Bradley A. Hancock

Association of Vascular Endothelial Growth Factor and Vascular Endothelial Growth Factor Receptor-2 Genetic Polymorphisms With Outcome in a Trial of Paclitaxel Compared With Paclitaxel Plus Bevacizumab in Advanced Breast Cancer: ECOG 2100

PURPOSE No biomarkers have been identified to predict outcome with the use of an antiangiogenesis agent for cancer. Vascular endothelial growth factor (VEGF) genetic variability has been associated with altered risk of breast cancer and variable promoter activity. Therefore, we evaluated the association of VEGF genotype with efficacy and toxicity in E2100, a phase III study comparing paclitaxel versus paclitaxel plus bevacizumab as initial chemotherapy for metastatic breast cancer. PATIENTS AND METHODS DNA was extracted from tumor blocks of patients from E2100. Three hundred sixty-three samples were available to evaluate associations between genotype and outcome. Genotyping was performed for selected polymorphisms in VEGF and VEGF receptor 2. Testing for associations between each polymorphism and efficacy and toxicity was performed. RESULTS The VEGF-2578 AA genotype was associated with a superior median overall survival (OS) in the combination arm when compared with the alternate genotypes combined (hazard ratio = 0.58; 95% CI, 0.36 to 0.93; P = .023). The VEGF-1154 A allele also demonstrated a superior median OS with an additive effect of each active allele in the combination arm but not the control arm (hazard ratio = 0.62; 95% CI, 0.46 to 0.83; P = .001). Two additional genotypes, VEGF-634 CC and VEGF-1498 TT, were associated with significantly less grade 3 or 4 hypertension in the combination arm when compared with the alternate genotypes combined (P = .005 and P = .022, respectively). CONCLUSION Our data support an association between VEGF genotype and median OS as well as grade 3 or 4 hypertension when using bevacizumab in metastatic breast cancer.

Next-generation transcriptome sequencing of the premenopausal breast epithelium using specimens from a normal human breast tissue bank

IntroductionOur efforts to prevent and treat breast cancer are significantly impeded by a lack of knowledge of the biology and developmental genetics of the normal mammary gland. In order to provide the specimens that will facilitate such an understanding, The Susan G. Komen for the Cure Tissue Bank at the IU Simon Cancer Center (KTB) was established. The KTB is, to our knowledge, the only biorepository in the world prospectively established to collect normal, healthy breast tissue from volunteer donors. As a first initiative toward a molecular understanding of the biology and developmental genetics of the normal mammary gland, the effect of the menstrual cycle and hormonal contraceptives on DNA expression in the normal breast epithelium was examined.MethodsUsing normal breast tissue from 20 premenopausal donors to KTB, the changes in the mRNA of the normal breast epithelium as a function of phase of the menstrual cycle and hormonal contraception were assayed using next-generation whole transcriptome sequencing (RNA-Seq).ResultsIn total, 255 genes representing 1.4% of all genes were deemed to have statistically significant differential expression between the two phases of the menstrual cycle. The overwhelming majority (221; 87%) of the genes have higher expression during the luteal phase. These data provide important insights into the processes occurring during each phase of the menstrual cycle. There was only a single gene significantly differentially expressed when comparing the epithelium of women using hormonal contraception to those in the luteal phase.ConclusionsWe have taken advantage of a unique research resource, the KTB, to complete the first-ever next-generation transcriptome sequencing of the epithelial compartment of 20 normal human breast specimens. This work has produced a comprehensive catalog of the differences in the expression of protein-coding genes as a function of the phase of the menstrual cycle. These data constitute the beginning of a reference data set of the normal mammary gland, which can be consulted for comparison with data developed from malignant specimens, or to mine the effects of the hormonal flux that occurs during the menstrual cycle.

Resequencing of the Vascular Endothelial Growth Factor Promoter Reveals Haplotype Structure and Functional Diversity.

