Data Bank And BioRepository Research Articles

DNMT3A and TET2 mutant Clonal Hematopoiesis May Drive a Proinflammatory State and Predict Enhanced Response to Immune Checkpoint Inhibitors

DNMT3A and TET2 mutant Clonal Hematopoiesis May Drive a Proinflammatory State and Predict Enhanced Response to Immune Checkpoint Inhibitors

Clonal Hematopoiesis in Patients with Neuroendocrine Tumors Receiving Peptide Receptor Radionuclide Therapy (PRRT)

Background. Clonal hematopoiesis (CH) prevalence increases with age. Elderly (>70 years) healthy individuals have a 10% incidence of CH. Cancer cohorts are at increased risk of harboring CH mutations (~25%). Specific tumor types such as thyroid, ovarian, and bladder, have previously been shown to be at the highest risk of CH due to increased exposure to radionuclide and chemo-radiotherapies. Radionuclide therapies in particular are associated with a high incidence of subsequent myeloid disorders. Exposure to peptide receptor radionuclide therapy (PRRT, Lu 177 dotatate) leads to an increased risk (~2-5%) of therapy related myeloid neoplasms (MNs). It is not known if baseline CH architecture of neuroendocrine tumors (NETs) contributes to the increased risk or the protracted course of NETs or exposure to leukemogenic therapies. With a broader view to understand emergence of therapy induced myeloid neoplasms in various malignancies, we aimed to characterize baseline rates of CH in patients (pts) with NETs and assess clonal evolution in serial blood samples procured prior to and after exposure to PRRT. Methods. The NET Biobank housed at Roswell Park Comprehensive Cancer Center contains banked serial samples for pts receiving PRRT since its FDA approval in 2018. In this retrospective analysis, we identified pre-PRRT blood samples from 13 pts with NET treated at our institute from 2018-19. Serial samples (post PRRT exposure) were available for 6 of 13 pts. Genomic DNA collected from pts before and after PRRT treatment initiation was analyzed for CH mutations using a custom panel targeting 93 genes. A VAF cut off of 1% was used to define putative CH mutations. Relationships among clinical, laboratory and mutational variables were examined using chi-square test, at a significance level of 0.05. Results. Pt characteristics (n=13) are shown in Table 1. Median age was 58 years. 70% were men. Only 1/13 pt had prior chemotherapy exposure, and 3/13 had prior RT exposure. The primary location of NETs was small bowel (46%) followed by pancreas (23%) and others (cecal, rectal or unknown - 31%). All pts had stage IV disease. Over half (54%) harbored CH mutations, despite their relatively young age. This prevalence is much higher than previously reported CH prevalence of 25.1% in pts with other solid tumors.1TET2 (25%) was the most commonly mutated gene, followed by ASXL1 (12.5%), CHEK (12.5%), PPM1D (12.5%) and TP53 (12.5%) which together accounted for 50% of all mutations. Other mutations (DNMT3A, JAK2, DDX41, SRCAP) were less common (Figure 1). DNMT3A mutations (usually most prevalent mutation in cancer cohorts) were uncommon in this cohort and did not occur in the R882 AML/MDS hotspot, previously described in CH. Frameshift mutations and truncations comprised 53.3% of all mutations, and these mutations had higher mean VAFs (6.14%) than single nucleotide variants (1.93%; p = 0.001). This may suggest that frameshift mutations and truncations provide survival advantage to the affected clone. Mutations were more common in smokers than in non-smokers (100% vs 40%, p = 0.067). As expected, mutations were more common in individuals age 61 and above (75% harbored 1 or more mutation) vs only 20% mutation occurrence was noted in age 60 or below (p=0.05). Conclusion. CH with a distinct mutation profile occurs in NET patients, and in higher prevalence (54%) than observed in other solid tumors (25%). High baseline prevalence of putative CH mutations in NET patients may be an important contributor to heightened risk of MN development after PRRT exposure. Ongoing serial sample evaluation will provide further insights into clonal evolution of the above detected CH mutations after exposure to PRRT. The results regarding clonal evolution may have implications in predicting risk of MN associated with PRRT therapy and influence treatment selection in pts planned for PRRT. References Coombs CC, Zehir A, Devlin SM, et al. Therapy-Related Clonal Hematopoiesis in Patients with Non-hematologic Cancers Is Common and Associated with Adverse Clinical Outcomes. Cell Stem Cell. 2017;21(3):374-382.e374. Acknowledgement: Data and samples for this study were provided by the Data Bank and BioRepository (DBBR), which is funded by the National Cancer Institute (P30 CA016056) and is a Roswell Park Cancer Institute Cancer Center Support Grant shared resource. Disclosures Iyer: Advanced Accelerator Applications: Consultancy. Guzman:SeqRx: Honoraria; Cellectis: Research Funding. Wang:Abbvie: Consultancy; Jazz Pharmaceuticals: Consultancy; Genentech: Consultancy; Stemline: Speakers Bureau; PTC Therapeutics: Consultancy; Astellas: Consultancy; Macrogenics: Consultancy; Pfizer: Speakers Bureau; Bristol Meyers Squibb (Celgene): Consultancy.

