Duvelisib, an oral dual PI3K-δ,γ inhibitor, shows clinical and pharmacodynamic activity in chronic lymphocytic leukemia and small lymphocytic lymphoma in a phase 1 study.

Duvelisib (IPI-145), an oral, dual inhibitor of phosphoinositide-3-kinase (PI3K)-δ and -γ, was evaluated in a Phase 1 study in advanced hematologic malignancies, which included expansion cohorts in relapsed/refractory (RR) chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL) and treatment-naïve (TN) CLL. Per protocol, TN patients were at least 65 years old or had a del(17p)/TP53 mutation. Duvelisib was administered twice daily (BID) in 28-day cycles at doses of 8-75 mg in RR patients (n = 55) and 25 mg in TN patients (n = 18.) Diarrhea was the most common nonhematologic AE (TN 78%, RR 47%); transaminase elevations the most frequent lab-abnormality AE (TN 33.3%, RR 30.9%); and neutropenia the most common ≥grade 3 AE (RR 44%, TN 33%). The overall response rates were 56.4% for RR patients (1.8% CR, 54.5% PR) and 83.3% for TN patients (all PRs); median response duration was 21.0 months in RR patients but was not reached for TN patients. Based upon phase 1 efficacy, pharmacodynamics, and safety, duvelisib 25 mg BID was selected for further investigation in a phase 3 study in RR CLL/SLL.

Diverse somatic mutation patterns and pathway alterations in human cancers.

The systematic characterization of somatic mutations in cancer genomes is essential for understanding the disease and for developing targeted therapeutics. Here we report the identification of 2,576 somatic mutations across approximately 1,800 megabases of DNA representing 1,507 coding genes from 441 tumours comprising breast, lung, ovarian and prostate cancer types and subtypes. We found that mutation rates and the sets of mutated genes varied substantially across tumour types and subtypes. Statistical analysis identified 77 significantly mutated genes including protein kinases, G-protein-coupled receptors such as GRM8, BAI3, AGTRL1 (also called APLNR) and LPHN3, and other druggable targets. Integrated analysis of somatic mutations and copy number alterations identified another 35 significantly altered genes including GNAS, indicating an expanded role for galpha subunits in multiple cancer types. Furthermore, our experimental analyses demonstrate the functional roles of mutant GNAO1 (a Galpha subunit) and mutant MAP2K4 (a member of the JNK signalling pathway) in oncogenesis. Our study provides an overview of the mutational spectra across major human cancers and identifies several potential therapeutic targets.

The effects of hepatitis B virus integration into the genomes of hepatocellular carcinoma patients.

Hepatitis B virus (HBV) infection is a leading risk factor for hepatocellular carcinoma (HCC). HBV integration into the host genome has been reported, but its scale, impact and contribution to HCC development is not clear. Here, we sequenced the tumor and nontumor genomes (>80× coverage) and transcriptomes of four HCC patients and identified 255 HBV integration sites. Increased sequencing to 240× coverage revealed a proportionally higher number of integration sites. Clonal expansion of HBV-integrated hepatocytes was found specifically in tumor samples. We observe a diverse collection of genomic perturbations near viral integration sites, including direct gene disruption, viral promoter-driven human transcription, viral-human transcript fusion, and DNA copy number alteration. Thus, we report the most comprehensive characterization of HBV integration in hepatocellular carcinoma patients. Such widespread random viral integration will likely increase carcinogenic opportunities in HBV-infected individuals.

Genome and transcriptome sequencing of lung cancers reveal diverse mutational and splicing events.