Our group has previously reported that SNPs in the VEGF promoter are strongly associated with efficacy and toxicity to the anti-VEGF antibody bevacizumab in breast cancer. In order to better understand the biologic mechanism for our previously reported biomarkers, we embarked on a comprehensive evaluation of genetic variation in the VEGF promoter coupled with a study of its intrinsic function. We resequenced 48 Caucasians and 48 African-Americans for the VEGF promoter to identify SNPs and elucidate its haplotype structure. We further cloned the haplotypes into reporter constructs and assessed the role of SNPs on promoter function in breast cancer cell lines. SNPs that were identified included twenty previously reported SNPs/insertions/deletions, one novel SNP, and one novel deletion. Among these variants, we identified five SNPs that tag six haplotypes capturing 74% of the genetic variation of the promoter. Subsequently, assessment of the haplotypes in reporter constructs demonstrates significant variation in promoter induced expression among the haplotypes. In particular, two haplotypes had higher expression and one haplotype had lower expression across cell lines. Haplotypes containing SNPs previously reported to be associated with increased survival with the use of bevacizumab are high-expressing haplotypes, thus lending putative functional evidence to the prior clinical finding.

A large microRNA cluster on chromosome 19 is a transcriptional hallmark of WHO type A and AB thymomas.

Background:Thymomas are one of the most rarely diagnosed malignancies. To better understand its biology and to identify therapeutic targets, we performed next-generation RNA sequencing.Methods:The RNA was sequenced from 13 thymic malignancies and 3 normal thymus glands. Validation of microRNA expression was performed on a separate set of 35 thymic malignancies. For cell-based studies, a thymoma cell line was used.Results:Hierarchical clustering revealed 100% concordance between gene expression clusters and WHO subtype. A substantial differentiator was a large microRNA cluster on chr19q13.42 that was significantly overexpressed in all A and AB tumours and whose expression was virtually absent in the other thymomas and normal tissues. Overexpression of this microRNA cluster activates the PI3K/AKT/mTOR pathway. Treatment of a thymoma AB cell line with a panel of PI3K/AKT/mTOR inhibitors resulted in marked reduction of cell viability.Conclusions:A large microRNA cluster on chr19q13.42 is a transcriptional hallmark of type A and AB thymomas. Furthermore, this cluster activates the PI3K pathway, suggesting the possible exploration of PI3K inhibitors in patients with these subtypes of tumour. This work has led to the initiation of a phase II clinical trial of PI3K inhibition in relapsed or refractory thymomas (http://clinicaltrials.gov/ct2/show/NCT02220855).

Dual PI3K and Wnt pathway inhibition is a synergistic combination against triple negative breast cancer

Triple negative breast cancer accounts for 15–20% of all breast cancer cases, but despite its lower incidence, contributes to a disproportionately higher rate of mortality. As there are currently no Food and Drug Administration-approved targeted agents for triple negative breast cancer, we embarked on a genomic-guided effort to identify novel targeted modalities. Analyses by our group and The Cancer Genome Atlas have identified activation of the PI3K-pathway in the majority of triple negative breast cancers. As single agent therapy is commonly subject to resistance, we investigated the use of combination therapy against compensatory pathways. Herein, we demonstrate that pan-PI3K inhibition in triple negative breast cancers results in marked activation of the Wnt-pathway. Using the combination of two inhibitors currently in clinical trial as single agents, buparlisib(pan-PI3K) and WNT974(WNT-pathway), we demonstrate significant in vitro and in vivo synergy against triple negative breast cancer cell lines and xenografts. Taken together, these observations provide a strong rationale for testing dual targeting of the PI3K and WNT-pathways in clinical trials.Therapeutics: Dual therapy shrinks triple negative breast tumorsTwo experimental drugs currently in clinical trials as single agents might work better together against triple negative breast cancer. Jeffrey Solzak and Milan Radovich, and colleagues from the Indiana University School of Medicine in Indianapolis, USA, analyzed gene expression data from cancerous and healthy breast tissues and from published genomic datasets. They found the PI3K pathway consistently activated in the majority of breast cancers that test negative for the three most common receptors known to fuel breast tumor growth. Inhibiting PI3K in these cells led to a marked increase in the activity of another critical pathway called Wnt, an observation that prompted the researchers to test a combination of a PI3K inhibitor, buparlisib, and a Wnt inhibitor, WNT974, in cell lines and xenograft models. Tumor cells shrunk more and mice lived longer than treatment with either agent alone.