Clonal Hematopoiesis in Patients Receiving Immune Checkpoint Inhibitor Therapy

Background. Clonal hematopoiesis (CH) is an aging associated phenomenon with potential to transform to overt myeloid disease at a rate of 1% per year. CH is hypothesized to represent a precursor state for leukemogenesis. CH is detected in 10% of healthy, elderly (>70 years) individuals at variant allele frequencies (VAFs) above 2%, but prevalence is higher in patients (pts) with malignancies (~25%). We recently demonstrated in a large population database study that recent changes in therapy for melanoma and non-small lung cancer (NSCLC) are associated with a decreased incidence of therapy-related myeloid neoplasms (tMNs).1 This trend may reflect declining utilization of chemo-radiotherapies or even regression of CH clones in the context of immune checkpoint inhibitor therapy (ICI). To improve our understanding of the evolving clonal architecture in patients with CH receiving ICI we analyzed blood samples from pts with melanoma and NSCLC at baseline and after exposure to ICIs. We aimed to characterize (i) baseline prevalence of CH in pts with melanoma and NSCLC (ii) alterations in clonal architecture associated with ICI treatment in serial blood samples from these pts. Methods. In this retrospective analysis, stored blood samples from pts with melanoma (N=32) and NSCLC (N= 109) treated with ICIs were analyzed. To detect CH, genomic DNA collected before treatment initiation and serially on ICI were analyzed using a custom panel targeting 93 genes. A 1% VAF cutoff was used to define CH. Relationships among clinical, laboratory and mutational variables were examined using chi-square test, at a significance level of 0.05. Results. We present preliminary results on 25 pts with melanoma (Table 1). Median age was 61 years. 52% (13/25) were men. One/25 pts had previously received chemotherapy and 4/25 had prior radiation therapy exposure. 64% (16/25) had stage IV disease and 36% (9/25) had stage III disease. 20% (5/25) had a history of autoimmune disease. CH was present in 32% of cohort, consistent with prior reports in solid tumor pts. Mutations in DNMT3A (36%) were most common, followed by TET2 (18%). Mutations in PPM1D, ARID1A, SF3B1, LPA and TGFBR2 accounted for 46% of the mutations (Figure 1). The mutational pattern was similar to prior reports in other cancer cohorts. DNMT3A mutations were most common in this cohort, with only one patient harboring a mutation in the R882 AML/MDS hotspot, previously described in CH. Most common mutation type was single nucleotide variants (55%), followed by frameshift (27%), and splice site mutations and truncations. The mean VAF for somatic mutations was 9%, much higher than the usual VAF cutoff for CH. Mutations were more common in smokers (40% vs 26.7%), and in individuals older than 60 years (37.5% vs 22%). Pts with autoimmune disease did not demonstrate CH mutations (0/5), whereas 40% (8/20) of pts without autoimmune diseases harbored mutations, (p=0.08). Conclusions. Our findings demonstrate a high baseline prevalence of putative CH mutations and high mean VAFs in a cohort of melanoma patients, even in the setting of minimal prior chemotherapy and radiation exposure. Despite this high CH prevalence, risk of tMN development after melanoma is declining with the use of newer therapies, suggesting a possible immune clearance or halt in progression of clonal hematopoiesis with newer therapies (ICI). Lack of CH mutations in individuals with autoimmune disease is an interesting finding and warrants further investigation. Ongoing serial sample analysis of the melanoma cohort after exposure to ICI will provide insight into the impact of ICI on the underlying clonal architecture. These findings are also being tested in the NSCLC validation cohort. References Singh A, Mrad C, Faber MG, et al. Evolving risk of therapy-related myelodysplastic syndromes and acute myeloid leukemia (tMDS/AML) following modern cancer therapies. Journal of Clinical Oncology. 2020;38(15_suppl):7516-7516. Acknowledgement: Data and samples for this study were provided by the Data Bank and BioRepository (DBBR), which is funded by the National Cancer Institute (P30 CA016056) and is a Roswell Park Cancer Institute Cancer Center Support Grant shared resource. Disclosures Griffiths: Persimmune: Research Funding; Boston Biomedical: Honoraria; Genentech Inc: Research Funding; Astex Pharmceuticals: Research Funding; Alexion Pharmaceuticals: Honoraria, Research Funding; Celgene/BMS: Honoraria, Research Funding; AbbVie Inc: Honoraria; Novartis: Honoraria, Research Funding. Guzman:Cellectis: Research Funding; SeqRx: Honoraria. Wang:Stemline: Speakers Bureau; PTC Therapeutics: Consultancy; Macrogenics: Consultancy; Astellas: Consultancy; Pfizer: Speakers Bureau; Abbvie: Consultancy; Jazz Pharmaceuticals: Consultancy; Bristol Meyers Squibb (Celgene): Consultancy; Genentech: Consultancy.

