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.

Cytokine levels in breast cancer are highly dependent on cytomegalovirus (CMV) status.

PURPOSE: Breast cancer accounts for 30% of all female cancers in the US. Cytomegalovirus (CMV), a herpesvirus that establishes lifelong infection, may play a role in breast cancer. CMV is not oncogenic, yet viral DNA and proteins have been detected in breast tumors, indicating possible contribution to tumor development. CMV encodes cmvIL-10, a homolog of human cellular IL-10 (cIL-10) with potent immunosuppressive activities. We investigated the relationship between CMV infection, cytokines, and breast cancer. METHODS: We evaluated CMV serostatus and cytokine levels in plasma of women with benign breast disease (n = 38), in situ carcinoma (n = 41), invasive carcinoma, no lymph node involvement (Inv/LN-; n = 41), and invasive with lymph node involvement (Inv/LN+; n = 37). RESULTS: Fifty percent of the patient samples (n = 79) were CMV seropositive. There was no correlation between CMV status and diagnosis (p = 0.75). For CMV+ patients, there was a trend toward higher CMV IgG levels in invasive disease (p = 0.172). CmvIL-10 levels were higher in CMV+ in situ patients compared to the Inv/LN- and Inv/LN+ groups (p = 0.020). Similarly, cIL-10 levels were higher in CMV+ in situ patients compared to the Inv/LN- and Inv/LN+ groups (p = 0.043). The results were quite different in CMV- patients where cIL-10 levels were highest in Inv/LN- compared to benign, in situ, or Inv/LN+ (p = 0.019). African American patients were significantly associated with CMV+ status (p = 0.001) and had lower cmvIL-10 levels than Caucasian patients (p = 0.046). CONCLUSION: No association was observed between CMV IgG and diagnosis, but CMV infection influences cytokine production and contributes to altered cytokine profiles in breast cancer.

The Future of Biobanking: A Conceptual Look at How Biobanks Can Respond to the Growing Human Biospecimen Needs of Researchers.

Biobanking of human biological specimens has evolved from the simple private collection of often poorly annotated residual clinical specimens, to well annotated and organized collections setup by commercial and not-for-profit organizations. The activities of biobanks is now the focus of international and government agencies in recognition of the need to adopt best practices and provide scientific, ethical and legal guidelines for the industry. The demand for more, high quality and clinically annotated biospecimens will increase, primarily due to the unprecedented level of genomic, post genomic and personalized medicine research activities going on. Demand for more biospecimens provides new challenges and opportunities for developing strategies to build biobanking into a business that is better able to supply the biospecimen needs of the future. A paradigm shift is required particularly in organization and funding, as well as in how and where biospecimens are collected, stored and distributed. New collection sites, organized as Research Ready Hospitals (RRHs) and new public-private partnership models are needed for sustainability and increased biospecimen availability. Biobanks will need to adopt industry-wide standard operating procedures, better and "non-destructive" methods for quality assessment, less expensive methods for sample storage/distribution, and objective methods to manage scarce biospecimens. Ultimately, the success of future biobanks will rely greatly on the success of public-private partnerships, number and diversity of available biospecimens, cost management and the realization that an effective biobank is one that provides high quality and affordable biospecimens to drive research that leads to better health and quality of life for all.

Towards Preanalytical Best Practices for Liquid Biopsy Studies: A BLOODPAC Landscape Analysis.

BLOODPAC is a public-private consortium that develops best practices, coordinates clinical and translational research, and manages the BLOODPAC Data Commons to broadly support the liquid biopsy community and accelerate regulatory review to aid patient accessibility. BLOODPAC previously recommended 11 preanalytical minimal technical data elements (MTDEs) for BLOODPAC-sponsored studies and data submitted to BLOODPAC Data Commons. The current landscape analysis evaluates the overlap of the BLOODPAC MTDEs with current best practices, guidelines, and standards documents related to clinical and research liquid biopsy applications. Our findings indicate an existing high degree of concordance among these documents. Where differences exist, the BLOODPAC preanalytical MTDEs can be considered a minimal practicable set for organizations to utilize. These MTDEs were developed following extensive examination of best practices and iterative conversations with the U.S. FDA. BLOODPAC recommends the use of these MTDEs in submissions to data commons and to support liquid biopsy clinical trials and research globally.