Next-generation sequencing of circulating tumor DNA to predict recurrence in triple-negative breast cancer patients with residual disease after neoadjuvant chemotherapy

Next-generation sequencing to detect circulating tumor DNA is a minimally invasive method for tumor genotyping and monitoring therapeutic response. The majority of studies have focused on detecting circulating tumor DNA from patients with metastatic disease. Herein, we tested whether circulating tumor DNA could be used as a biomarker to predict relapse in triple-negative breast cancer patients with residual disease after neoadjuvant chemotherapy. In this study, we analyzed samples from 38 early-stage triple-negative breast cancer patients with matched tumor, blood, and plasma. Extracted DNA underwent library preparation and amplification using the Oncomine Research Panel consisting of 134 cancer genes, followed by high-coverage sequencing and bioinformatics. We detected high-quality somatic mutations from primary tumors in 33 of 38 patients. TP53 mutations were the most prevalent (82%) followed by PIK3CA (16%). Of the 33 patients who had a mutation identified in their primary tumor, we were able to detect circulating tumor DNA mutations in the plasma of four patients (three TP53 mutations, one AKT1 mutation, one CDKN2A mutation). All four patients had recurrence of their disease (100% specificity), but sensitivity was limited to detecting only 4 of 13 patients who clinically relapsed (31% sensitivity). Notably, all four patients had a rapid recurrence (0.3, 4.0, 5.3, and 8.9 months). Patients with detectable circulating tumor DNA had an inferior disease free survival (p < 0.0001; median disease-free survival: 4.6 mos. vs. not reached; hazard ratio = 12.6, 95% confidence interval: 3.06–52.2). Our study shows that next-generation circulating tumor DNA sequencing of triple-negative breast cancer patients with residual disease after neoadjuvant chemotherapy can predict recurrence with high specificity, but moderate sensitivity. For those patients where circulating tumor DNA is detected, recurrence is rapid.Diagnostics: Liquid biopsy detects recurrence but lacks sensitivityA blood test can detect disease recurrence among women with triple-negative breast cancer (TNBC), but lacks sensitivity for distant relapse. A team led by Milan Radovich from Indiana University School of Medicine in Indianapolis, USA, analyzed tumor and blood plasma samples from 38 patients with TNBC who underwent neoadjuvant chemotherapy to see whether they could find evidence of mutated tumor DNA circulating in the blood after surgery that might predict cancer relapse. This kind of “liquid biopsy” has shown promise for patients with metastatic disease, but it has been tested less extensively in people with earlier-stage disease. The researchers’ blood test picked up tumor DNA in four patients after surgery, all of whom experienced disease recurrence. However, it missed nine other cases of relapse—indicating that the test has high specificity, but only moderate sensitivity.

EZH2 Modifies Sunitinib Resistance in Renal Cell Carcinoma by Kinome Reprogramming

Acquired and intrinsic resistance to receptor tyrosine kinase inhibitors (RTKi) represents a major hurdle in improving the management of clear cell renal cell carcinoma (ccRCC). Recent reports suggest that drug resistance is driven by tumor adaptation via epigenetic mechanisms that activate alternative survival pathways. The histone methyl transferase EZH2 is frequently altered in many cancers, including ccRCC. To evaluate its role in ccRCC resistance to RTKi, we established and characterized a spontaneously metastatic, patient-derived xenograft model that is intrinsically resistant to the RTKi sunitinib, but not to the VEGF therapeutic antibody bevacizumab. Sunitinib maintained its antiangiogenic and antimetastatic activity but lost its direct antitumor effects due to kinome reprogramming, which resulted in suppression of proapoptotic and cell-cycle-regulatory target genes. Modulating EZH2 expression or activity suppressed phosphorylation of certain RTKs, restoring the antitumor effects of sunitinib in models of acquired or intrinsically resistant ccRCC. Overall, our results highlight EZH2 as a rational target for therapeutic intervention in sunitinib-resistant ccRCC as well as a predictive marker for RTKi response in this disease. Cancer Res; 77(23); 6651-66. ©2017 AACR.