Abstract 5254: The associations of habitual physical inactivity with cancer outcomes: Evidence from the Roswell Park Cancer Institute Data Bank and BioRepository

Abstract Despite recent position statements asserting that a physically active lifestyle reduces cancer mortality, inverse associations of physical activity (PA) with cancer outcomes are only well established for breast, colorectal, and prostate cancers. Further, the independent associations of physical inactivity with cancer outcomes are rarely reported. Given the prevalence of physical inactivity at the population level and among cancer survivors, we examined the associations of habitual physical inactivity with clinical outcomes in a large patient cohort at Roswell Park Cancer Institute (RPCI). We utilized data from 7,827 patients enrolled in the Data Bank and BioRepository (DBBR) at RPCI between 2003 and 2016. Pre-diagnostic PA, spanning the decade prior to diagnosis, and post-diagnostic PA, representing PA at study enrollment, were assessed by self-administered questionnaire. Patients reporting no regular PA in the decade prior to diagnosis (i.e., walking for exercise, moderate or vigorous PA, or weight training) and no PA after diagnosis were categorized as habitually inactive, our primary analytic exposure of interest. Multivariable Cox proportional hazards models adjusted for age, sex, stage, and tumor site were used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) representing associations of habitual inactivity with all-cause mortality, cancer mortality, and disease recurrence. Additional variables (e.g., race, education, smoking, BMI, diabetes, tumor grade, treatment, occupational PA, etc.) were assessed for confounding utilizing the 10% change-in-estimate method. During the follow-up period, median time to follow-up was 54 months and 2,504 (32%) patients died. Collectively, 1,372 patients (17.5%) self-reported habitual inactivity. In comparison to patients who engaged in regular PA before and after diagnosis, habitually inactive patients experienced increased hazards of all-cause mortality (HR=1.41, 95% CI: 1.27-1.56, p<0.001), cancer mortality (HR=1.34, 95% CI: 1.19-1.52, p<0.001) and cancer recurrence (HR=1.15, 95% CI: 1.05-1.26, p=0.004). Associations remained robust and consistent in subgroup analyses by sex, stage and BMI. In sensitivity analyses, associations were strengthened when excluding patients with <3 years of follow-up and remained significant when excluding breast, colorectal and prostate cancer patients for all-cause mortality (HR=1.33, 95% CI: 1.18-1.50, p<0.001), cancer-specific mortality (HR=1.32, 95% CI: 1.14-1.52, p<0.001) and disease recurrence (HR =1.17, 95% CI: 1.04-1.30, p=0.007). We observed consistent evidence that habitual inactivity associates with increased hazards of cancer recurrence and mortality across multiple tumor types. Well-designed survivorship studies are needed to confirm how much mortality can be reduced among regularly active cancer patients. Citation Format: Rikki A. Cannioto, Shruti Dighe, Karen Hulme, Alexandra Drake, Susan E. McCann, Christine B. Ambrosone. The associations of habitual physical inactivity with cancer outcomes: Evidence from the Roswell Park Cancer Institute Data Bank and BioRepository [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5254.

