Leonid Kvecher

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.

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.

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.

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.

Abstract P5-01-07: Fibroadenomatoid changes are more prevalent in middle-aged women and have a positive association with invasive breast cancer

Background: The role of benign breast diseases (BBDs) in the development of invasive breast cancers (IBCs) has been studied for many years. Some BBDs have been studied comprehensively (e.g., fibrocystic changes (FCC)) while less is known about other BBDs (e.g., fiboadenomatoid changes (FAC)). FAC has been considered by some researchers as a precursor of fibroadenoma (FA). Conclusions from different studies vary, partially due to different interpretation methods and diagnostic criteria when multiple hospitals and pathologists were involved. In this study, we used subjects in the Clinical Breast Care Project (CBCP) from a military medical center where pathology slides were reviewed by a single breast pathologist to study FAC, FA, and FCC in comparison to the published literature. Methods: Subjects were enrolled in the study following IRB-approved, HIPAA-compliant protocols. All the clinicopathologic data are available from the CBCP data warehouse (DW4TR). In the CBCP, FCC is composed of 4 components: stromal fibrosis, cysts, apocrine metaplasia, and sclerosing adenosis. Two modeling studies were performed. i) For the BBDs and IBC association study, two groups of subjects were identified: 1136 subjects diagnosed with “Benign” or “Atypical” diseases, and 619 cases diagnosed with IBCs. A logistic regression model was developed for the prediction of IBCs by the 3 BBDs and 2 well-established risk factors (RF): age (younger, 60) and race (Caucasian, African American, Asian, and other). ii) For the RF association study with the BBDs, 6 additional RFs reported to be associated with these BBDs were identified from the literature: current use of oral contraceptives, number of live births, education, body mass index, hormonal replacement therapy, and IBC family history. These 8 RFs were used to develop a logistic regression model for each of the BBDs. The analyses were performed in SAS. Results: In the first study, age and race were confirmed as RFs for IBCs. FAC was positively associated with IBC (OR = 3.04, 95%CI=2.06 to 4.50). FA was negatively associated with IBC, and the level of the association was stronger in women without FCC (OR = 0.15, 95%CI=0.08 to 0.28), compared to women with FCC (OR = 0.40, 95%CI=0.24 to 0.65). FCC was not significantly associated with IBC. Results from the second study indicated that, age was significantly associated with FAC (p = 0.015), specifically the middle-aged women were more likely to have FAC compared to younger women (OR = 2.03, 95%CI=1.23 to 3.34), while the older women were at a non-significantly increased risk. Trends of association with FAC were also noted for the number of live birth (p = 0.095), ethnicity (p = 0.096), and current oral contraceptive pill use (p = 0.077). The FCC model results were in general consistent with the literature, and we also confirmed that age was negatively associated with the diagnosis of FA. Discussion: Our study was consistent with FCC findings in the literature. We observed that FAC was positively associated with IBC, whereas FA was negatively associated. Also, FAC occurred more often in middle-aged women while FAs occurrence was higher in younger women. Our results suggest that FAC and FA may be two different diseases. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-01-07.

P1-03-06: Fibroadenomatoid Changes Have a Higher Occurrence Rate in Middle-Aged Benign Breast Disease Patients with the Trend Retained in Cancer Patients.

Background: Fibroadenoma (FA) is a common benign breast lesion known to have a high incidence rate in younger women. There are controversial reports whether FA elevates the risk of developing breast cancers. In clinical practice, FA may be surgically removed due to multiple reasons making it complicated to study its impact on the development of breast cancers that have a higher incidence rate in older women. Fibroadenomatoid change (FAC), also known as fibroadenomatous hyperplasia, is an uncommon lesion with histologic features similar to that of FA but lacking well-defined borders and usually discovered incidentally on breast biopsy specimens. FAC is not surgically targeted. The Walter Reed Army Medical Center, through the Clinical Breast Care Project, has enrolled over 2000 subjects undergoing a biopsy; all the pathology was reviewed by a single pathologist. These subjects provide an opportunity to study the age-dependent pattern of FAC in different patient populations. Methods: Subjects were enrolled following IRB-approved protocols with data collected through two comprehensive questionnaires, a Core Questionnaire and a Pathology Checklist. A total of 1964 female subjects were identified for this study, including 1135 benign/atypical, 192 in situ, and 637 invasive cancer patients. Patients were divided into three age groups: =66 years. Chi-Square test in the SAS was used for statistical analysis. Results: As shown in the table, FA occurrence rate decreases significantly with increasing age in benign disease patients. FAC, on the other hand, shows a significantly higher occurrence rate in middle-aged patients with benign findings, and this trend is retained in the invasive or in situ cancer populations. FAC rate is also significantly higher in patients with cancer (invasive, or invasive and in situ combined) compared to benign patients in each age group with p-values ranging from 0.0001 to 0.019 (not shown). Discussion: Our preliminary results suggest that FAC occurs more often in middle-aged patients. It9s significantly lower occurrence in patients with benign findings may be partially explained by the fact that breast cancer patients undergo more extensive surgeries, thus providing more breast tissue for pathologic evaluation. Otherwise, the increased FAC rate may suggest its role as a risk factor for cancer development. Since FAC may be considered a miniature FA that is not surgically targeted, it may be used as a window for the study of FA on its impact in cancer development. Further study needs to be performed to explain why FA and FAC have different age-dependent patterns and whether FA or FAC is a risk factor for breast cancer development. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-03-06.

