Detection of breast cancer stem cell gene mutations in circulating free DNA during the evolution of metastases.

PURPOSE: Limited knowledge exists on the detection of breast cancer stem cell (BCSC)-related mutations in circulating free DNA (cfDNA) from patients with advanced cancers. Identification of new cancer biomarkers may allow for earlier detection of disease progression and treatment strategy modifications. METHODS: We conducted a prospective study to determine the feasibility and prognostic utility of droplet digital polymerase chain reaction (ddPCR)-based BCSC gene mutation analysis of cfDNA in patients with breast cancer. RESULTS: Detection of quantitative BCSC gene mutation in cfDNA by ddPCR mirrors disease progression and thus may represent a valuable and cost-effective measure of tumor burden. We have previously shown that hematological and neurological expressed 1-like (HN1L), ribosomal protein L39 (RPL39), and myeloid leukemia factor 2 (MLF2) are novel targets for BCSC self-renewal, and targeting these genetic alterations could be useful for personalized genomic-based therapy. CONCLUSION: BCSC mutation detection in cfDNA may have important implications for diagnosis, prognosis, and serial monitoring.

Novel algorithmic approach predicts tumor mutation load and correlates with immunotherapy clinical outcomes using a defined gene mutation set.

BACKGROUND: While clinical outcomes following immunotherapy have shown an association with tumor mutation load using whole exome sequencing (WES), its clinical applicability is currently limited by cost and bioinformatics requirements. METHODS: We developed a method to accurately derive the predicted total mutation load (PTML) within individual tumors from a small set of genes that can be used in clinical next generation sequencing (NGS) panels. PTML was derived from the actual total mutation load (ATML) of 575 distinct melanoma and lung cancer samples and validated using independent melanoma (n = 312) and lung cancer (n = 217) cohorts. The correlation of PTML status with clinical outcome, following distinct immunotherapies, was assessed using the Kaplan-Meier method. RESULTS: PTML (derived from 170 genes) was highly correlated with ATML in cutaneous melanoma and lung adenocarcinoma validation cohorts (R2 = 0.73 and R2 = 0.82, respectively). PTML was strongly associated with clinical outcome to ipilimumab (anti-CTLA-4, three cohorts) and adoptive T-cell therapy (1 cohort) clinical outcome in melanoma. Clinical benefit from pembrolizumab (anti-PD-1) in lung cancer was also shown to significantly correlate with PTML status (log rank P value < 0.05 in all cohorts). CONCLUSIONS: The approach of using small NGS gene panels, already applied to guide employment of targeted therapies, may have utility in the personalized use of immunotherapy in cancer.

Dynamic clonal remodelling in breast cancer metastases is associated with subtype conversion.

BACKGROUND: Changes in the clinical subtype (CS) and intrinsic subtype (IS) between breast cancer (BC) metastases and corresponding primary tumours have been reported. However, their relationship with tumour genomic changes remains poorly characterised. Here, we analysed the association between genomic remodelling and subtype conversion in paired primary and metastatic BC samples. METHODS: A total of 57 paired primary and metastatic tumours from GEICAM/2009-03 (ConvertHER, NCT01377363) study participants with centrally assessed CS (n = 57) and IS (n = 46) were analysed. Targeted capture and next-generation sequencing of 202 genes on formalin-fixed paraffin-embedded samples was performed. The cancer cell fraction (CCF) of mutations in primary and metastatic pairs was estimated as a surrogate of tumour clonal architecture. Changes in mutation CCF between matched primary and metastatic tumours were analysed in the presence or absence of subtype conversion. FINDINGS: CS conversion occurred in 24.6% and IS conversion occurred in 36.9% of metastases. Primary tumours and metastases had a median of 11 (range, 3-29) and 9 (range, 1-38) mutations, respectively (P = 0.05). Overall, mutations in metastases showed a higher estimated CCF than in primary tumours (median CCF, 0.51 and 0.47, respectively; P = 0.042), consistent with increased clonal homogeneity. The increase in mutation CCF was significant in CS-converted (P = 0.04) but not in IS-converted (P = 0.48) metastases. Clonal remodelling was highest in metastases from hormone receptor-positive and human epidermal growth factor 2 (HER2)-positive tumours (P = 0.006). CONCLUSIONS: Mutations in BC metastases showed significantly higher estimated CCF than primary tumours. CCF changes were more prominent in metastases with CS conversion. Our findings suggest that changes in BC subtypes are linked to clonal remodelling during BC evolution.

Differential expression of 12 histone deacetylase (HDAC) genes in astrocytomas and normal brain tissue: class II and IV are hypoexpressed in glioblastomas.

