Jeffrey P. Solzak

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.

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.

Abstract P2-01-01: Next generation sequencing of circulating tumor DNA to predict recurrence in triple-negative breast cancer patients with residual disease after neoadjuvant chemotherapy

Background: Incorporation of next-generation sequencing to detect plasma-derived tumor DNA (ptDNA) is emerging as a popular method for tumor genotyping and for monitoring therapeutic response. The vast majority of studies so far have focused on detecting ptDNA from patients with metastatic disease. Herein, we tested whether ptDNA could be used as a biomarker to predict relapse in triple-negative breast cancer (TNBC) patients with residual disease after neoadjuvant chemotherapy and surgery. Methods: BRE09-146 was a Phase II clinical trial that randomized TNBC patients with residual disease after neoadjuvant chemotherapy to Cisplatin or Cisplatin+Rucaparib. From the combination arm, 1ml of plasma was collected at four predefined time points post-surgery. In total, 39 patients with matched tumor, blood, and plasma were analyzed. Extracted DNA underwent library preparation and amplification using the Ion Ampliseq Oncomine Research Panel which consists of 134 cancer genes that are well-known to be mutated in cancer. Samples were then sequenced on an Ion Proton next-generation sequencer to at least 2500X coverage followed by bioinformatic analyses using the Torrent Variant Caller. Results: We first detected high-quality somatic mutations in primary tumors. TP53 mutations were the most prevalent (70%) followed by AKT1 (8%). Somatic mutation frequencies in our trial were congruent with publically-available mutation data of TNBCs from The Cancer Genome Atlas. Using these somatic mutations, we then analyzed the plasma-sequencing data to detect the same mutations in the circulation. Out of 39 patients, 14 patients had a clinical relapse (median follow-up for disease free survival = 24 months). Of the 14 patients, we were able to detect somatic ptDNA in 4 patients (3 TP53 mutations, 1 AKT mutation). Notably, all 4 patients had a rapid recurrence (0.3, 4.0, 5.3, and 8.9 months). ptDNA-sequencing was unable to detect distant recurrence. The combination of a paucity of ptDNA molecules in the circulation of patients who have no evidence of disease along with a limited amount of plasma available per patient are potential factors for the inability to detect distant recurrence. Conclusions: Next-generation ptDNA-sequencing of triple-negative breast cancer patients after neoadjuvant chemotherapy and surgery can detect rapid-recurrence but sensitivity to detect distant recurrence is limited. Studies to increase sensitivity by incorporating mutation calling from ptRNA along with ptDNA are currently underway. Citation Format: Chen Y-H, Hancock BA, Solzak JP, Miller KD, Radovich M. Next generation sequencing of circulating tumor DNA to predict recurrence in triple-negative breast cancer patients with residual disease after neoadjuvant chemotherapy. [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 P2-01-01.

Abstract 4403: Next-generation RNA sequencing identifies UHRF1 as a key regulatory protein in triple-negative breast cancer survival.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Introduction: Triple-negative breast cancer (TNBC) is a particularly aggressive form of breast cancer that presently lacks specific treatment. Using next-generation RNA sequencing of 94 TNBC tumors and 20 normal breast tissues, we have identified UHRF1, an E3 ubiquitin ligase, as significantly overexpressed. UHRF1 is involved in DNA methylation, is upregulated via mutated p53, and plays a role in the repression of RB1 & BRCA1. Epigallocatechin gallate (EGCG), a polyphenol found in green tea, has been previously investigated in its role of downregulating UHRF1. Methods: cDNA libraries from 20 normal breast tissues from the Susan G. Komen Tissue Bank at the IU Simon Cancer Center and 10 TNBC tumors were sequenced on an Applied Biosystems SOLiD3 sequencer. Mapping of reads to the human genome (hg19) was performed using the LifeScope software. RNA-seq data from an additional 84 TNBCs was downloaded from The Cancer Genome Atlas (TCGA) data portal. Differential gene expression was analyzed using Partek Genomics Suite; and network, pathway, and transcription factor analysis was performed using Ingenuity Systems IPA 9.0. To investigate the effects of EGCG a known downregulator of UHRF1 in vitro, nine TNBC cell lines were utilized representing the known TNBC molecular subtypes. TNBC cell lines were chosen based on differing subtypes which include different genetic and morphological characteristics. Cells were seeded in 96 well plates at 10,000 cells per well and dosed with increasing concentrations of EGCG from 0-400μM, in increments of 50μM. CellTiter-Glo was used to assess the viability of all cells lines after 72 hours. The IC50 was ascertained using percent changes from control luminosity of untreated cells. Results: Transcription factor analysis of RNA-seq data identified RB1 activity as significantly inhibited in TNBC. A search for upstream inhibitors identified UHRF1 as highly overexpressed in TNBC (Fold-change =7.96, p-value=8.34x10−6). Further network analysis revealed that upregulation of UHRF1 is mediated by mutated p53 (which is mutated >80% of TNBCs) and is an inhibitor of BRCA1 which is known to be suppressed in TNBC. Using a publically available gene expression database, high expression of UHRF1 is correlated with poor survival in breast cancer (p<0.001). As EGCG is a known downregulator of UHRF1, we tested EGCG on nine TNBC cell lines and assessed for cell viability. EGCG resulted in significant cell death with a median LD50 of 109μM. Furthermore, in two of the cell lines, HCC70 and HCC1143, 100% cell death was observed at 250μM. Conclusions: UHRF1 is a key member of the p53/RB1/BRCA1 network and is important in TNBC survival. EGCG, a natural compound that downregulates UHRF1, significantly decreases the viability of TNBC cell lines in-vitro. Investigation into more potent small molecule inhibitors of UHRF1 is currently underway. Citation Format: Jeffrey P. Solzak, Rutuja Atale, Milan Radovich. Next-generation RNA sequencing identifies UHRF1 as a key regulatory protein in triple-negative breast cancer survival. [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 4403. doi:10.1158/1538-7445.AM2013-4403