Usual Consumption of Specific Dairy Foods Is Associated with Breast Cancer in the Roswell Park Cancer Institute Data Bank and BioRepository

BackgroundDairy foods are complex mixtures which include nutrients and non-nutrient substances that could potentially influence cancer etiology, including breast cancer. ObjectiveThe purpose of this study was to examine associations between the types and quantity of dairy foods consumed and the risk of breast cancer among women participating in the Roswell Park Cancer Institute Data Bank and BioRepository (DBBR) between 2003 and 2014. MethodsArchived clinical and questionnaire data were obtained from the DBBR from 1941 women diagnosed with breast cancer between December 2003 and October 2014, and 1237 control participants. Intakes of dairy foods were queried with a self-administered food-frequency questionnaire and grouped into monthly intakes of total dairy, milk, yogurt, low-fat cheese, other cheese, and sweet dairy. ORs and 95% CIs were estimated with unconditional logistic regression adjusting for age, race, body mass index, menopausal status, energy intake, type of milk usually consumed, cigarette smoking status, and family history of breast cancer. ResultsTotal dairy intakes were associated with a non-significant 15% reduction in breast cancer risk (P = 0.11). Higher intakes of yogurt were associated with reduced risk of breast cancer (OR: 0.61; 95% CI: 0.46, 0.82) and higher intakes of American, cheddar, and cream cheeses were associated with a marginally significant increased risk (OR: 1.53; 95% CI: 0.99, 2.34; P = 0.05). Associations with dairy foods were mixed when stratified by estrogen receptor (ER) status, and in general reflected those of overall breast cancer. However, we observed positive associations between milk intake and risk of ER− breast cancer (OR: 1.58; 95% CI: 1.05, 2.37) and inverse associations between sweet dairy and ER+ breast cancer (OR: 0.52; 95% CI: 0.29, 0.95). ConclusionsSpecific dairy foods may contribute to breast cancer risk in women, although the risk varies by source of dairy. Future studies are warranted to confirm the protective potential of yogurt in this type of cancer.

Engaging diverse populations in biospecimen donation: results from the Hoy y Mañana study.

The disproportionately lower number of certain subpopulations participating in clinical and prevention research has a significant impact on the representativeness of scientific outcomes. The Hoy y Mañana program (Today and Tomorrow) was developed as a culturally and linguistically appropriate education program to engage diverse medically underserved populations without a cancer diagnosis in biospecimen donation for cancer genomic research. Participants were recruited to in-depth community-based educational programs (∼45-60-min duration) or during open events in the community based on a convenience sampling. Programs were offered in English and Spanish. An on-site mobile lab along with phlebotomy services was provided at all programs and events to collect participant biospecimen (blood) samples to be stored at the cancer center's Data Bank and BioRepository (DBBR). The distributions for education, race/ethnicity, and gender were similar across the event types. Most of the participants were women. The analysis sample had a total of 311 participants, including 231 from the education programs and 80 participants from open events. Those with a higher education (college or more) were more likely to donate than those with a lower level of education (high school or less) (45 vs 28%, p = 0.007). Actual donation status was not associated with age or race. Willingness to donate a biospecimen and biospecimen donation rates followed the same pattern with respect to participants with higher levels of education being more willing to donate and giving a blood donation. Prior to outreach efforts, less than 6% of specimens donated to DBBR from healthy/non-cancer patients were from minority participants.

Abstract IA24: Building capacity and testing the impact of methods and variables for enhancing biospecimen donations with diverse communities

Abstract IA24: Building capacity and testing the impact of methods and variables for enhancing biospecimen donations with diverse communities

Dietary lignan intake and androgen receptor expression in breast tumors.