Lung cancer is a highly heterogeneous disease in terms of both underlying genetic lesions and response to therapeutic treatments. We performed deep whole-genome sequencing and transcriptome sequencing on 19 lung cancer cell lines and three lung tumor/normal pairs. Overall, our data show that cell line models exhibit similar mutation spectra to human tumor samples. Smoker and never-smoker cancer samples exhibit distinguishable patterns of mutations. A number of epigenetic regulators, including KDM6A, ASH1L, SMARCA4, and ATAD2, are frequently altered by mutations or copy number changes. A systematic survey of splice-site mutations identified 106 splice site mutations associated with cancer specific aberrant splicing, including mutations in several known cancer-related genes. RAC1b, an isoform of the RAC1 GTPase that includes one additional exon, was found to be preferentially up-regulated in lung cancer. We further show that its expression is significantly associated with sensitivity to a MAP2K (MEK) inhibitor PD-0325901. Taken together, these data present a comprehensive genomic landscape of a large number of lung cancer samples and further demonstrate that cancer-specific alternative splicing is a widespread phenomenon that has potential utility as therapeutic biomarkers. The detailed characterizations of the lung cancer cell lines also provide genomic context to the vast amount of experimental data gathered for these lines over the decades, and represent highly valuable resources for cancer biology.

Disruption of PH-kinase domain interactions leads to oncogenic activation of AKT in human cancers.

The protein kinase v-akt murine thymoma viral oncogene homolog (AKT), a key regulator of cell survival and proliferation, is frequently hyperactivated in human cancers. Intramolecular pleckstrin homology (PH) domain-kinase domain (KD) interactions are important in maintaining AKT in an inactive state. AKT activation proceeds after a conformational change that dislodges the PH from the KD. To understand these autoinhibitory interactions, we generated mutations at the PH-KD interface and found that most of them lead to constitutive activation of AKT. Such mutations are likely another mechanism by which activation may occur in human cancers and other diseases. In support of this likelihood, we found somatic mutations in AKT1 at the PH-KD interface that have not been previously described in human cancers. Furthermore, we show that the AKT1 somatic mutants are constitutively active, leading to oncogenic signaling. Additionally, our studies show that the AKT1 mutants are not effectively inhibited by allosteric AKT inhibitors, consistent with the requirement for an intact PH-KD interface for allosteric inhibition. These results have important implications for therapeutic intervention in patients with AKT mutations at the PH-KD interface.

Immunohistochemistry and fluorescence in situ hybridization assessment of HER2 in clinical trials of adjuvant therapy for breast cancer (NCCTG N9831, BCIRG 006, and BCIRG 005).

A comprehensive, blinded, pathology evaluation of HER2 testing in HER2-positive/negative breast cancers was performed among three central laboratories. Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) analyses were performed on 389 tumor blocks from three large adjuvant trials: N9831, BCIRG-006, and BCIRG-005. In 123 cases, multiple blocks were examined. HER2 status was defined according to FDA-approved guidelines and was independently re-assessed at each site. Discordant cases were adjudicated at an on-site, face-to-face meeting. Results across three independent pathologists were concordant by IHC in 351/381 (92 %) and FISH in 343/373 (92 %) blocks. Upon adjudication, consensus was reached on 16/30 and 18/30 of discordant IHC and FISH cases, respectively, resulting in overall concordance rates of 96 and 97 %. Among 155 HER2-negative blocks, HER2 status was confirmed in 153 (99 %). In the subset of 102 HER2-positive patients from N9831/BCIRG-006, primary blocks from discordant cases were selected, especially those with discordant test between local and central laboratories. HER2 status was confirmed in 73 (72 %) of these cases. Among 118 and 113 cases with IHC and FISH results and >1 block evaluable, block-to-block variability/heterogeneity in HER2 results was seen in 10 and 5 %, respectively. IHC-/FISH- was confirmed for 57/59 (97 %) primary blocks from N9831 (locally positive, but centrally negative); however, 5/22 (23 %) secondary blocks showed HER2 positivity. Among 53 N9831 patients with HER2-normal disease adjudicated as IHC-/FISH-(although locally positive), there was a non-statistically significant improvement in disease-free survival with concurrent trastuzumab compared to chemotherapy alone (adjusted hazard ratio 0.34; 95 % CI, 0.11-1.05; p = 0.06). There were similar agreements for IHC and FISH among pathologists (92 % each). Agreement was improved at adjudication (96 %). HER2 tumor heterogeneity appears to partially explain discordant results in cases initially tested as positive and subsequently called negative.

