A B S T R A C T PurposeWe report the experience with 2,000 consecutive patients with advanced cancer who underwent testing on a genomic testing protocol, including the frequency of actionable alterations across tumor types, subsequent enrollment onto clinical trials, and the challenges for trial enrollment. Patients and MethodsStandardized hotspot mutation analysis was performed in 2,000 patients, using either an 11-gene (251 patients) or a 46-or 50-gene (1,749 patients) multiplex platform. Thirty-five genes were considered potentially actionable based on their potential to be targeted with approved or investigational therapies. ResultsSeven hundred eighty-nine patients (39%) had at least one mutation in potentially actionable genes. Eighty-three patients (11%) with potentially actionable mutations went on genotypematched trials targeting these alterations. Of 230 patients with PIK3CA/AKT1/PTEN/BRAF mutations that returned for therapy, 116 (50%) received a genotype-matched drug. Forty patients (17%) were treated on a genotype-selected trial requiring a mutation for eligibility, 16 (7%) were treated on a genotype-relevant trial targeting a genomic alteration without biomarker selection, and 40 (17%) received a genotype-relevant drug off trial. Challenges to trial accrual included patient preference of noninvestigational treatment or local treatment, poor performance status or other reasons for trial ineligibility, lack of trials/slots, and insurance denial. ConclusionBroad implementation of multiplex hotspot testing is feasible; however, only a small portion of patients with actionable alterations were actually enrolled onto genotype-matched trials. Increased awareness of therapeutic implications and access to novel therapeutics are needed to optimally leverage results from broad-based genomic testing.

Rapidly improving understanding of molecular oncology, emerging novel therapeutics, and increasingly available and affordable next-generation sequencing have created an opportunity for delivering genomically informed personalized cancer therapy. However, to implement genomically informed therapy requires that a clinician interpret the patient's molecular profile, including molecular characterization of the tumor and the patient's germline DNA. In this Commentary, we review existing data and tools for precision oncology and present a framework for reviewing the available biomedical literature on therapeutic implications of genomic alterations. Genomic alterations, including mutations, insertions/deletions, fusions, and copy number changes, need to be curated in terms of the likelihood that they alter the function of a "cancer gene" at the level of a specific variant in order to discriminate so-called "drivers" from "passengers." Alterations that are targetable either directly or indirectly with approved or investigational therapies are potentially "actionable." At this time, evidence linking predictive biomarkers to therapies is strong for only a few genomic markers in the context of specific cancer types. For these genomic alterations in other diseases and for other genomic alterations, the clinical data are either absent or insufficient to support routine clinical implementation of biomarker-based therapy. However, there is great interest in optimally matching patients to early-phase clinical trials. Thus, we need accessible, comprehensive, and frequently updated knowledge bases that describe genomic changes and their clinical implications, as well as continued education of clinicians and patients.

Background: Next-generation sequencing in cancer research may reveal germline variants of clinical significance. We report patient preferences for return of results and the prevalence of incidental pathogenic germline variants (PGVs).Patients and methods: Targeted exome sequencing of 202 genes was carried out in 1000 advanced cancers using tumor and normal DNA in a research laboratory. Pathogenic variants in 18 genes, recommended for return by The American College of Medical Genetics and Genomics, as well as PALB2, were considered actionable. Patient preferences of return of incidental germline results were collected. Return of results was initiated with genetic counseling and repeat CLIA testing.Results: Of the 1000 patients who underwent sequencing, 43 had likely PGVs: APC (1), BRCA1 (11), BRCA2 (10), TP53 (10), MSH2 (1), MSH6 (4), PALB2 (2), PTEN (2), TSC2 (1), and RB1 (1). Twenty (47%) of 43 variants were previously known based on clinical genetic testing. Of the 1167 patients who consented for a germline testing protocol, 1157 (99%) desired to be informed of incidental results. Twenty-three previously unrecognized mutations identified in the research environment were confirmed with an orthogonal CLIA platform. All patients approached decided to proceed with formal genetic counseling; in all cases where formal genetic testing was carried out, the germline variant of concern validated with clinical genetic testing. Conclusions:In this series, 2.3% patients had previously unrecognized pathogenic germline mutations in 19 cancerrelated genes. Thus, genomic sequencing must be accompanied by a plan for return of germline results, in partnership with genetic counseling.

Estrogens play a crucial role in regulating the growth and differentiation of breast cancers, with approximately two-thirds of all breast tumours expressing the estrogen receptor alpha (ERα). Therefore, therapeutic strategies directed at inhibiting the action of ERα by using antiestrogens such as tamoxifen, or reducing estrogens levels by using aromatase inhibitors (AIs), such as letrozole, anastrozole, or exemestane, are the standard treatments offered to women with ERα-positive cancer. However, not all patients respond to endocrine therapies (termed de novo resistance), and a large number of patients who do respond will eventually develop disease progression or recurrence while on therapy (acquired resistance). Recently variant forms of the receptor due to alternative splicing or gene mutation have been identified. This article reviews these variant receptors and their clinical relevance in resistance to endocrine therapy, by addressing their molecular cross-talk with growth factor receptors and signaling components. Understanding the complexity of receptor-mediated signaling has promise for new combined therapeutic options which focus on more efficient blockade of receptor cross-talk.