Abstract 3372: Differential exon usage in the HER2 subtype of breast cancer identified with RNA-seq and proteomic data

Background: Heterogeneity between different breast cancer tumors plays an important role in patients’ survival outcomes, and such tumor heterogeneity unveiled by transcriptome and proteome are often in disagreement. We hypothesize that alternative splicing of mRNA could explain heterogeneity within intrinsic breast cancer subtypes. Methods: Cases used in this study were drawn from the Clinical Breast Care Project where breast cancer patients were consented using an IRB-approved protocol. Fifty breast tumors were selected and processed by laser microdissection. RNA and protein were extracted from tissues using the Illustra triplePrep kit (GE Healthcare). Paired-end mRNA sequencing was performed using the Illumina HiSeq platform. Paired-end reads were preprocessed using PRINSEQ and splice-aligned to the genome using GSNAP software. Gene counts were measured using HTSeq while Exon counts used the DEXSeq. Differential expression was called at 10% false discovery rate. Proteome Discoverer with Byonic node was used for analyzing the quantitative global proteomics dataset, and we were able to quantitate 8600 proteins. All other analyses were performed using Perl and R. Results: The number of sequencing reads for the 50 cases ranged from 60 million to 410 million reads. After preprocessing, an average of 93% of reads was mapped, and 36,700 genes were identified among 50 tumor samples. PAM50 algorithm was used for intrinsic subtype calls, yielding 16 Basal, 8 Her2+, 19 Luminal A (LA) and 7 Luminal B tumors. Unsupervised clustering of the 8 Her2+ cases using the PAM50 genes and highly varying proteins resulted in different clustering patterns, with the latter clustering 6 of the 8 Her2+ cases with two distinct 3-case sub-clusters. Between these two sub-clusters, there were 10 differentially expressed (DEX) genes and 25 DEX proteins, but none of the 25 proteins were mapped to the 10 DEX genes. This motivated us to investigate the DEX exons, and we found 7,076 DEX exons between the Her2+ sub-clusters, and 9 of the 25 DEX proteins were matched to genes bearing DEX exons. For comparison, we performed the same analyses between similarly clustered Basal and LA sub-clusters. Even though the number of DEX genes between Basal (8) and LA (18) sub-clusters were comparable to that between the Her2+ sub-clusters, DEX exons were much lower for both subtypes (616 & 157), and none of the DEX proteins (3 & 7) mapped to DEX exons or genes for either subtype. Conclusions: Our findings imply that there is more proteomic-level heterogeneity in the Her2+ subtype than in Basal and LA subtypes, which could be due to alternative usage of exons (alternative splicing). If such heterogeneity is associated with patients’ survival outcomes then our results will further stress the importance of alternative splicing in breast cancer. The views expressed in this article are those of the author and do not reflect the official policy of the Department of Defense, or U.S. Government. Citation Format: Praveen Kumar Raj Kumar, Tao Liu, Lori A. Sturtz, Albert Kovatich, Marina A. Gritsenko, Vladislav A. Petyuk, Brenda Deyarmin, Viswanadham Sridhara, James Craig, Jason E. McDermott, Anil K. Shukla, Ronald J. Moore, Matthew E. Monroe, Bobbie-Jo M. Webb-Robertson, Jeffrey A. Hooke, Leigh Fantacone-Campbell, Leonid Kvecher, Jianfang Liu, Jennifer Kane, Jennifer Melley, Stella Somiari, Joji Iida, Stephen C. Benz, Justin Golovato, Shahrooz Rabizadeh, Patrick Soon-Shiong, Richard D. Smith, Richard J. Mural, Karin D. Rodland, Craig D. Shriver, Hai Hu. Differential exon usage in the HER2 subtype of breast cancer identified with RNA-seq and proteomic data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3372. doi:10.1158/1538-7445.AM2017-3372