Contrived Materials and a Data Set for the Evaluation of Liquid Biopsy Tests: A Blood Profiling Atlas in Cancer (BLOODPAC) Community Study.

The Blood Profiling Atlas in Cancer (BLOODPAC) Consortium is a collaborative effort involving stakeholders from the public, industry, academia, and regulatory agencies focused on developing shared best practices on liquid biopsy. This report describes the results from the JFDI (Just Freaking Do It) study, a BLOODPAC initiative to develop standards on the use of contrived materials mimicking cell-free circulating tumor DNA, to comparatively evaluate clinical laboratory testing procedures. Nine independent laboratories tested the concordance, sensitivity, and specificity of commercially available contrived materials with known variant-allele frequencies (VAFs) ranging from 0.1% to 5.0%. Each participating laboratory utilized its own proprietary evaluation procedures. The results demonstrated high levels of concordance and sensitivity at VAFs of >0.1%, but reduced concordance and sensitivity at a VAF of 0.1%; these findings were similar to those from previous studies, suggesting that commercially available contrived materials can support the evaluation of testing procedures across multiple technologies. Such materials may enable more objective comparisons of results on materials formulated in-house at each center in multicenter trials. A unique goal of the collaborative effort was to develop a data resource, the BLOODPAC Data Commons, now available to the liquid-biopsy community for further study. This resource can be used to support independent evaluations of results, data extension through data integration and new studies, and retrospective evaluation of data collection.

Assessing the quality of RNA isolated from human breast tissue after ambient room temperature exposure.

High quality human tissue is essential for molecular research, but pre-analytical conditions encountered during tissue collection could degrade tissue RNA. We evaluated how prolonged exposure of non-diseased breast tissue to ambient room temperature (22±1°C) impacted RNA quality. Breast tissue received between 70 to 190 minutes after excision was immediately flash frozen (FF) or embedded in Optimal Cutting Temperature (OCT) compound upon receipt (T0). Additional breast tissue pieces were further exposed to increments of 60 (T1 = T0+60 mins), 120 (T2 = T0+120 mins) and 180 (T3 = T0+180 mins) minutes of ambient room temperature before processing into FF and OCT. Total exposure, T3 (T0+180 mins) ranged from 250 minutes to 370 minutes. All samples (FF and OCT) were stored at -80°C before RNA isolation. The RNA quality assessment based on RNA Integrity Number (RIN) showed RINs for both FF and OCT samples were within the generally acceptable range (mean 7.88±0.90 to 8.52±0.66). No significant difference was observed when RIN at T0 was compared to RIN at T1, T2 and T3 (FF samples, p = 0.43, 0.56, 0.44; OCT samples, p = 0.25, 0.82, 1.0), or when RIN was compared between T1, T2 and T3. RNA quality assessed by quantitative real-time PCR (qRT-PCR) analysis of beta-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cyclophilin A (CYPA), and porphobilinogen deaminase (PBGD) transcripts showed threshold values (Ct) that indicate abundant and intact target nucleic acid in all samples (mean ranging from 14.1 to 25.3). The study shows that higher RIN values were obtained for non-diseased breast tissue up to 190 minutes after resection and prior to stabilization. Further experimental exposure up to 180 minutes had no significant effect on RIN values. This study strengthens the rationale for assessing RIN and specific gene transcript levels as an objective method for determining how suitable RNA will be for a specific research purpose ("fit-for purpose").

Proteogenomic analysis of lung adenocarcinoma reveals tumor heterogeneity, survival determinants, and therapeutically relevant pathways.