Germline and Somatic DNA Damage Repair Gene Mutations and Overall Survival in Metastatic Pancreatic Adenocarcinoma Patients Treated with FOLFIRINOX

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer with lack of predictive biomarkers. We conducted a study to assess DNA damage repair (DDR) gene mutations as a predictive biomarker in PDAC patients treated with FOLFIRINOX. Experimental Design: Indiana University Simon Cancer Center pancreatic cancer database was used to identify patients with metastatic PDAC, treated with FOLFIRINOX and had tissue available for DNA sequencing. Baseline demographic, clinical, and pathologic information was gathered. DNA isolation and targeted sequencing was performed using the Ion AmpliSeq protocol. Overall survival (OS) analysis was conducted using Kaplan–Meier, logistic regression and Cox proportional hazard methods. Multivariate models were adjusted for age, gender, margin status, CA 19-9, adjuvant chemotherapy, tumor and nodal stage. Results: Overall, 36 patients were sequenced. DDR gene mutations were found in 12 patients. Mutations were seen in BRCA1 (N = 7), BRCA2 (N = 5), PALB2 (N = 3), MSH2 (N = 1), and FANCF (N = 1) of all the DDR genes sequenced. Median age was 65.5 years, 58% were male, 97.2% were Caucasian and 51.4% had any family history of cancer. The median OS was near significantly superior in those with DDR gene mutations present vs. absent [14 vs. 5 months; HR, 0.58; 95% confidence interval (CI), 0.29–1.14; log-rank P = 0.08]. Multivariate logistic (OR, 1.47; 95% CI, 1.04–2.06; P = 0.04) and Cox regression (HR, 0.37; 95% CI, 0.15–0.94; P = 0.04) showed presence of DDR gene mutations was associated with improved OS. Conclusions: In a single institution, retrospective study, we found that the presence of DDR gene mutations are associated with improved OS in PDAC patients treated with FOLFIRINOX.

Abstract 4250: Molecular alterations in the breast associated with early menarche

Background: Menarche, the onset of the female menstruation, is a marker of pubertal timing. Age at menarche varies widely between girls and is highly dependent on nutritional status and body fat accumulation. The occurrence of menarche at an early age is linked to an increased risk of several adverse health conditions later in life, such as obesity, type-2 diabetes, breast and endometrial cancer, and cardiovascular disease. Indeed, for every one year decrease in age at menarche (from an average age at menarche of 12.5 years), breast cancer risk is increased by 5%. Several genome wide association studies (GWAS) have identified genetic variants (i.e. in the LIN28B gene) that are associated with early age at menarche, however little is known about the changes occurring in the breast tissue of women with early menarche. We hypothesize that early age at menarche results in permanent molecular alterations in the breast tissue and that those abnormalities may contribute to the tissue’s susceptibility to carcinogens and breast cancer development. Methods: To test our hypothesis we used the resources available at the Susan G. Komen Tissue Bank at the Indiana University Simon Cancer Center (KTB). We selected histologically normal breast tissue from healthy, young women with either early (age ≤ 10 years) or late menarche (age ≥ 15 years), and matched for age, race, BMI, and menstrual phase. Breast tissue biopsies from these women were microdissected to isolate the breast epithelium and next generation RNA-sequencing was used to generate a transcriptome profile for each sample. Differential expression was performed using DESeq2 in R. The tissue samples were also evaluated using immunostaining. Results/Conclusions: Preliminary data show significant differences when comparing the transcriptome profiles of the microdissected breast epithelium from the early and late menarche sample cohorts. The tissue from women with early menarche had upregulation of genes associated with defense against oxidative stress and/or infectious bacteria (lactotransferrin [LTF], ceruloplasmin [CP]), cell adhesion, (ITGα11, ITGαX, ITGαM, ITGαL, ITGβ2), immune response (CARD9, LAIR1), and had downregulation of ubiquitination pathways (USP40, AMFR) and lipoprotein metabolism (OSBPL1A, LIPH, PIGN). Immunohistochemical evaluation of markers of oxidative stress (LTF, CP), cell proliferation (Ki67), and immune infiltrates (CD45, CD20, CD8, CD68) is underway. Together, this information will give us the opportunity to better understand early age at menarche as a breast cancer risk factor and advance research for women’s health. Citation Format: Mariah L. Johnson, Natascia Marino, Anna Maria V. Storniolo, Bradley A. Hancock, Milan Radovich, George E. Sandusky. Molecular alterations in the breast associated with early menarche [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 4250. doi:10.1158/1538-7445.AM2017-4250