Cancer genetics and drug discovery in the zebrafish.

Fish have a long history of use in cancer toxicology studies, because they develop neoplasms that are histologically similar to human cancers. Because of considerable progress in zebrafish genetics and genomics over the past few years, the zebrafish system has provided many useful tools for studying basic biological processes. These tools include forward genetic screens, transgenic models, specific gene disruptions and small-molecule screens. By combining carcinogenesis assays, genetic analyses and small-molecule screening techniques, the zebrafish is emerging as a powerful system for identifying novel cancer genes and for cancer drug discovery.

Zebrafish as a cancer model system.

The zebrafish, with its combination of forward genetics and vertebrate biology, has great potential as a cancer model system.

Genetic alterations and oncogenic pathways associated with breast cancer subtypes.

Breast cancers can be divided into subtypes with important implications for prognosis and treatment. We set out to characterize the genetic alterations observed in different breast cancer subtypes and to identify specific candidate genes and pathways associated with subtype biology. mRNA expression levels of estrogen receptor, progesterone receptor, and HER2 were shown to predict marker status determined by immunohistochemistry and to be effective at assigning samples to subtypes. HER2(+) cancers were shown to have the greatest frequency of high-level amplification (independent of the ERBB2 amplicon itself), but triple-negative cancers had the highest overall frequencies of copy gain. Triple-negative cancers also were shown to have more frequent loss of phosphatase and tensin homologue and mutation of RB1, which may contribute to genomic instability. We identified and validated seven regions of copy number alteration associated with different subtypes, and used integrative bioinformatics analysis to identify candidate oncogenes and tumor suppressors, including ERBB2, GRB7, MYST2, PPM1D, CCND1, HDAC2, FOXA1, and RASA1. We tested the candidate oncogene MYST2 and showed that it enhances the anchorage-independent growth of breast cancer cells. The genome-wide and region-specific differences between subtypes suggest the differential activation of oncogenic pathways.

Construction and application of a zebrafish array comparative genomic hybridization platform.

The zebrafish is emerging as a prominent model system for studying the genetics of human development and disease. Genetic alterations that underlie each mutant model can exist in the form of single base changes, balanced chromosomal rearrangements, or genetic imbalances. To detect genetic imbalances in an unbiased genome-wide fashion, array comparative genomic hybridization (CGH) can be used. We have developed a 5-Mb resolution array CGH platform specifically for the zebrafish. This platform contains 286 bacterial artificial chromosome (BAC) clones, enriched for orthologous sequences of human oncogenes and tumor suppressor genes. Each BAC clone has been end-sequenced and cytogenetically assigned to a specific location within the zebrafish genome, allowing for ease of integration of array CGH data with the current version of the genome assembly. This platform has been applied to three zebrafish cancer models. Significant genomic imbalances were detected in each model, identifying different regions that may potentially play a role in tumorigenesis. Hence, this platform should be a useful resource for genetic dissection of additional zebrafish developmental and disease models as well as a benchmark for future array CGH platform development.

Oncogenic BRAF is required for tumor growth and maintenance in melanoma models.

The usual paradigm for developing kinase inhibitors in oncology is to use a high-affinity proof-of-concept inhibitor with acceptable metabolic properties for key target validation experiments. This approach requires substantial medicinal chemistry and can be confounded by drug toxicity and off-target activities of the test molecule. As a better alternative, we have developed inducible short-hairpin RNA xenograft models to examine the in vivo efficacy of inhibiting oncogenic BRAF. Our results show that tumor regression resulting from BRAF suppression is inducible, reversible, and tightly regulated in these models. Analysis of regressing tumors showed the primary mechanism of action for BRAF to be increased tumor cell proliferation and survival. In a metastatic melanoma model, conditional BRAF suppression slowed systemic tumor growth as determined by in vivo bioluminescence imaging. Taken together, gain-of-function BRAF signaling is strongly associated with in vivo tumorigenicity, confirming BRAF as an important target for small-molecule and RNA interference-based therapeutics.