Abstract P4-09-14: PhosphohistoneH3 as a prognostic marker in breast cancer: High expression is associated with younger age, triple negative subtype, and disease specific survival

BACKGROUND PhosphohistoneH3 (PPH3) is an emerging marker in breast cancer and has been linked to both patient survival and age. Phosphorylation of HistoneH3 is an important step during the cell cycle leading to proper compaction of the chromatin during late G2 and early mitosis. Here we assessed the use of PPH3 as a prognostic marker within a group of invasive breast cancers in the Clinical Breast Care Project (CBCP). METHODS CBCP participants and their samples were collected following IRB-approved, HIPAA-compliant protocols. Samples from 157 CBCP patients were selected for tissue whole section immunohistochemistry (IHC), using antibodies to PPH3, ER, PR, Ki67, and Her2. For each sample, staining of PPH3 was assessed across 5 high powered microscope fields and was considered positive if there was on average >2 stained cells per field. ER and PR were considered positive when there was >5% nuclear staining, and Ki67 was positive when there was >15% nuclear staining. Her2 was considered positive with an IHC score of 3+ or 2+ with a FISH score above 2.2. The samples were subtyped as Luminal A (LA: ER+/HER2-/Ki67-), Luminal B1 (LB1: ER+/HER2-/Ki67+), Luminal B2 (LB2: ER+/HER2+), Her2+ (ER-/PR-/HER2+), and Triple Negative (TN: ER-/PR-/HER2-). PPH3 was tested for associations with age and subtype using a stratified univariate Wilcoxon rank-sum analysis and a multivariate analysis controlling for subtype. To test the efficacy of PPH3 as a prognostic marker, Kaplan-Meier curves for disease specific survival were analyzed and the cox proportional hazard regression model was calculated. Further analysis addressing population demographics and additional cancer characteristics is ongoing. RESULTS Wilcoxon analysis revealed an association between higher PPH3 levels and younger age (P=.0038). Subtype was also found to be associated with PPH3, with the TN subtype 6.26 times more likely to have higher PPH3 expression than LA (P=.005). The association with age was confirmed by repeating the analysis and stratifying into non-TN subtypes (P=.05) and TN only subtype (P=.017). Non-TN subtypes positive for PPH3 expression had median age of 53.18 at diagnosis and 63.29 for negative PPH3 expression; TN subtypes that were positive for PPH3 had a median age of 50.44 and 72.9 for negative PPH3. Multivariate analysis with age and subtype as the variables also supported these results (age P=.017; TN vs LA P=.022). Disease specific survival analysis showed that a shorter survival time was associated with positive PPH3 protein levels (P=0.03; hazard ratio=6.97). CONCLUSIONS High expression of PPH3 is associated with a younger age, poorer survival rate, and the TN subtype. These results corroborate the use of PPH3 as a prognostic marker for breast cancer patients. The views expressed in this article are those of the author and do not reflect the official policy of the Department of Defense, or U.S. Government. Citation Format: Craig J, Kovatich AJ, Hooke JA, Kvecher L, Liu J, Fantacone-Campbell JL, Rui H, Shriver CD, Hu H. PhosphohistoneH3 as a prognostic marker in breast cancer: High expression is associated with younger age, triple negative subtype, and disease specific survival. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-09-14.

Abstract P4-09-24: CD163 expression is associated with young age, triple negative subtype, and poor outcome in breast cancer

