Sergey Klimov

Biomarkers Predicting Pathologic Complete Response to Neoadjuvant Chemotherapy in Breast Cancer

OBJECTIVES Recent studies have shown strong correlation of pathologic complete response (pCR) to neoadjuvant chemotherapy with survival and prognosis in breast cancers. METHODS Clinical data from 237 breast cancer patients who received neoadjuvant chemotherapy between 2012 and 2014 were reviewed. Correlations were sought between pCR and estrogen receptor (ER), progesterone receptor (PR), and HER2 status; Nottingham and nuclear grades; tumor tubule formation; mitotic score; Ki67 index; and tumoral and stromal lymphocytic infiltration (TLI and SLI, respectively). RESULTS Of the 237 cases, 104 (43.9%) achieved pCR. The HER2+ and triple negative breast cancer (TNBC) subtypes had higher pCR rates compared with the luminal subtype (ER+ or PR+ and HER2-). ER and PR negativity, HER2 positivity, Nottingham grade 3, increased TLI and SLI, high mitotic count and Ki67 score correlated significantly with pCR in the overall cohort. TLI and SLI correlated significantly with pCR in the HER2+ and TNBC subtypes in multivariate analysis, whereas no biomarkers correlated with pCR in the luminal subtype. CONCLUSIONS In addition to the pathologic parameters and biomarkers already routinely assessed, evaluation of TLI and SLI may help to better select patients with HER2+ and TNBC for neoadjuvant chemotherapy.

Multi-institutional study of nuclear KIFC1 as a biomarker of poor prognosis in African American women with triple-negative breast cancer

Nuclear KIFC1 (nKIFC1) predicts worse outcomes in breast cancer, but its prognostic value within racially distinct triple-negative breast cancer (TNBC) patients is unknown. Thus, nKIFC1 expression was assessed by immunohistochemistry in 163 African American (AA) and 144 White TNBC tissue microarrays (TMAs) pooled from four hospitals. nKIFC1 correlated significantly with Ki67 in White TNBCs but not in AA TNBCs, suggesting that nKIFC1 is not merely a surrogate for proliferation in AA TNBCs. High nKIFC1 weighted index (WI) was associated with significantly worse overall survival (OS), progression-free survival (PFS), and distant metastasis-free survival (DMFS) (Hazard Ratios [HRs] = 3.5, 3.1, and 3.8, respectively; P = 0.01, 0.009, and 0.007, respectively) in multivariable Cox models in AA TNBCs but not White TNBCs. Furthermore, KIFC1 knockdown more severely impaired migration in AA TNBC cells than White TNBC cells. Collectively, these data suggest that nKIFC1 WI an independent biomarker of poor prognosis in AA TNBC patients, potentially due to the necessity of KIFC1 for migration in AA TNBC cells.

Distinctions in Breast Tumor Recurrence Patterns Post-Therapy among Racially Distinct Populations

Background Clinical studies have revealed a higher risk of breast tumor recurrence in African-American (AA) patients compared to European-American (EA) patients, contributing to the alarming inequality in clinical outcomes among the ethnic groups. However, distinctions in recurrence patterns upon receiving hormone, radiation, and/or chemotherapy between the races remain poorly characterized. Methods We compared patterns and rates (per 1000 cancer patients per 1 year) of recurrence following each form of treatment between AA (n = 1850) and EA breast cancer patients (n = 7931) from a cohort of patients (n = 10504) treated between 2005–2015 at Northside Hospital in Atlanta, GA. Results Among patients who received any combination of adjuvant therapy, AA displayed higher overall rates of recurrence than EA (p = 0.015; HR: 1.699; CI: 1.108–2.606). Furthermore, recurrence rates were higher in AA than EA among stage I (p = 0.031; HR: 1.736; CI: 1.052–2.864) and T1 classified patients (p = 0.003; HR: 2.009; CI: 1.263–3.197). Interestingly, among patients who received neoadjuvant chemotherapy, AA displayed higher rates of local recurrence than EA (p = 0.024; HR: 7.134; CI: 1.295–39.313). Conclusion Our analysis revealed higher incidence rates of recurrence in AA compared to EA among patients that received any combination of adjuvant therapy. Moreover, our data demonstrates an increased risk of tumor recurrence in AA than EA among patients diagnosed with minimally invasive disease. This is the first clinical study to suggest that neoadjuvant chemotherapy improves breast cancer recurrence rates and patterns in AA.