Abstract P2-08-23: TP53 mutation is a biomarker for prognosis in triple-negative breast cancer patients treated with post-neoadjuvant cisplatin

Introduction: Patients with Triple-Negative Breast Cancer (TNBC) who have residual disease (RD) after neoadjuvant chemotherapy are at an increased risk of relapse and have a poor prognosis. No adjuvant therapies are currently indicated for this group. BRE09-146 was a Phase II post-neoadjuvant clinical trial testing Cisplatin or Cisplatin + Rucaparib in TNBC patients with RD after neoadjuvant chemotherapy. As TP53 is mutated in 70-80% of TNBCs, and is well known to play a role in the DNA damage response, we sought to determine the prognostic capability of mutated TP53 in BRE09-146. Methods: We performed full sequence and copy number analysis of 134 genes in 76 tumors from BRE09-146 using the Oncomine Research Panel along with Ion Proton Next Generation Sequencing. All patients included had RD. Somatic mutations were called by identifying mutations that were present in the tumor that were not present in the germ line DNA from a normal blood sample. Mutations were annotated using the IARC TP53 somatic mutation database. Gene copy numbers in tumors were identified using the Ion Reporter system from Thermo-Fisher Scientific and called as copy number loss, normal, or gain based upon a comparison to a reference range established from the normal blood samples. Survival analyses were generated using the Log-Rank and Kaplan-Meier methods. Results: 84% (64/76) of our TNBC tumors harbored a somatic mutation in the TP53 gene. The majority were missense mutations (particularly in the DNA binding and tetramerization domains) followed by frameshift insertions/deletions, and copy number loss. Patients whose tumors harbored somatic TP53 mutations were observed to have a significantly inferior disease free survival (DFS) compared to non-mutated tumors (events = 29/64 vs. 1/12; median = 25.9 mos vs. NR (Not Reached); p=0.021, HR=7.28 (95% C.I.: 2.98-17.79). The same was observed for overall survival (OS) (events = 23/64 vs. 0/12, median = 33.78 vs. NR; p=0.017, HR = Not evaluable). There was no difference in DFS or OS when comparing the nature of the mutation (point mutation vs. indel vs. copy loss) at a p=0.88 and p=0.91, respectively. We then sought to determine if clonal status of TP53 mutations was also associated with survival. Cases were divided into non-mutated, subclonal (mutations present in a fraction of cells), or truncal (mutation present in most or all cells). Interestingly, tumors that harbored subclonal TP53 mutations had a superior OS compared to truncal mutations (events = 1/9 vs. 22/55; median = NR vs. 29.1, p=0.036, HR =0.16 (95% C.I.:0.06-0.42). OS for subclonal mutations was highly similar to non-mutated tumors (p=0.25). Conclusions: While RD after neoadjuvant chemotherapy in TNBC is a well-known risk factor for poor prognosis, in our study, we observed a subset of RD patients defined by a lack of TP53 mutation or presence of a subclonal mutation that portended a superior survival outcome after post-neoadjuvant Cisplatin. If validated, these results reveal that the presence and clonal status of TP53 mutations is important to accurate prognostication and should be considered in decision-making algorithms for this patient population. Citation Format: Hancock BA, Chen Y-H, Solzak JP, Miller KD, Radovich M. TP53 mutation is a biomarker for prognosis in triple-negative breast cancer patients treated with post-neoadjuvant cisplatin. [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 P2-08-23.