Differential regulation of PD-L1 expression by immune and tumor cells in NSCLC and the response to treatment with atezolizumab (anti-PD-L1).

Programmed death-ligand 1 (PD-L1) expression on tumor cells (TCs) by immunohistochemistry is rapidly gaining importance as a diagnostic for the selection or stratification of patients with non-small cell lung cancer (NSCLC) most likely to respond to single-agent checkpoint inhibitors. However, at least two distinct patterns of PD-L1 expression have been observed with potential biological and clinical relevance in NSCLC: expression on TC or on tumor-infiltrating immune cells (ICs). We investigated the molecular and cellular characteristics associated with PD-L1 expression in these distinct cell compartments in 4,549 cases of NSCLC. PD-L1 expression on IC was more prevalent and likely reflected IFN-γ-induced adaptive regulation accompanied by increased tumor-infiltrating lymphocytes and effector T cells. High PD-L1 expression on TC, however, reflected an epigenetic dysregulation of the PD-L1 gene and was associated with a distinct histology described by poor immune infiltration, sclerotic/desmoplastic stroma, and mesenchymal molecular features. Importantly, durable clinical responses to atezolizumab (anti-PD-L1) were observed in patients with tumors expressing high PD-L1 levels on either TC alone [40% objective response rate (ORR)] or IC alone (22% ORR). Thus, PD-L1 expression on TC or IC can independently attenuate anticancer immunity and emphasizes the functional importance of IC in regulating the antitumor T cell response.

Profiling cancer gene mutations in clinical formalin-fixed, paraffin-embedded colorectal tumor specimens using targeted next-generation sequencing.

PURPOSE: The success of precision oncology relies on accurate and sensitive molecular profiling. The Ion AmpliSeq Cancer Panel, a targeted enrichment method for next-generation sequencing (NGS) using the Ion Torrent platform, provides a fast, easy, and cost-effective sequencing workflow for detecting genomic "hotspot" regions that are frequently mutated in human cancer genes. Most recently, the U.K. has launched the AmpliSeq sequencing test in its National Health Service. This study aimed to evaluate the clinical application of the AmpliSeq methodology. METHODS: We used 10 ng of genomic DNA from formalin-fixed, paraffin-embedded human colorectal cancer (CRC) tumor specimens to sequence 46 cancer genes using the AmpliSeq platform. In a validation study, we developed an orthogonal NGS-based resequencing approach (SimpliSeq) to assess the AmpliSeq variant calls. RESULTS: Validated mutational analyses revealed that AmpliSeq was effective in profiling gene mutations, and that the method correctly pinpointed "true-positive" gene mutations with variant frequency >5% and demonstrated high-level molecular heterogeneity in CRC. However, AmpliSeq enrichment and NGS also produced several recurrent "false-positive" calls in clinically druggable oncogenes such as PIK3CA. CONCLUSION: AmpliSeq provided highly sensitive and quantitative mutation detection for most of the genes on its cancer panel using limited DNA quantities from formalin-fixed, paraffin-embedded samples. For those genes with recurrent "false-positive" variant calls, caution should be used in data interpretation, and orthogonal verification of mutations is recommended for clinical decision making.

Development of a robust RNA-based classifier to accurately determine ER, PR, and HER2 status in breast cancer clinical samples.

Breast cancers are categorized into three subtypes based on protein expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2/ERBB2). Patients enroll onto experimental clinical trials based on ER, PR, and HER2 status and, as receptor status is prognostic and defines treatment regimens, central receptor confirmation is critical for interpreting results from these trials. Patients enrolling onto experimental clinical trials in the metastatic setting often have limited available archival tissue that might better be used for comprehensive molecular profiling rather than slide-intensive reconfirmation of receptor status. We developed a Random Forests-based algorithm using a training set of 158 samples with centrally confirmed IHC status, and subsequently validated this algorithm on multiple test sets with known, locally determined IHC status. We observed a strong correlation between target mRNA expression and IHC assays for HER2 and ER, achieving an overall accuracy of 97 and 96%, respectively. For determining PR status, which had the highest discordance between central and local IHC, incorporation of expression of co-regulated genes in a multivariate approach added predictive value, outperforming the single, target gene approach by a 10% margin in overall accuracy. Our results suggest that multiplexed qRT-PCR profiling of ESR1, PGR, and ERBB2 mRNA, along with several other subtype associated genes, can effectively confirm breast cancer subtype, thereby conserving tumor sections and enabling additional biomarker data to be obtained from patients enrolled onto experimental clinical trials.