Loss of Rho GDIα enhances metastasis and resistance to tamoxifen via effects on both ERα and MTA2 in models of ERα-positive breast cancer and in tumors of tamoxifen-treated patients.

Purpose Tamoxifen (Tam) resistance represents a significant clinical problem in estrogen receptor (ER) -positive breast cancer. We previously showed that decreased expression of Rho guanine nucleotide dissociation inhibitor (Rho GDI), a negative regulator of the Rho GTPase pathway, is associated with Tam resistance. We now discover that androgen receptor (AR) is overexpressed in cells with decreased Rho GDI and seek to determine AR’s contribution to resistance. Methods We engineered ER -positive cell lines with stable knock-down (KD) of Rho GDI (KD cells). Resistance mechanisms were examined using microarray profiling, protein-interaction studies, growth and reporter gene assays, and Western blot analysis combined with a specific AR antagonist and other signaling inhibitors. Results Tam-resistant tumors and cell lines with low Rho GDI levels exhibited upregulated AR expression. Microarray of Rho GDI KD cells indicated that activation of EGFR and ER was associated with Tam treatment. When AR levels were elevated interaction between AR and EGFR was detected. Constitutive and Tam-induced phosphorylation of EGFR and ERK1/2 was blocked by the AR antagonist Enzalutamide, suggesting that AR-mediated EGFR activation was a mechanism of resistance in these cells. Constitutive ERα phosphorylation and transcriptional activity was inhibited by Enzalutamide and the EGFR inhibitor gefitinib, demonstrating that AR-mediated EGFR signaling activated ER. Tam exhibited agonist activity in AR over-expressing cells, stimulating ERα transcriptional activity and proliferation, which was blocked by Enzalutamide and gefitinib. Conclusions We describe a novel model of AR-mediated Tam resistance through activation of EGFR signaling leading to ER activation in ER -positive cells with low expression of Rho GDI.

PurposeWe determined the frequency of recurrent hotspot mutations in 46 cancer-related genes across tumor histologies in patients with advanced cancer.MethodsWe reviewed data from 500 consecutive patients who underwent genomic profiling on an IRB-approved prospective clinical protocol in the Phase I program at the MD Anderson Cancer Center. Archival tumor DNA was tested for 740 hotspot mutations in 46 genes (Ampli-Seq Cancer Panel; Life Technologies, CA).ResultsOf the 500 patients, 362 had at least one reported mutation/variant. The most common likely somatic mutations were within TP53 (36%), KRAS (11%), and PIK3CA (9%) genes. Sarcoma (20%) and kidney (30%) had the lowest proportion of likely somatic mutations detected, while pancreas (100%), colorectal (89%), melanoma (86%), and endometrial (75%) had the highest. There was high concordance in 62 patients with paired primary tumors and metastases analyzed. 151 (30%) patients had alterations in potentially actionable genes. 37 tumor types were enrolled; both rare actionable mutations in common tumor types and actionable mutations in rare tumor types were identified.ConclusionMultiplex testing in the CLIA environment facilitates genomic characterization across multiple tumor lineages and identification of novel opportunities for genotype-driven trials.

Purpose: We sought to determine the significant genomic alterations in patients with metastatic breast cancer (MBC), and survival outcomes in common genotypes. Patients and Methods: High-depth next generation sequencing was performed for 202 genes in tumor and normal DNA from 257 patients with MBC, including 165 patients with ER/PR+ HER2- (hormone receptor positive, HR+ positive), 32 patients with HER2+ and 60 patients with triple negative (ER/PR/HER2-) cancer. Kaplan Meier survival analysis was performed in our discovery set, in breast cancer patients analyzed in The Cancer Genome Atlas, and in a separate cohort of 98 patients with MBC who underwent clinical genomic testing. Results: Significantly mutated genes (SMGs) varied by histology and tumor subtype, but TP53 was a SMG in all three subtypes. The most SMGs in HR+ patients included PIK3CA (32%), TP53 (29%), GATA3 (15%), CDH1 (8%), MAP3K1 (8%), PTEN (5%), TGFBR2 (4%), AKT1 (4%), and MAP2K4 (4%). TP53 mutations were associated with shorter recurrence-free survival (P=0.004), progression-free survival (P=0.00057) and overall survival (P=0.003). Further, TP53 status was prognostic among HR+ patients with PIK3CA mutations. TP53 mutations were also associated with poorer overall survival in the 442 HR+ breast cancer patients in the TCGA (P=0.042) and in an independent set of 96 HR+ MBC who underwent clinical sequencing (P=0.0004). Conclusions: SMGs differ by tumor subtype but TP53 is significantly mutated in all three breast cancer subtypes. TP53 mutations are associated with poor prognosis in HR+ breast cancer. TP53 mutations should be considered in the design and interpretation of precision oncology trials.