Lignans, a class of phytoestrogen commonly found in the Western diet, have been linked to decreased breast cancer risks in epidemiologic studies. Similar to estrogen receptors, the androgen receptor (AR), a prognostic factor in breast tumors, may be affected by lignans. However, few studies have investigated this link in the context of breast cancer etiology. We evaluated the relationship between dietary lignan intake and AR expression in incident breast tumors. Tumor tissue, epidemiological, and clinical data were collected from 216 women with incident, primary, histologically confirmed breast cancer enrolled in the Roswell Park Cancer Institute (RPCI) Data Bank and BioRepository (DBBR). On average, three tumor cores from each participant were assembled into a tissue micro array. After immunohistochemical staining, a trained RPCI pathologist determined AR status of each core. Lignan intake was calculated from a food frequency questionnaire collected upon enrollment into the DBBR. We observed a weak positive association between dietary lignans and AR expression [β (SE) 27.6 (17.0), p 0.10], and there was no significant difference in lignan intake across categories of AR expression (p=0.09, R (2)=0.35). Our results do not support a clear relationship between dietary lignan intake and AR expression. This investigation is the first, to our knowledge, to examine dietary lignan intake and AR expression in breast tumors. Further research is needed within a larger, more representative sample to determine whether lignan intake is truly associated with AR expression.

Effects of preanalytic variables on circulating microRNAs in whole blood.

Research in the last decade suggests the clinical potential of circulating microRNAs in whole blood as biomarkers for cancer detection. However, before applying the identified circulating microRNAs clinically, biospecimen-focused research has to be performed to identify possible preanalytic variables that may significantly affect the levels of circulating microRNAs. In this study, using a unique resource of the Data Bank and BioRepository (DBBR) at Roswell Park Cancer Institute, we conducted a two-step analysis to identify internal control circulating microRNAs in whole blood and then to study how selected major preanalytic variables (namely, processing delay, storage condition, storage time, and freeze/thaw cycles) might affect the detection of circulating microRNAs. In the discovery phase of the first step, we identified three microRNAs, including miR346, miR134, and miR934, whose levels exhibited the smallest variation between the case-control groups, as well as within each group interindividually. In the further validation analysis, the consistency was validated for miR346 and miR134 but not for miR934. At the second step, using miR346 and miR134 as internal controls, we observed that as the numbers of freeze/thaw cycles increased, levels of both miR346 and miR134 were significantly decreased (Ptrend < 0.0001); varying other processing and storage conditions did not affect miRNA levels. In the paralleled analysis in plasma samples, levels of miR16 were significantly decreased by increasing processing delay and increasing numbers of freeze/thaw cycles but not affected by storage condition and duration. The results from this study highlight the necessity of biospecimen-focused research on circulating microRNAs before clinical utilization. See all the articles in this CEBP Focus section, "Biomarkers, Biospecimens, and New Technologies in Molecular Epidemiology."

Dietary supplement use among participants of a databank and biorepository at a comprehensive cancer centre.

We assessed the prevalence, patterns and predictors of dietary supplement use among participants of the databank and biorepository (DBBR) at a comprehensive cancer centre in western New York. Archived epidemiological questionnaire data were obtained from the DBBR at Roswell Park Cancer Institute. Descriptive statistics and logistic regression explored the prevalence, patterns and predictors of lifetime use of four common supplements (multivitamins, vitamin C, vitamin E and calcium) and use of multivitamins, sixteen single vitamins/minerals and eighteen herbal/specialty supplements within the previous 10 years. Western New York, USA. DBBR participants (n 8096) enrolled between December 2003 and July 2012 were included in these analyses: 66.9 % (n 5418) with cancer, 65.6 % (n 5309) women, mean age for patients v. cancer-free controls 59.9 (SD 12.6) years and 50.7 (SD 15.4) years, respectively. Overall, 54.4 % of DBBR participants reported lifetime use of one or more supplements and 63.1 % reported use of one or more supplements within the previous 10 years (excluding multivitamins). Multivitamin use was high in this sample (lifetime: 64.1 %; 10 years: 71.3 %; current: 51.8 %). Supplementation was higher among cancer-free controls than cancer patients. Vitamin C, calcium and fish oil were the most common single vitamin, mineral and specialty product, respectively. A consistently high and increasing proportion of dietary supplement use over time remains clear. Supplementation is prevalent among cancer patients and may even be higher than predicted in cancer-free individuals. Further studies should assess the safety and efficacy of specific supplements in reducing disease risk.