BACKGROUND CD163 is a scavenger receptor specifically expressed by cells of monocyte/macrophage lineage. It is a biomarker in many clinical conditions, including coronary artery disease, anti-inflammatory response, and cancers. In breast cancer, high numbers of CD163-positive macrophages correlated with unfavorable outcome. Expression of CD163 in breast cancer was also related to early distant recurrence and poor survival. In this study we evaluated whether CD163 expression was associated with aggressive breast cancers from patients enrolled in the Clinical Breast Care Project (CBCP). METHODS Patients were enrolled into the CBCP following IRB-approved, HIPAA-compliant protocols. The study focused on 129 invasive breast cancer samples with CD163 immunohistochemistry (IHC) results. Expression of CD163, ER, PR, HER2, and Ki67 were assayed by IHC. CD163 was positive if IHC>0. ER and PR were positive if there was >5% nuclear staining. HER2 was negative if IHC=0 or 1+ and positive if IHC=3+. For HER2 IHC=2+, HER2 was negative if FISH was 2.2. Ki67 was positive if there was ≥15% nuclear staining. For subtyping, Luminal A (LA) was ER+/HER2-/Ki67-, Luminal B1 (LB1) was ER+/HER2-/Ki67+, Luminal B2 (LB2) was ER+/HER2+, Her2+ was ER-/PR-/HER2+, and triple negative (TN) was ER-/PR-/HER2-. Associations of CD163 IHC score with age, race, and IHC subtype were examined by Wilcoxon rank-sum tests and/or Fisher9s exact tests. Prognoses of CD163 in overall survival (OS), disease specific survival (DSS), disease free survival (DFS), and recurrence were studied using univariable and multivariable Cox proportional hazards regression models. CD163 score, age, race, AJCC stage, and subtype were included in the multivariable model. RESULTS CD163 IHC score displayed a significant negative correlation with age (R=-0.20, P=0.022). Patients with a CD163 score of 3+ were significantly younger than those with a score of 0 (P=0.019). CD163 score distributions were not statistically different between white and African American patients. CD163 scores of LA tumors were significantly lower than those of the tumors with all other subtypes except Her2+. Similarly, the CD163 scores of TN tumors were significantly higher than those of the tumors with all other subtypes but LB2. A higher CD163 score predicted worse DSS (HR=3.87 & P=0.0020 in univariable model; HR=4.21 & P=0.033 in multivariable model) and higher risk of recurrence (HR=2.85 & P=0.00016 in univariable model; HR=2.81 & P=0.012 in multivariable model). CONCLUSION Higher CD163 expression in breast cancer was significantly associated with younger age, the TN subtype, worse DSS, and higher risk of recurrence. These results highlight CD163 as a prognostic marker for breast cancer. The views expressed in this article are those of the author and do not reflect the official policy of the Department of Defense, or U.S. Government. Citation Format: Ru Y, Hu PT, Kovatich AJ, Hooke JA, Liu J, Kvecher L, Fantacone-Campbell JL, Deyarmin B, Kovatich AW, Cammarata F, Rui H, Davidson-Moncada J, Shriver CD, Hu H. CD163 expression is associated with young age, triple negative subtype, and poor outcome in breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-09-24.

Abstract P3-07-20: Survival comparative analysis of patients with invasive breast cancer treated by a military medical center and matched patients of the US general population

BACKGROUND U.S. military beneficiaries differ from the U.S. general population with regards to access to health care as care is provided at no or much lower cost in the military health system. Other differences also exist. Many of these differences are known factors affecting invasive breast cancer outcomes. Thus it is desirable to conduct a comparative analysis of breast cancer patient outcomes between these two populations to find out whether there is any outcome difference, and if yes what the contributing factors are. METHODS We compared overall survival (OS), disease-specific survival (DSS), and 5-year OS and DSS rates in breast cancers between 399 patients from the Clinical Breast Care Project at the Walter Reed National Military Medical Center (CBCP-WR) and 1,000 sets of 1596 matched patients from the Surveillance, Epidemiology, and End Results (SEER) Program of the National Cancer Institute. All patients were diagnosed between 2001 and 2010. Each CBCP-WR patient was randomly matched to four SEER patients on six demographic and clinicopathologic variables (age at diagnosis in 5-year groups, race, diagnosis year, estrogen receptor (ER), progesterone receptor, and AJCC stage). RESULTS The CBCP-WR cohort had better survival than the SEER population. At the whole cohort level, the mean hazard ratios (HRs) from 1,000 matched comparisons for OS and DSS were 0.774 and 0.708, with mean log-rank P-values of 0.124 and 0.125. The numbers of 175 and 141 comparisons showing a log-rank P-value CONCLUSION Overall, these results suggested that breast cancer patients, especially older patients seen in the CBCP-WR, carried more favorable outcomes than those from the general population. The findings warrant further analyses of the contributing factors, such as health care access, treatments, population characteristics, additional pathologic characteristics, and socioeconomic statuses, to this outcome disparity. The views expressed in this article are those of the author and do not reflect the official policy of the Department of Defense, or U.S. Government. Citation Format: Yuanbin Ru, Jianfang Liu, Jamie Leigh Campbell, Kangmin Zhu, Albert J Kovatich, Jeffrey A Hooke, Leonid Kvecher, Brenda Deyarmin, Audrey W Kovatich, Frank Cammarata, Hallgeir Rui, Richard J Mural, Craig D Shriver, Hai Hu. Survival comparative analysis of patients with invasive breast cancer treated by a military medical center and matched patients of the US general population [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-07-20.