We present a deep proteogenomic profiling study of 87 lung adenocarcinoma (LUAD) tumors from the United States, integrating whole-genome sequencing, transcriptome sequencing, proteomics and phosphoproteomics by mass spectrometry, and reverse-phase protein arrays. We identify three subtypes from somatic genome signature analysis, including a transition-high subtype enriched with never smokers, a transversion-high subtype enriched with current smokers, and a structurally altered subtype enriched with former smokers, TP53 alterations, and genome-wide structural alterations. We show that within-tumor correlations of RNA and protein expression associate with tumor purity and immune cell profiles. We detect and independently validate expression signatures of RNA and protein that predict patient survival. Additionally, among co-measured genes, we found that protein expression is more often associated with patient survival than RNA. Finally, integrative analysis characterizes three expression subtypes with divergent mutations, proteomic regulatory networks, and therapeutic vulnerabilities. This proteogenomic characterization provides a foundation for molecularly informed medicine in LUAD.

Biospecimen reporting for improved study quality (BRISQ).

Human biospecimens are subject to a number of different collection, processing, and storage factors that can significantly alter their molecular composition and consistency. These biospecimen preanalytical factors, in turn, influence experimental outcomes and the ability to reproduce scientific results. Currently, the extent and type of information specific to the biospecimen preanalytical conditions reported in scientific publications and regulatory submissions varies widely. To improve the quality of research utilizing human tissues, it is critical that information regarding the handling of biospecimens be reported in a thorough, accurate, and standardized manner. The Biospecimen Reporting for Improved Study Quality (BRISQ) recommendations outlined herein are intended to apply to any study in which human biospecimens are used. The purpose of reporting these details is to supply others, from researchers to regulators, with more consistent and standardized information to better evaluate, interpret, compare, and reproduce the experimental results. The BRISQ guidelines are proposed as an important and timely resource tool to strengthen communication and publications around biospecimen-related research and help reassure patient contributors and the advocacy community that the contributions are valued and respected.

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.

The Impact of Hormonal Contraceptives on Breast Cancer Pathology.

This retrospective case series study, using data obtained through questionnaires and histopathological diagnoses from 656 patients enrolled in the Department of Defense (DoD) Clinical Breast Care Project (CBCP), evaluated associations between hormonal contraceptive use and breast cancer pathology including benign breast pathologies. Three combination hormonal contraceptive agents (COCs) Lo Ovral (LO), Ortho Novum (ON), and Ortho Tri-Cyclen (OTC) were evaluated as they represented the most commonly used hormonal contraceptives in our cohort. The results of this study suggest that the ever use of LO + ON + OTC does not influence the overall incidence of benign breast condition or malignant disease compared to other COCs; however, patients that have used OTC had an association with a diagnosis of benign or luminal A pathologies whereas ON was associated with a diagnosis of benign and DCIS; LO showed no association with any diagnosis-benign or malignant. Patients that have used LO or ON were more likely to be diagnosed with breast cancer at age ≥ 40 years whereas patients that had ever used OTC were likely to be diagnosed before the age of 40. Caucasians were less likely to have used OTC and more likely to have used ON; however, use of either hormonal agent positively correlated with premenopausal status at diagnosis and having a benign condition. Age at diagnosis, ethnicity, BMI, family history, menstruation status, and duration of use were all independent predictors of different histopathological subtypes. We conclude that patient-specific variables should be considered when deciding on which type of hormonal contraceptive to use to minimize the risk of developing breast cancer or a breast-related pathology.

Screening for Multiple Autoantibodies in Plasma of Patients with Breast Cancer.