BACKGROUND: Glioblastoma is the most lethal primary malignant brain tumor. Although considerable progress has been made in the treatment of this aggressive tumor, the clinical outcome for patients remains poor. Histone deacetylases (HDACs) are recognized as promising targets for cancer treatment. In the past several years, HDAC inhibitors (HDACis) have been used as radiosensitizers in glioblastoma treatment. However, no study has demonstrated the status of global HDAC expression in gliomas and its possible correlation to the use of HDACis. The purpose of this study was to evaluate and compare mRNA and protein levels of class I, II and IV of HDACs in low grade and high grade astrocytomas and normal brain tissue and to correlate the findings with the malignancy in astrocytomas. METHODS: Forty-three microdissected patient tumor samples were evaluated. The histopathologic diagnoses were 20 low-grade gliomas (13 grade I and 7 grade II) and 23 high-grade gliomas (5 grade III and 18 glioblastomas). Eleven normal cerebral tissue samples were also analyzed (54 total samples analyzed). mRNA expression of class I, II, and IV HDACs was studied by quantitative real-time polymerase chain reaction and normalized to the housekeeping gene beta-glucuronidase. Protein levels were evaluated by western blotting. RESULTS: We found that mRNA levels of class II and IV HDACs were downregulated in glioblastomas compared to low-grade astrocytomas and normal brain tissue (7 in 8 genes, p < 0.05). The protein levels of class II HDAC9 were also lower in high-grade astrocytomas than in low-grade astrocytomas and normal brain tissue. Additionally, we found that histone H3 (but not histone H4) was more acetylated in glioblastomas than normal brain tissue. CONCLUSION: Our study establishes a negative correlation between HDAC gene expression and the glioma grade suggesting that class II and IV HDACs might play an important role in glioma malignancy. Evaluation of histone acetylation levels showed that histone H3 is more acetylated in glioblastomas than normal brain tissue confirming the downregulation of HDAC mRNA in glioblastomas.

A Population of Heterogeneous Breast Cancer Patient-Derived Xenografts Demonstrate Broad Activity of PARP Inhibitor in BRCA1/2 Wild-Type Tumors.

Background: Breast cancer patients who do not respond to neoadjuvant therapy have a poor prognosis. There is a pressing need for novel targets and models for preclinical testing. Here we report characterization of breast cancer patient-derived xenografts (PDX) largely generated from residual tumors following neoadjuvant chemotherapy.Experimental Design: PDXs were derived from surgical samples of primary or locally recurrent tumors. Normal and tumor DNA sequencing, RNASeq, and reverse phase protein arrays (RPPA) were performed. Phenotypic profiling was performed by determining efficacy of a panel of standard and investigational agents.Results: Twenty-six PDXs were developed from 25 patients. Twenty-two were generated from residual disease following neoadjuvant chemotherapy, and 24 were from triple-negative breast cancer (TNBC). These PDXs harbored a heterogeneous set of genomic alterations and represented all TNBC molecular subtypes. On RPPA, PDXs varied in extent of PI3K and MAPK activation. PDXs also varied in their sensitivity to chemotherapeutic agents. PI3K, mTOR, and MEK inhibitors repressed growth but did not cause tumor regression. The PARP inhibitor talazoparib caused dramatic regression in five of 12 PDXs. Notably, four of five talazoparib-sensitive models did not harbor germline BRCA1/2 mutations, but several had somatic alterations in homologous repair pathways, including ATM deletion and BRCA2 alterations.Conclusions: PDXs capture the molecular and phenotypic heterogeneity of TNBC. Here we show that PARP inhibition can have activity beyond germline BRCA1/2 altered tumors, causing regression in a variety of molecular subtypes. These models represent an opportunity for the discovery of rational combinations with targeted therapies and predictive biomarkers. Clin Cancer Res; 23(21); 6468-77. ©2017 AACR.

ClinSeK: a targeted variant characterization framework for clinical sequencing.

Applying genomics to patient care demands sensitive, unambiguous and rapid characterization of a known set of clinically relevant variants in patients' samples, an objective substantially different from the standard discovery process, in which every base in every sequenced read must be examined. Further, the approach must be sufficiently robust as to be able to detect multiple and potentially rare variants from heterogeneous samples. To meet this critical objective, we developed a novel variant characterization framework, ClinSeK, which performs targeted analysis of relevant reads from high-throughput sequencing data. ClinSeK is designed for efficient targeted short read alignment and is capable of characterizing a wide spectrum of genetic variants from single nucleotide variation to large-scale genomic rearrangement breakpoints. Applying ClinSeK to over a thousand cancer patients demonstrated substantively better performance, in terms of accuracy, runtime and disk storage, for clinical applications than existing variant discovery tools. ClinSeK is freely available for academic use at http://bioinformatics.mdanderson.org/main/clinsek.