Regulation of ERK3/MAPK6 expression by BRAF.

Several forms of cancer are characterized by frequent activating mutations in the serine/threonine kinase, BRAF. Substitution of glutamic acid for valine at codon 600 (V600E) accounts for approximately 90% of all BRAF activating mutations and leads to stimulation of kinase activity, downstream signaling, and cell transformation. To better understand the molecular pathogenesis induced by oncogenic BRAF signaling, we used microarray gene expression profiling to comprehensively analyze the BRAF-directed transcriptional program of cells expressing a conditionally active form of BRAFV600E. Several novel genes that affect proliferation, cell survival, angiogenesis and immune surveillance were identified as possible mediators of BRAF-induced oncogenic signaling. Moreover, we show that a MAPK family member, extracellular signal-regulated kinase-3 (ERK3/MAPK6) is highly expressed in response to BRAF signaling in this system. Cellular ERK3 protein is highly unstable and pharmacological inhibition of BRAF activity resulted in rapid ERK3 degradation. In melanoma cells, RNAi-mediated knockdown of endogenous BRAF or treatment with MEK inhibitors that prevent ERK1/2 activation led to a reduction in ERK3 levels, indicating that elevated ERK3 expression is mediated through MEK1/2 signaling. These results provide strong evidence for another mode by which BRAF can regulate the ERK protein kinase family and suggest ERK3 to be a potential pharmacodynamic marker for targeting BRAF signaling in melanoma.

PTEN Loss Is Associated with Worse Outcome in HER2-Amplified Breast Cancer Patients but Is Not Associated with Trastuzumab Resistance.

PURPOSE: To investigate the clinical relevance of PTEN in HER2-amplified and HER2-nonamplified disease. EXPERIMENTAL DESIGN: We assessed PTEN status in two large adjuvant breast cancer trials (BCIRG-006 and BCIRG-005) using a PTEN immunohistochemical (IHC) assay that was previously validated in a panel of 33 breast cancer cell lines and prostate cancer tissues with known PTEN gene deletion. RESULTS: In the HER2-positive patient population, absence of tumor cell PTEN staining occurred at a rate of 5.4% and was independent of ER/PR status. In contrast, 15.9% of HER2-negative patients exhibited absence of PTEN staining with the highest frequency seen in triple-negative breast cancer (TNBC) subgroup versus ER/PR-positive patients (35.1% vs. 10.9%). Complete absence of PTEN staining in tumor cells was associated with poor clinical outcome in HER2-positive disease. Those patients whose cancers demonstrated absent PTEN staining had a significant decrease in disease-free survival (DFS) and overall survival (OS) compared with patients with tumors exhibiting any PTEN staining patterns (low, moderate, or high). Trastuzumab appeared to provide clinical benefit even for patients lacking PTEN staining. In the HER2-negative population, there were no statistically significant differences in clinical outcome based on PTEN status. CONCLUSIONS: This study is the largest to date examining PTEN status in breast cancer and the data suggest that the rate and significance of PTEN status differ between HER2-positive and HER2-negative disease. Furthermore, the data clearly suggest that HER2-positive patients with PTEN loss still benefit from trastuzumab.

Integrated exome and transcriptome sequencing reveals ZAK isoform usage in gastric cancer.

Gastric cancer is the second leading cause of worldwide cancer mortality, yet the underlying genomic alterations remain poorly understood. Here we perform exome and transcriptome sequencing and SNP array assays to characterize 51 primary gastric tumours and 32 cell lines. Meta-analysis of exome data and previously published data sets reveals 24 significantly mutated genes in microsatellite stable (MSS) tumours and 16 in microsatellite instable (MSI) tumours. Over half the patients in our collection could potentially benefit from targeted therapies. We identify 55 splice site mutations accompanied by aberrant splicing products, in addition to mutation-independent differential isoform usage in tumours. ZAK kinase isoform TV1 is preferentially upregulated in gastric tumours and cell lines relative to normal samples. This pattern is also observed in colorectal, bladder and breast cancers. Overexpression of this particular isoform activates multiple cancer-related transcription factor reporters, while depletion of ZAK in gastric cell lines inhibits proliferation. These results reveal the spectrum of genomic and transcriptomic alterations in gastric cancer, and identify isoform-specific oncogenic properties of ZAK.