075 Biospecimen banking research with circulating microRNAs: internal controls and pre-analytic variables

Because of the significance of microRNA (miRNA) in carcinogenesis, circulating miRNAs offer unique opportunities as minimal invasive biomarkers for cancer diagnosis and prognosis. To date, more than 200 studies have been reported. However, no single miRNA has been consistently observed across the studies. The wide inconsistency might be due to the fact that a significant amount of pre-analytic variations occurs from blood collection to the molecular analysis, and these variations may significantly contribute to observed inter-individual and intra-individual variations in circulating miRNAs. Little is known about how pre-analytic variations might affect the levels of circulating miRNAs, and there are no reliable internal controls currently available. Therefore, using high-quality biospecimens collected from the DataBank and BioRepository (DBBR) of Roswell Park Cancer Institute (RPCI), we performed a study to identify circulating miRNAs which can be used internal controls and then use them to study the effect of selected pre-analytic variables on circulating miRNAs in EDTA-plasma and whole blood collected using PaxGene tubes. Our criteria for internal control miRNAs include: they occur in all tested samples; their expression levels are not significantly different between cancer cases and controls; they show little inter-individual variations among cases and controls. Using exiqon miRNA PCR panel analysis, we performed miRNA profiling in 40 cancer cases and 40 healthy controls. A total of 4 miRNAs in whole blood (miR-134, miR-346, miR-934 and miR-16) and one miRNA in plasma (miR-16) fit our selection criteria for internal control miRNAs. These candidates were further validated in additional 100 cancer cases and 100 healthy controls. Using these identified internal control miRNAs, we next studied the effect of selected pre-analytic variables, including processing delay time, storage conditions, storage durations, and cycles of freeze/thaw. Twenty-four hour processing delay did not affect three tested miRNAs in whole blood, but significantly decreased the levels of miR-16 in plasma (P < 0.01). Increasing cycles of freeze/thaw from 0 to 1, 2 in whole blood, and 0 to 1, 2, 4 in plasma significantly decreased the levels of tested circulating miRNAs in both whole blood and plasma. Inverse dose response relationships were observed (P < 0.01 for both whole blood and plasma). Storage condition variations (Plasma: cryovials at −80°C vs straws in liquid nitrogen; Whole blood: −20°C vs −80°C) and storage duration variations (0 vs 6 months) seemed to have no effect on levels of tested circulating miRNAs in both whole blood and plasma. Our data clearly demonstrate the impact of pre-analytic variables on circulating miRNAs. Further biospecimen research is urgently needed to optimize the potential use of circulating miRNAs in cancer management. Source of funding: None declared. Conflict of interest: None declared. hzhao@mdanderson.org

Abstract 4583: Biospecimen banking research with circulating microRNAs: internal controls and pre-analytic variables.

Abstract Because of the significance of microRNA (miRNA) in carcinogenesis, circulating miRNAs offer unique opportunities as minimal invasive biomarkers for cancer diagnosis and prognosis. To date, more than 200 studies have been reported. However, no single miRNA has been consistently observed across the studies. The wide inconsistency might be due to the fact that a significant amount of pre-analytic variations occurs from blood collection to the molecular analysis, and these variations may significantly contribute to observed inter-individual and intra-individual variations in circulating miRNAs. Little is known about how pre-analytic variations might affect the levels of circulating miRNAs, and there are no reliable internal controls currently available. Therefore, using high-quality biospecimens collected from the DataBank and BioRepository (DBBR) of Roswell Park Cancer Institute (RPCI), we performed a study to identify circulating miRNAs which can be used internal controls and then use them to study the effect of selected pre-analytic variables on circulating miRNAs in EDTA-plasma and whole blood collected using PaxGene tubes. Our criteria for internal control miRNAs include: they occur in all tested samples; their expression levels are not significantly different between cancer cases and controls; they show little inter-individual variations among cases and controls. Using exiqon miRNA PCR panel analysis, we performed miRNA profiling in 40 cancer cases and 40 healthy controls. A total of 4 miRNAs in whole blood (miR-134, miR-346, miR-934 and miR-16) and one miRNA in plasma (miR-16) fit our selection criteria for internal control miRNAs. These candidates were further validated in additional 100 cancer cases and 100 healthy controls. Using these identified internal control miRNAs, we next studied the effect of selected pre-analytic variables, including processing delay time, storage conditions, storage durations, and cycles of freeze/thaw. Twenty-four hour processing delay did not affect three tested miRNAs in whole blood, but significantly decreased the levels of miR-16 in plasma (P&amp;lt;0.01). Increasing cycles of freeze/thaw from 0 to 1, 2 in whole blood, and 0 to 1, 2, 4 in plasma significantly decreased the levels of tested circulating miRNAs in both whole blood and plasma. Inverse dose response relationships were observed (P&amp;lt;0.01 for both whole blood and plasma). Storage condition variations (Plasma: cryovials at -80°C vs straws in liquid nitrogen; Whole blood: -20°C vs -80°C) and storage duration variations (0 vs 6 months) seemed to have no effect on levels of tested circulating miRNAs in both whole blood and plasma. Our data clearly demonstrate the impact of pre-analytic variables on circulating miRNAs. Further biospecimen research is urgently needed to optimize the potential use of circulating miRNAs in cancer management. Funded by NCI Contract No. HHSN261200800001E Citation Format: Hua Zhao, Song Liu, Qiang Hu, Li Yan, Warren Davis, Mary Nesline, Christine Ambrosone. Biospecimen banking research with circulating microRNAs: internal controls and pre-analytic variables. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4583. doi:10.1158/1538-7445.AM2013-4583