Novel immunohistochemistry-based signatures to predict metastatic site of triple-negative breast cancers

Background:Although distant metastasis (DM) in breast cancer (BC) is the most lethal form of recurrence and the most common underlying cause of cancer related deaths, the outcome following the development of DM is related to the site of metastasis. Triple negative BC (TNBC) is an aggressive form of BC characterised by early recurrences and high mortality. Athough multiple variables can be used to predict the risk of metastasis, few markers can predict the specific site of metastasis. This study aimed at identifying a biomarker signature to predict particular sites of DM in TNBC.Methods:A clinically annotated series of 322 TNBC were immunohistochemically stained with 133 biomarkers relevant to BC, to develop multibiomarker models for predicting metastasis to the bone, liver, lung and brain. Patients who experienced metastasis to each site were compared with those who did not, by gradually filtering the biomarker set via a two-tailed t-test and Cox univariate analyses. Biomarker combinations were finally ranked based on statistical significance, and evaluated in multivariable analyses.Results:Our final models were able to stratify TNBC patients into high risk groups that showed over 5, 6, 7 and 8 times higher risk of developing metastasis to the bone, liver, lung and brain, respectively, than low-risk subgroups. These models for predicting site-specific metastasis retained significance following adjustment for tumour size, patient age and chemotherapy status.Conclusions:Our novel IHC-based biomarkers signatures, when assessed in primary TNBC tumours, enable prediction of specific sites of metastasis, and potentially unravel biomarkers previously unknown in site tropism.

CancerVis: An interactive exploratory tool for cancer biomarker analysis

A biomarker is a measurable substance that can be used as an indicator of a particular disease. In the research field of cancer biomarker analysis, data visualization has generally been utilized as a technique for presenting results, with very little application of integrating data visualization as a tool for biological data exploration or pattern discovery. To address this issue, we have developed a visualization platform, CancerVis, for visual and interactive analysis of cancer data and pattern discovery. CancerVis provides multiple interactive views from different prospective of a dataset. The views are synchronized so that users can easily link the same data entry in multiple views. CancerViss visualization technologies also supports common data mining techniques for pattern discovery in cancer biomarker research, such as visualization of optimal cutoff point and its variability over time. The usability of CancerVis is verified by a clinical dataset.

Abstract PR02: β-Catenin overexpression underlies the aggressive disease course in African American triple-negative breast cancer patients who lack androgen receptor

Background: Androgen receptor (AR) has emerged as a new target for treating TNBC. AR is expressed in 10-43% of TNBCs. Although there are conflicting reports in the literature about the effect of AR status on TNBC prognosis, agents targeting AR signaling (enzalutamide) are already being evaluated in AR-positive TNBCs in early-stage clinical trials. However, no study so far has evaluated the association/correlation of AR status with ethnicity in TNBCs and downstream effects of AR loss in TNBCs. Given the association of AR loss with poor prognosis in breast cancer and that the African American (AA) with TNBC suffers aggressive disease course when compared to European American (EA) TNBCs, we hypothesized that AR loss might be an underlying cause of aggressive disease course in AR-negative TNBCs. Thus, in this project we aimed to study if loss or gain of AR in AA and EA TNBCs regulates the expression of β-catenin and leads to more aggressive disease course by activating downstream canonical Wnt-beta catenin signaling. Methods: We evaluated AR expression immunohistochemically in 424 formalin-fixed, paraffin-embedded samples from TNBC patients for whom complete clinicopathologic and overall survival (OS) data were available. Samples with Results: IHC staining of AR indicated that 79.5% of AA TNBCs (n=214) and 70% of EA TNBCs (n=210) were AR negative. Loss of AR was associated with poor overall survival in adjuvant-treated high Ki67 (>14%) (HR=1.72; p=0.095) AA TNBC (n=98) when compared to EA TNBCs (n=80). These data were validated by our in silico findings, which suggested that EA TNBCs (n=81) exhibited higher levels of AR mRNA compared to AA TNBCs (n=41) (p Conclusion: This study suggests that increased expression of β-catenin coupled with AR loss in AAs may underlie the ethnic disparity in outcomes among TNBC patients and strongly supports the prognostic role of AR and β-catenin in this breast cancer subtype. Citation Format: Karuna Mittal, Shristi Bhattarai, Sergey Klimov, Uma Krishnamurthi, Xiaoxian Li, Ceyda Sonmez Wetherilt, Mohammad A. Aleskandaran, Andrew A. Green, Emad A. Rakha, Ian O. Ellis, Guilherme Cantuaria, Guanhao Wei, Remus Mihai Osan, Meenakshi V. Gupta, Upender Manne, Padmashree C.G Rida, Ritu Aneja. β-Catenin overexpression underlies the aggressive disease course in African American triple-negative breast cancer patients who lack androgen receptor [abstract]. In: Proceedings of the Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr PR02.