Oncogenic ERBB3 mutations in human cancers.

The human epidermal growth factor receptor (HER) family of tyrosine kinases is deregulated in multiple cancers either through amplification, overexpression, or mutation. ERBB3/HER3, the only member with an impaired kinase domain, although amplified or overexpressed in some cancers, has not been reported to carry oncogenic mutations. Here, we report the identification of ERBB3 somatic mutations in ~11% of colon and gastric cancers. We found that the ERBB3 mutants transformed colonic and breast epithelial cells in a ligand-independent manner. However, the mutant ERBB3 oncogenic activity was dependent on kinase-active ERBB2. Furthermore, we found that anti-ERBB antibodies and small molecule inhibitors effectively blocked mutant ERBB3-mediated oncogenic signaling and disease progression in vivo.

Improving treatment of HER2-positive cancers: opportunities and challenges.

Amplification of the ERBB2 gene, which encodes human epidermal growth factor receptor 2 (HER2), causes the overexpression of a major proliferative driver for a subset of breast and gastric cancers. Treatments for patients with HER2-positive cancer include the monoclonal antibody trastuzumab and, in the case of metastatic breast cancer, the tyrosine kinase inhibitor lapatinib. Despite significant improvement in patient outcome as a result of these therapies, challenges remain. This Review focuses on proposed mechanisms of action and resistance in the context of potential new therapeutic options. Therapeutic approaches currently in development likely will yield additional clinically meaningful improvements for patients with HER2-positive cancer.

Analysis of Fcγ receptor IIIa and IIa polymorphisms: lack of correlation with outcome in trastuzumab-treated breast cancer patients.

PURPOSE: The mechanisms by which trastuzumab imparts clinical benefit remain incompletely understood. Antibody-dependent cellular cytotoxicity via interactions with Fcγ receptors (FcγR) on leukocytes may contribute to its antitumor effects. Single-nucleotide polymorphisms (SNP) in FCGR3A and FCGR2A genes lead to amino acid substitutions at positions 158 and 131, respectively, and affect binding of antibodies to FcγR such that 158V/V and 131H/H bind with highest affinity. This study aimed to determine whether high-affinity SNPs are associated with disease-free survival (DFS) among patients with HER2-positive nonmetastatic breast cancer. EXPERIMENTAL DESIGN: Genomic DNA was isolated from 1,286 patients enrolled in a trial of adjuvant trastuzumab-based chemotherapy. Genotyping was conducted using Sanger sequencing and Sequenom mass spectrometry. RESULTS: Patient samples (N = 1,189) were successfully genotyped for FCGR3A and 1,218 for FCGR2A. Compared with the overall results of the BCIRG006 study, in the subset of patients genotyped in this analysis, a less robust improvement in DFS was observed for the trastuzumab arms than control arm (HR, 0.842; P = 0.1925). When stratified for prognostic features, the HR in favor of trastuzumab was consistent with that of the overall study (HR, 0.74; P = 0.036). No correlation between DFS and FCGR3A/2A genotypes was seen for trastuzumab-treated patients (158V/V vs. V/F vs. F/F, P = 0.98; 131H/H vs. H/R vs. R/R, P = 0.76; 158V/V and/or 131H/H vs. others, P = 0.67). CONCLUSION: This analysis evaluating the association between FCGR3A/2A genotypes and trastuzumab efficacy in HER2-positive breast cancer did not show a correlation between FCGR3A-V/F and FCGR2A-H/R SNPs and DFS in patients treated with trastuzumab.