Predictors of Immunosuppressive Regulatory T Lymphocytes in Healthy Women

Immunosuppressive regulatory T (Treg) cells play an important role in antitumor immunity, self-tolerance, transplantation tolerance, and attenuation of allergic response. Higher proportion of Treg cells has been observed in peripheral blood of cancer cases compared to controls. Little is known about potential epidemiological predictors of Treg cell levels in healthy individuals. We conducted a cross-sectional study including 75 healthy women, between 20 and 80 years of age, who participated in the Data Bank and BioRepository (DBBR) program at Roswell Park Cancer Institute (RPCI), Buffalo, NY, USA. Peripheral blood levels of CD4+CD25+FOXP3+ Treg cells were measured using flow cytometric analysis. A range of risk factors was evaluated using Wilcoxon Rank-Sum test, Kruskal-Wallis test, and linear regression. Age, smoking, medications for treatment of osteoporosis, postmenopausal status, body mass index (BMI), and hormone replacement therapy (HRT) were found to be significant positive predictors of Treg cell levels in peripheral blood (P ≤ 0.05). Higher education, exercise, age at first birth, oral contraceptives, and use of Ibuprofen were found be significant (P < 0.05) negative predictors of Treg levels. Thus, various epidemiological risk factors might explain interindividual variation in immune response to pathological conditions, including cancer.

P1-09-06: Single Nucleotide Polymorphisms in the BRMS1 and SIPA1 Metastasis Suppressor Genes as Prognostic Markers in Breast Cancer Patients.

Abstract Introduction: Single nucleotide polymorphisms (SNPs) in the metastasis suppressors BRMS1 and SIPA1 may affect metastatic efficiency. BRMS1 affects apoptosis, colonization, cell adhesion, and invasive potential. Loss of BRMS1 expression has been correlated with younger age at diagnosis and reduced survival time in patients with progesterone-receptor (PR) negative, HER2−positive tumors. SIPA1 affects extracellular matrix gene expression and cell adhesion, and while SNPs have been associated with node positive, estrogen-receptor (ER)/PR negative tumors, evidence for a relationship with survival has been conflicting. Identifying SNPs that affect risk of recurrence and survival may improve the ascertainment of patients who require aggressive adjuvant therapy following a diagnosis of breast cancer. We evaluated associations between seven BRMS1 and SIPA1 SNPs and recurrence and survival in patients with primary breast cancer. Methods: We identified 1,015 incident breast cancer patients who received surgery at Roswell Park Cancer Institute (RPCI) and participated in the DataBank and BioRepository (DBBR) resource. Participants completed an epidemiologic questionnaire and provided a blood sample prior to surgery or other treatment. Clinical and pathologic data were linked to de-identified participant data in the DBBR database. SNPs in BRMS1 (rs11537993, rs3116068, and rs1052566) and SIPA1 (rs75894763, rs746429, rs3741378, and rs2306364) were genotyped by RPCI's Genomics facility using Sequenom® iPLEX Gold and Taqman® real-time PCR assays. Cox proportional hazards regression was used to estimate hazard ratios and 95% confidence intervals. Results: The median follow-up time was 33 months, and 49 deaths and 42 recurrences occurred. Tumors were more likely to be larger, node positive, ER/PR negative, and high grade among patients who experienced a recurrence or death. Recurrence was less likely in older patients and those with higher body mass index, although the latter association was nonsignificant (p=0.06). Patients with at least one variant allele of the BRMS1 rs3116068 genotype experienced shorter overall survival compared to patients with the homozygous common genotype (HR=2.05, 95% CI 1.15−3.63, rs3116068 AG+AA compared to GG). The remaining SNPs were not associated with overall survival, and none of the SNPs were associated with recurrence. Conclusions: In our data, the variant allele of rs3116068 was more common among women whose breast cancer was node negative and HER2−positive, compared to those with the common rs3116068 allele. The rs3116068 variant allele is also associated with poorer survival. While our findings do not support a role for common SNPs in the SIPA1 gene in breast cancer prognosis, BRMS1 rs3116068 may be a useful prognostic biomarker. Future goals are to examine additional SNPs in BRMS1 and other metastasis-related genes in a larger, racially diverse population. Funding: This research was supported by a gift from the Jayne and Phil Hubbell Family. The DBBR and Genomics Facility are RPCI Cancer Center Support Grant shared resources, supported by P30 CA016056-32. Ms. Roberts is a DOD Predoctoral Award recipient (BC10068) and Dr. Ambrosone is funded by the Breast Cancer Research Foundation. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-09-06.