BACKGROUND/AIM: Autoantibodies have potential as circulating biomarkers for early cancer detection. This study aimed to screen for known autoantibodies in human plasma using an Autoantibody Profiling System (APS) and quantify the levels in plasma of donors with/without breast cancer. MATERIALS AND METHODS: Plasma from nine female donors diagnosed with breast cancer (test group) and nine matched donors with no personal history of cancer (reference group) were screened with an APS containing probes for 30 autoantibodies. Autoantibody levels ≥1.5 times the mean concentration of the group were considered elevated, and test/reference ratios ≥1.3 were considered higher in the test group compared to the reference group. RESULTS: Twenty percent of the probes detected elevated levels of autoantibodies against proteins involved in different cancer mechanisms. Amongst these, the levels of autoantibodies against interleukin 29 (IL29), osteoprotegerin (OPG), survivin (SUR), growth hormone (GRH) and resistin (RES) were significantly higher in the cancer group compared to the reference group (p<0.05), whereas the level of autoantibody against cytotoxic T-lymphocyte associated antigen-4 (CTLA4) was not significantly different between the two groups (p=0.38). CONCLUSION: Disease-relevant autoantibodies were detected in the plasma of patients with breast cancer and donors without breast cancer. This means that identifying the type and level of autoantibodies in samples will be important in determining their significance in the disease process. A microtiter plate-based array system could be a fast and inexpensive screening method for identifying and quantifying autoantibodies in human plasma.

Plasma concentration and activity of matrix metalloproteinase 2 and 9 in patients with breast disease, breast cancer and at risk of developing breast cancer.

Matrix metalloproteinases (MMPs) are involved in extracellular matrix modification and associated with invasive and metastatic behavior of human malignant tumors. Specifically, MMP2 and MMP9 are implicated in both early and late processes of tumor development. It is reported that MMPs occur as inactive precursors, active enzymes or enzyme inhibitor complexes in biological samples. However, there is limited knowledge on the role of each form in disease and/or the significance of changes in the plasma concentration and/or activity in breast cancer patients. The aim of this study was to determine if patients with breast cancer, benign disease and at risk for developing breast cancer display characteristic levels of active and/or total MMP2 and MMP9 in plasma. Concentration and activity of MMP2 and MMP9 were determined quantitatively in the plasma of 124 female volunteers diagnosed with breast cancer (n=31), benign disease (n=38), or determined by the Gail Model to be at high risk (n=31) or low risk (controls, n=24) of developing breast cancer. Data obtained was statistically analyzed to search for differences/patterns characteristic of each category. Concentration of total MMP2 was significantly lower in control individuals than benign, high risk (P<0.001 respectively) and breast cancer patients (P=0.002). Activity of total MMP2 was significantly lower in controls compared to cancer, benign and high risk patients (P<0.001 respectively). Attempts to build a predictive/descriptive model using canonical discriminant analysis (utilizing all eight features; concentrations and activity levels of active/total MMP2 and MMP9) enabled the distinction of the controls from the high risk, benign and cancer groups. Our results suggest that preoperative plasma concentration and activity of MMP2 and MMP9 may permit sub-classification of female patients with breast disorders.

A Low-density Antigen Array for Detection of Disease-associated Autoantibodies in Human Plasma.

BACKGROUND/AIM: The ability to easily detect autoantibodies will help in the early diagnosis and treatment of certain diseases. Currently, available methods for autoantibody detection are time-consuming and cumbersome. The present study aimed to evaluate the performance of an easy-to-use antigen array developed for autoantibody detection. MATERIALS AND METHODS: Plasma from 9 female donors diagnosed with ovarian cancer (test group) and 9 matched donors with no history of cancer (reference group) were screened and results were compared. Autoantibody levels ≥1.5-times the background were classified as positive. RESULTS: A total of 29 autoantibodies were detected, out of which the autoantibody against osteoprotegerin was found to be significantly higher in the "test" group (p<0.001) while those against macrophage migration inhibitor factor, interleukin-2 and vascular endothelial growth factor were lower (p<0.05). CONCLUSION: The evaluated antigen array has potential as a simple method for determining the presence/absence of up to 90 disease-associated autoantibodies in a plasma specimen.

Assays for Qualification and Quality Stratification of Clinical Biospecimens Used in Research: A Technical Report from the ISBER Biospecimen Science Working Group.

This technical report presents quality control (QC) assays that can be performed in order to qualify clinical biospecimens that have been biobanked for use in research. Some QC assays are specific to a disease area. Some QC assays are specific to a particular downstream analytical platform. When such a qualification is not possible, QC assays are presented that can be performed to stratify clinical biospecimens according to their biomolecular quality.