Functional consequence of the MET-T1010I polymorphism in breast cancer.

Major breast cancer predisposition genes, only account for approximately 30% of high-risk breast cancer families and only explain 15% of breast cancer familial relative risk. The HGF growth factor receptor MET is potentially functionally altered due to an uncommon germline single nucleotide polymorphism (SNP), MET-T1010I, in many cancer lineages including breast cancer where the MET-T1010I SNP is present in 2% of patients with metastatic breast cancer. Expression of MET-T1010I in the context of mammary epithelium increases colony formation, cell migration and invasion in-vitro and tumor growth and invasion in-vivo. A selective effect of MET-T1010I as compared to wild type MET on cell invasion both in-vitro and in-vivo suggests that the MET-T1010I SNP may alter tumor pathophysiology and should be considered as a potential biomarker when implementing MET targeted clinical trials.

Mediators of glioblastoma resistance and invasion during antivascular endothelial growth factor therapy.

PURPOSE: Vascular endothelial growth factor (VEGF) has been identified as a critical regulator of angiogenesis. Currently, several different strategies are being used to target the VEGF-VEGF receptor signal transduction pathway in glioblastoma. Although anti-VEGF therapy seems be effective in normalizing abnormal tumor vasculature, leading to an enhanced response to radiation and chemotherapy, tumors eventually become resistant to the therapy and adopt a highly infiltrative and invasive phenotype. EXPERIMENTAL DESIGN: In the present study, we evaluated the effects of anti-VEGF therapy (bevacizumab) on glioblastoma invasion both in vitro and in vivo and evaluated the angiogenesis- and invasion-related mediators of developed resistance to this therapy. RESULTS: We found that glioblastoma tumors escaped from antiangiogenic treatment by (a) reactivating angiogenesis through up-regulation of other proangiogenic factors and (b) invading normal brain areas, which was seen in association with up-regulation of matrix metalloproteinase (MMP)-2, MMP-9, and MMP-12; secreted protein, acidic, cysteine-rich; and tissue inhibitor of metalloproteinase 1. In addition to the paracrine effects of VEGF on endothelial cells, autocrine VEGF signaling seemed to regulate glioblastoma invasion because anti-VEGF therapy increased tumor invasiveness in vitro. CONCLUSIONS: Collectively, these findings reinforce the importance of VEGF in regulating tumor invasion and identify potential mediators of resistance to targeted VEGF therapy. These results will be important for developing novel combination therapies to overcome this resistance phenotype.

Gene expression profile analysis of primary glioblastomas and non-neoplastic brain tissue: identification of potential target genes by oligonucleotide microarray and real-time quantitative PCR.

The prognosis of glioblastomas is still extremely poor and the discovery of novel molecular therapeutic targets can be important to optimize treatment strategies. Gene expression analyses comparing normal and neoplastic tissues have been used to identify genes associated with tumorigenesis and potential therapeutic targets. We have used this approach to identify differentially expressed genes between primary glioblastomas and non-neoplastic brain tissues. We selected 20 overexpressed genes related to cell cycle, cellular movement and growth, proliferation and cell-to-cell signaling and analyzed their expression levels by real time quantitative PCR in cDNA obtained from microdissected fresh tumor tissue from 20 patients with primary glioblastomas and from 10 samples of non-neoplastic white matter tissue. The gene expression levels were significantly higher in glioblastomas than in non-neoplastic white matter in 18 out of 20 genes analyzed: P < 0.00001 for CDKN2C, CKS2, EEF1A1, EMP3, PDPN, BNIP2, CA12, CD34, CDC42EP4, PPIE, SNAI2, GDF15 and MMP23b; and NFIA (P: 0.0001), GPS1 (P: 0.0003), LAMA1 (P: 0.002), STIM1 (P: 0.006), and TASP1 (P: 0.01). Five of these genes are located in contiguous loci at 1p31-36 and 2 at 17q24-25 and 8 of them encode surface membrane proteins. PDPN and CD34 protein expression were evaluated by immunohistochemistry and they showed concordance with the PCR results. The present results indicate the presence of 18 overexpressed genes in human primary glioblastomas that may play a significant role in the pathogenesis of these tumors and that deserve further functional investigation as attractive candidates for new therapeutic targets.