Comprehensive genomic analysis identifies SOX2 as a frequently amplified gene in small-cell lung cancer.

Small-cell lung cancer (SCLC) is an exceptionally aggressive disease with poor prognosis. Here, we obtained exome, transcriptome and copy-number alteration data from approximately 53 samples consisting of 36 primary human SCLC and normal tissue pairs and 17 matched SCLC and lymphoblastoid cell lines. We also obtained data for 4 primary tumors and 23 SCLC cell lines. We identified 22 significantly mutated genes in SCLC, including genes encoding kinases, G protein-coupled receptors and chromatin-modifying proteins. We found that several members of the SOX family of genes were mutated in SCLC. We also found SOX2 amplification in ∼27% of the samples. Suppression of SOX2 using shRNAs blocked proliferation of SOX2-amplified SCLC lines. RNA sequencing identified multiple fusion transcripts and a recurrent RLF-MYCL1 fusion. Silencing of MYCL1 in SCLC cell lines that had the RLF-MYCL1 fusion decreased cell proliferation. These data provide an in-depth view of the spectrum of genomic alterations in SCLC and identify several potential targets for therapeutic intervention.

TRPS1 targeting by miR-221/222 promotes the epithelial-to-mesenchymal transition in breast cancer.

The basal-like subtype of breast cancer has an aggressive clinical behavior compared to that of the luminal subtype. We identified the microRNAs (miRNAs) miR-221 and miR-222 (miR-221/222) as basal-like subtype-specific miRNAs and showed that expression of miR-221/222 decreased expression of epithelial-specific genes and increased expression of mesenchymal-specific genes, and increased cell migration and invasion in a manner characteristic of the epithelial-to-mesenchymal transition (EMT). The transcription factor FOSL1 (also known as Fra-1), which is found in basal-like breast cancers but not in the luminal subtype, stimulated the transcription of miR-221/222, and the abundance of these miRNAs decreased with inhibition of the epidermal growth factor receptor (EGFR) or MEK (mitogen-activated or extracellular signal-regulated protein kinase kinase), placing miR-221/222 downstream of the RAS pathway. Furthermore, miR-221/222-mediated reduction in E-cadherin abundance depended on their targeting the 3' untranslated region of the GATA family transcriptional repressor TRPS1 (tricho-rhino-phalangeal syndrome type 1), which inhibited EMT by decreasing ZEB2 (zinc finger E-box-binding homeobox2) expression. We conclude that by promoting EMT, miR-221/222 may contribute to the more aggressive clinical behavior of basal-like breast cancers.

miR-221/222 targeting of trichorhinophalangeal 1 (TRPS1) promotes epithelial-to-mesenchymal transition in breast cancer.

Compared with the luminal subtype, the basal-like subtype of breast cancer has an aggressive clinical behavior, but the reasons for this difference between the two subtypes are poorly understood. We identified microRNAs (miRNAs) miR-221 and miR-222 (miR-221/222) as basal-like subtype-specific miRNAs that decrease expression of epithelial-specific genes and increase expression of mesenchymal-specific genes. In addition, expression of these miRNAs increased cell migration and invasion, which collectively are characteristics of the epithelial-to-mesenchymal transition (EMT). The basal-like transcription factor FOSL1 (also known as Fra-1) directly stimulated the transcription of miR-221/222, and the abundance of these miRNAs decreased with inhibition of MEK (mitogen-activated or extracellular signal-regulated protein kinase kinase), placing miR-221/222 downstream of the RAS pathway. The miR-221/222-mediated reduction in E-cadherin abundance depended on their targeting of the 3' untranslated region (3'UTR) of TRPS1 (trichorhinophalangeal syndrome type 1), which is a member of the GATA family of transcriptional repressors. TRPS1 inhibited EMT by directly repressing expression of ZEB2 (Zinc finger E-box-binding homeobox 2). Therefore, miR-221/222 may contribute to the aggressive clinical behavior of basal-like breast cancers.