Abstract B13: A novel metric illuminates disparities in cell cycling kinetics between ethnically-distinct breast tumors and enhances prediction of metastatic risk

Breast tumors in African-Americans (AAs) exhibit higher recurrence rates and faster kinetic progression to metastasis than those in European-Americans (EAs). This results in a stark ethnic disparity in breast cancer outcomes. Hence, enhancing understanding of cell cycle kinetics within breast tumors may illuminate hitherto overlooked and fundamental tumor biological characteristics of breast tumors underpinning racial differences in metastatic propensities. Current clinico-pathological prognostic markers that evaluate cell proliferation in breast carcinomas include mitotic index (MI) and Ki67 proliferation index (KI). However, as autonomous prognosticators measured on distinct scales, MI and KI lack the ability to capture information about the cycling kinetics of proliferating tumor cells-a key driver of intratumoral heterogeneity; this diminishes and undermines their prognostic accuracy. We performed a three-color immunofluorescence staining on paraffin-embedded AA (n=83) and EA (n=151) breast tumor tissue specimens from Northside Hospital to integrate mitotic cells and cycling cells into the same measurement scale. Phospho histone H3 was used as a mitotic marker and Ki-67 as a cell proliferation marker. Stained samples were examined in confocal microscopy to determine the proportion of mitotic cells among Ki-67 positive proliferative cells to yield the Mitosis:Proliferation (M:P) Ratio, a measure of the turnover rate of proliferating tumor cells. We observed higher M:P ratio in AA compared to grade-matched and stage-matched EA tumor tissue specimens. AA displayed significantly higher M:P ratio than EA among early stage tumors (p=0.015). Furthermore, among the clinico-pathological parameters, age, race, grade, stage, and receptor status, a multivariate analysis revealed that race was the only variable that exhibited a significant confounding influence on M:P ratio (p=0.042). A higher M:P Ratio likely reflects an increased mitotic propensity and higher risk of developing intratumoral heterogeneity and producing aggressive clones; thus, a higher M:P ratio may underlie the observed greater metastatic propensity exhibited in AA compared to EA patients. Thus, our novel metric provides new insights into the KI-MI relationship in tumors, exposes previously unrecognized differences in cycling kinetics among early stage AA and EA breast tumors, and proffers additional metastatic risk predictive information currently unavailable in the clinic. Citation Format: Nikita Wright, Sergey Klimov, Mildred Jones, Guilherme H. Cantuaria, Padmashree C. G. Rida, Ritu Aneja. A novel metric illuminates disparities in cell cycling kinetics between ethnically-distinct breast tumors and enhances prediction of metastatic risk. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr B13.

Abstract B09: Multivariable Models for Predicting Likely Metastatic Sites for Triple Negative Breast Cancers

Background: Unique organ microenvironment may preferentially support growth of specific tumor clones because of which different breast cancer subtypes show distinct tropisms for sites of metastasis. While a few gene expressionbased signatures are known to predict sitespecific metastasis of breast cancer, little work has focused on identification of clinically facile immunohistochemical predictors of metastasis to specific sites, especially for triple negative breast cancers (TNBCs). Methods: Primary tumor samples from 322 TNBC patients were stained for 133 biomarkers and assessed by immunohistochemistry. Differences in average levels of these biomarkers were compared between patients with or without metastasis to specific sites (brain, bone, lungs, liver, lymph nodes). Significantly different biomarkers were then analyzed within a Cox regression model to evaluate their prognostic value when patients with metastasis to the site of interest were compared to patients with no metastasis. Ideal thresholds, based on maximizing model fit, stratified cohorts that show high and low expression of each biomarker. A combination of a biomarker found high for each site, low for each site, and the Nottingham Prognostic Index (NPI) was used to stratify patients. Results: Our analysis uncovered several biomarkers whose expression levels in primary tumors can predict the site of future metastasis in TNBCs. Our models for brain (PARP1 & BRCA2), bone (MTA1 T 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr B09.