Genetic Susceptibility as a Possible Risk Factor for Breast Cancer.

Abstract Background: Established risk factors for breast cancer (BC) only explain 25% to 47% of BC incidence. The Gail model is commonly used to assess BC risk and determine eligibility for prevention trials. Unfortunately, it has a discriminatory accuracy of 58% at the individual level. A greater understanding of BC risk factors is needed to offer improved risk stratification and prevention. It is plausible that BC risk may be related to deficiencies or abnormalities in DNA repair and cell cycle checkpoints because of their importance in maintaining genomic integrity. Recent epidemiologic studies support this hypothesis. In this pilot project, the bleomycin mutagen sensitivity assay (MSS), a measure of genetic susceptibility was performed on women of low, intermediate, and high risk based on commonly used risk models to determine if there was any correlation between MSS and established BC risk factors.Methods: Women in the Prevention Clinic at RPCI consented to donate blood for this study as part of the Data Bank and BioRepository (DBBR). Gail and Claus Models were calculated using CancerGene. Women were stratified into three groups low (&amp;lt;20%), intermediate (20%-35%), and high (&amp;gt; 35%) lifetime BC risk. For logistic regression analysis, women were grouped into low risk (&amp;lt;20%) and high risk (≥20%). MSS in blood lymphocytes were performed at LCCC using fresh blood samples. The mean values of MSS were compared between low, intermediate, and high risk groups using Wilcoxon-Mann-Whitney tests. Multivariate logistic regression was used to analyze the relationship between BC risk groups and MSS.Results: A total of 78 women were enrolled in the study. MSS were performed on 70 blood samples and 67 had evaluable slides. There were 30, 26 and 11 women in the low, intermediate, and high risk groups respectively. The mean age was 49.1 years. There was no statistical difference between the three groups with respect to age, race, menopausal status, proportion of patients with a family history of BC or another cancer, and smoking status (never, former, or current). The mean MSS score was not significantly different among the three risk groups: mean ±SD = 0.86±0.37, 0.84±0.37 (p=0.565), and 0.76+0.44 (0.361) for low, intermediate and high risk groups respectively. Spearman correlation revealed that MSS score did not correlate with Gail lifetime BC risk score (r=-0.054, p=0.668) or Claus model-10 year risk score (r=-0.057, p=0.722). Using the median in low risk women as a cut point, when women who had higher MSS score were compared with women had lower MSS score, the adjusted OR was 1.16 (95% CI, 0.29 to 4.57), adjusted for age, race, smoking status, BMI and menopausal status. When the MSS score were categorized into quartiles, no significant dose-response relationship was observed.Conclusions: In this pilot study, there was no correlation between commonly used breast cancer risk assessment models based on hormonal, family and biopsy history and the MSS in our study population. Previous case-control studies have consistently shown that MSS is significantly associated with BC risk. The lack of correlation between mutagen sensitivity and Gail or Claus risk score suggests that mutagen sensitivity may measure the host susceptibility factors that are not considered by Gail or Claus risk models. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6067.