Proteomics of breast carcinoma.

Beast cancer is the most diagnosed cancer in women, accounting for approximately 40,000 deaths annually in the USA. Significant advances have been made in the areas of detection and treatment, but a significant number of breast cancers are detected late. The advent of proteomics provides the hope of discovering novel biological markers that can be used for early detection, disease diagnosis, prognostication and prediction of response to therapy. Several proteomics technologies including 2D-PAGE, 2D-DIGE, ICAT, SELDI-TOF, MudPIT and protein arrays have been used to uncover molecular mechanisms associated with breast carcinoma at the global level, and a number of these technologies, particularly the SELDI-TOF hold promise as a proteomic approach that can be applied at the bedside for discovering protein patterns that distinguish disease and disease-free states with high sensitivity and specificity. Laser microdissection, a method for selection of homogenous cell populations, coupled to 2D-DIGE or MudPIT constitute a new proteomics-based paradigm for detecting disease in pathology specimens and monitoring disease response to therapy. This review describes proteomics technologies, and their application in the proteomic analysis of breast carcinoma.

Biomedical informatics: development of a comprehensive data warehouse for clinical and genomic breast cancer research.

The Windber Research Institute is an integrated high-throughput research center employing clinical, genomic and proteomic platforms to produce terabyte levels of data. We use biomedical informatics technologies to integrate all of these operations. This report includes information on a multi-year, multi-phase hybrid data warehouse project currently under development in the Institute. The purpose of the warehouse is to host the terabyte-level of internal experimentally generated data as well as data from public sources. We have previously reported on the phase I development, which integrated limited internal data sources and selected public databases. Currently, we are completing phase II development, which integrates our internal automated data sources and develops visualization tools to query across these data types. This paper summarizes our clinical and experimental operations, the data warehouse development, and the challenges we have faced. In phase III we plan to federate additional manual internal and public data sources and then to develop and adapt more data analysis and mining tools. We expect that the final implementation of the data warehouse will greatly facilitate biomedical informatics research.

Global search for chromosomal abnormalities in infiltrating ductal carcinoma of the breast using array-comparative genomic hybridization.

Array-comparative genomic hybridization (a-CGH) is a molecular cytogenetic technique for detection of multiple chromosomal abnormalities in genomic DNA samples. Using an a-CGH with 287 probes, we examined 14 cases of breast infiltrating ductal carcinoma (IDCA) that had previously been classified by fluorescent in situ hybridization (FISH) as either human epidermal growth factor receptor-2 positive (HER2+) or HER2- and analyzed the data by hierarchical, K-means, and principal component analyses. The aim of the study was to identify the genetic abnormalities that are present in breast IDCAs and determine if the global status of 287 cytogenetic locations could be used as a more objective method for breast IDCA classification. Concordance between FISH and a-CGH at the HER2 locus was 78.6% (11/14). In general, a-CGH detected more abnormalities in HER2+ cases. In HER 2+ cases, chromosomes 1, 2, 3, 7, 9, 17, and 20 had more regions that showed statistically significant (P < or = 0.01) changes in DNA copy number. Among all the aberrant cytogenetic locations detected, 20q13, 7p12.3 approximately p12.1, and 17q23.2 approximately q25.3, which contain among others, genes for TNFRSF6B, EGFR, and TK1 showed statistically significant gains (P < or = 0.01) in 83, 66.7, and 50% of the HER2+ IDCA cases, respectively. Chromosome location 8q24.12 approximately q24.13 was the only region that showed consistent amplification in approximately 50% of the HER2- cases. Unsupervised hierarchical and K-means cluster analyses and principal component analysis using the DNA copy number status of 287 cytogenetic locations or the 177 cytogenetic locations that showed statistically significant differences revealed a cluster consisting of mainly HER2- IDCA cases. Even though this study demonstrates the usefulness of a-CGH in the rapid identification of aberrant DNA regions in tumor samples, we conclude that an array-CGH with more than 287 probes will be needed for a more precise mapping of DNA aberrations at the global level.