Abstract B12: Racial differences in Androgen Receptor status among triple-negative breast cancers

Background: Triple Negative Breast Cancer (TNBC) is an aggressive subtype of breast cancer with high recurrence and mortality rates. Currently, there are no effective targeted therapies available for TNBC. Emerging data suggest that androgen receptor (AR), a nuclear steroid hormone receptor expressed in 10-43% of TNBCs, has emerged as a promising new biomarker with potential predictive value for certain TNBC subtypes. African American (AA) women suffer from earlier onset of the breast cancer along with significant aggressive disease course when compared to European American (EA) women. This study aimed to determine the prognostic value of AR in a large TNBC cohort and evaluate racewise trends in AR expression status among TNBCs. Methods: We evaluated AR expression immunohistochemically in formalin-fixed paraffin-embedded samples from 822 TNBC patients (142, 95 and 264 patients from Emory, Northside and Grady Memorial Hospitals, respectively, in Atlanta, and 321 from Nottingham Hospital, UK) for whom complete clinicopathologic and overall survival (OS) data were available. Ethnicity data were available for 138 AA and 675 EA TNBC cases. Samples with Results: In this study, 45.6% of the TNBC cases were QN and AAs had a much higher proportion (80.8%) of QN cases than EAs (40.1%, p 14%) proliferative index (p Conclusion: This study suggest that a higher prevalence of QN breast cancer among AAs may be a plausible reason for the ethnic disparity in outcomes among TNBC patients and strongly support the prognostic role of AR in this breast cancer subtype. Citation Format: Bhattarai Shristi, Jun Xia, Ceyda Sonmez Wetherilt, Sergey Klimov, Ansa Riaz, Sonal Pattni, Mohammad A. Aleskandarany, Andrew R. Green, Emad A. Rakha, Ian O. Ellis, Guilherme Cantuaria, Xiaoxian (Bill) Li, Uma Krishnamurthi, Remus Mihai Osan, Padmashree CG Rida, Ritu Aneja. Racial differences in Androgen Receptor status among triple-negative breast cancers. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr B12.

Abstract B08: Identifying high-risk triple negative breast cancer patients using a novel cycling kinetics metric

Background Ki67 Index (KI) and Mitotic Index (MI) are proliferation markers with established prognostic value in breast cancer. These indices are evaluated individually and on disparate measurement scales; they thereforefail to capture information about cell cycling kinetics of proliferating cells. Within the triple negative (TNBC) subtype , we rationally integrate the two markers to identify high-risk patients whose proliferative cells exhibit fast cycling kinetics. Methods Pathology reports of breast cancer patients (n=10,504 from Northside Hospital, Atlanta and n=1560 from Nottingham Hospital, UK) were retrospectively analyzed for mitotic scores, KI and clinical outcomes. Mitotic counts in 267 HE n=322 from Nottingham Hospital, UK, and n=108 from OlabisiOnabanjo University, Nigeria). Stratification of KAMS, KI and MI were performed onthe thresholdsthat produced the lowest AIC (best model fit).Slow-cycling and fast-cycling TNBC subgroups from Nottingham Hospital were analyzed for biomarker expression. Results Kaplan-Meier survival analyses, AIC andc2 values showed that KAMS-based stratification of TNBCs into two subgroups was superior to that by either KI or MI, regardless of hospital, and KAMS retained its significance in multivariate analyses, controlling for stage and age. Fast-cycling TNBCs have poorer prognosis than slow-cycling TNBCs, perhaps due to higher intratumoral heterogeneity in fast cycling tumors. Fast-cycling TNBCs showed high expression of proteins implicated in DNA damage response, sumoylation, EGFR signaling and metastasis. By contrast, slow-cycling TNBCs showed extensive chromatin modification. Conclusion KAMS quantifies cell cycling kinetics, stratifies TNBCs and yields new risk-predictive information that is not revealed by either KI or MI. KAMS reveals the underlying heterogeneity in cycling kinetics among TNBCs and helps identify TNBCs who might benefit from treatments that target the cell cycle machinery. Citation Format: Sergey Klimov, Guanhao Wei, Andrew Green, Mohammed Aleskandarany, Emad Rakha, Ian Ellis, Guilherme Cantuaria, Ayodeji O. Agboola, Michelle Reid, Li Xiaoxian, Rida C. G. Padmashree, Remus Osan, Ritu Aneja. Identifying high-risk triple negative breast cancer patients using a novel cycling kinetics metric. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr B08.