identifies structural variants in the human genome.

Numerous types of DNA variation exist, ranging from SNPs to larger structural alterations such as copy number variants (CNVs) and inversions. Alignment of DNA sequence from different sources has been used to identify SNPs and intermediate-sized variants (ISVs). However, only a small proportion of total heterogeneity is characterized, and little is known of the characteristics of most smaller-sized (<50 kb) variants. Here we show that genome assembly comparison is a robust approach for identification of all classes of genetic variation. Through comparison of two human assemblies (Celera's R27c compilation and the Build 35 reference sequence), we identified megabases of sequence (in the form of 13,534 putative non-SNP events) that were absent, inverted or polymorphic in one assembly. Database comparison and laboratory experimentation further demonstrated overlap or validation for 240 variable regions and confirmed >1.5 million SNPs. Some differences were simple insertions and deletions, but in regions containing CNVs, segmental duplication and repetitive DNA, they were more complex. Our results uncover substantial undescribed variation in humans, highlighting the need for comprehensive annotation strategies to fully interpret genome scanning and personalized sequencing projects.

Using shared genomic synteny and shared protein functions to enhance the identification of orthologous gene pairs.

MOTIVATION: The identification of orthologous gene pairs is generally based on sequence similarity. Gene pairs that are mutually 'best hits' between the genomes being compared are asserted to be orthologs. Although this method identifies most orthologous gene pairs with high confidence, it will miss a fraction of them, especially genes in duplicated gene families. In addition, the approach depends heavily on the completeness and quality of gene annotation. When the gene sequences are not correctly represented the approach is unlikely to find the correct ortholog. To overcome these limitations, we have developed an approach to identify orthologous gene pairs using shared chromosomal synteny and the annotation of protein function. RESULTS: Assembled mouse and human genomes were used to identify the regions of conserved synteny between these genomes. 'Syntenic anchors' are conserved non-repetitive locations between mouse and human genomes. Using these anchors, we identified blocks of sequences that contain consistently ordered anchors between the two genomes (syntenic blocks). The synteny information has been used to help us identify orthologous gene pairs between mouse and human genomes. The approach combines the mutual selection of the best tBlastX hits between human and mouse transcripts, and inferring gene orthologous relationships based on sharing syntenic anchors, collocating in the same syntenic blocks and sharing the same annotated protein function. Using this approach, we were able to find 19,357 orthologous gene pairs between human and mouse genomes, a 20% increase in the number of orthologs identified by conventional approaches.

Use of a dense single nucleotide polymorphism map for in silico mapping in the mouse.

Rapid expansion of available data, both phenotypic and genotypic, for multiple strains of mice has enabled the development of new methods to interrogate the mouse genome for functional genetic perturbations. In silico mapping provides an expedient way to associate the natural diversity of phenotypic traits with ancestrally inherited polymorphisms for the purpose of dissecting genetic traits. In mouse, the current single nucleotide polymorphism (SNP) data have lacked the density across the genome and coverage of enough strains to properly achieve this goal. To remedy this, 470,407 allele calls were produced for 10,990 evenly spaced SNP loci across 48 inbred mouse strains. Use of the SNP set with statistical models that considered unique patterns within blocks of three SNPs as an inferred haplotype could successfully map known single gene traits and a cloned quantitative trait gene. Application of this method to high-density lipoprotein and gallstone phenotypes reproduced previously characterized quantitative trait loci (QTL). The inferred haplotype data also facilitates the refinement of QTL regions such that candidate genes can be more easily identified and characterized as shown for adenylate cyclase 7.

Genome-wide single-nucleotide polymorphism analysis defines haplotype patterns in mouse.

The nature and organization of polymorphisms, or differences, between genomes of individuals are of great interest, because these variations can be associated with or even underlie phenotypic traits, including disease susceptibility. To gain insight into the genetic and evolutionary factors influencing such biological variation, we have examined the arrangement (haplotype) of single-nucleotide polymorphisms across the genomes of eight inbred strains of mice. These analyses define blocks of high or low diversity, often extending across tens of megabases that are delineated by abrupt transitions. These observations provide a striking contrast to the haplotype structure of the human genome.

A comparison of whole-genome shotgun-derived mouse chromosome 16 and the human genome.

The high degree of similarity between the mouse and human genomes is demonstrated through analysis of the sequence of mouse chromosome 16 (Mmu 16), which was obtained as part of a whole-genome shotgun assembly of the mouse genome. The mouse genome is about 10% smaller than the human genome, owing to a lower repetitive DNA content. Comparison of the structure and protein-coding potential of Mmu 16 with that of the homologous segments of the human genome identifies regions of conserved synteny with human chromosomes (Hsa) 3, 8, 12, 16, 21, and 22. Gene content and order are highly conserved between Mmu 16 and the syntenic blocks of the human genome. Of the 731 predicted genes on Mmu 16, 509 align with orthologs on the corresponding portions of the human genome, 44 are likely paralogous to these genes, and 164 genes have homologs elsewhere in the human genome; there are 14 genes for which we could find no human counterpart.

The Celera Discovery System.

The Celera Discovery System (CDS) is a web-accessible research workbench for mining genomic and related biological information. Users have access to the human and mouse genome sequences with annotation presented in summary form in BioMolecule Reports for genes, transcripts and proteins. Over 40 additional databases are available, including sequence, mapping, mutation, genetic variation, mRNA expression, protein structure, motif and classification data. Data are accessible by browsing reports, through a variety of interactive graphical viewers, and by advanced query capability provided by the LION SRS search engine. A growing number of sequence analysis tools are available, including sequence similarity, pattern searching, multiple sequence alignment and Hidden Markov Model search. A user workspace keeps track of queries and analyses. CDS is widely used by the academic research community and requires a subscription for access. The system and academic pricing information are available at http://cds.celera.com.