Development of a robust flow cytometry-based pharmacodynamic assay to detect phospho-protein signals for phosphatidylinositol 3-kinase inhibitors in multiple myeloma.

BACKGROUND: The phosphatidylinositol 3-kinase (PI3K) pathway plays an important role in multiple myeloma (MM), a blood cancer associated with uncontrolled proliferation of bone marrow plasma cells. This study aimed to develop a robust clinical pharmacodynamic (PD) assay to measure the on-target PD effects of the selective PI3K inhibitor GDC-0941 in MM patients. METHODS: We conducted an in vitro drug wash-out study to evaluate the feasibility of biochemical approaches in measuring the phosphorylation of S6 ribosomal protein (S6), one of the commonly used PD markers for PI3K pathway inhibition. We then developed a 7-color phospho-specific flow cytometry assay, or phospho flow assay, to measure the phosphorylation state of intracellular S6 in bone marrow aspirate (BMA) and peripheral blood (PB). Integrated mean fluorescence intensity (iMFI) was used to calculate fold changes of phosphorylation. Assay sensitivity was evaluated by comparing phospho flow with Meso Scale Discovery (MSD) and immunohistochemistry (IHC) assays. Finally, a sample handling method was developed to maintain the integrity of phospho signal during sample shipping and storage to ensure clinical application. RESULTS: The phospho flow assay provided single-cell PD monitoring of S6 phosphorylation in tumor and surrogate cells using fixed BMA and PB, assessing pathway modulation in response to GDC-0941 with sensitivity similar to that of MSD assay. The one-shot sample fixation and handling protocol herein demonstrated exceptional preservation of protein phosphorylation. In contrast, the IHC assay was less sensitive in terms of signal quantification while the biochemical approach (MSD) was less suitable to assess PD activities due to the undesirable impact associated with cell isolation on the protein phosphorylation in tumor cells. CONCLUSIONS: We developed a robust PD biomarker assay for the clinical evaluation of PI3K inhibitors in MM, allowing one to decipher the PD response in a relevant cell population. To our knowledge, this is the first report of an easily implemented clinical PD assay that incorporates an unbiased one-shot sample handling protocol, all (staining)-in-one (tube) phospho flow staining protocol, and an integrated modified data analysis for PD monitoring of kinase inhibitors in relevant cell populations in BMA and PB. The methods described here ensure a real-time, reliable and reproducible PD readout, which can provide information for dose selection as well as help to identify optimal combinations of targeted agents in early clinical trials.

Profiling bladder cancer organ site-specific metastasis identifies LAMC2 as a novel biomarker of hematogenous dissemination.

Little is known about which genes mediate metastasis in bladder cancer, which accounts for much of the mortality of this disease. We used human bladder cancer cell lines to develop models of two clinically common metastatic sites, lung and liver, and evaluated their gene expression with respect to human tumor tissues. Parental cells were injected into either the murine spleen to generate liver metastases or tail vein to generate lung metastases with sequential progeny derived by re-injection and comparisons made of their organ-specific nature by crossed-site injections. Both genomic and transcriptomic analyses of organ-selected cell lines found salient differences and shared core metastatic profiles, which were then screened against gene expression data from human tumors. The expression levels of laminin V gamma 2 (LAMC2) contained in the core metastatic signature were increased as a function of human tumor stage, and its genomic location was in an area of gain as measured by comparative genomic hybridization. Using immunohistochemistry in a human bladder cancer tissue microarray, LAMC2 expression levels were associated with tumor grade, but inversely with nodal status. In contrast, in node-negative patients, LAMC2 expression was associated with visceral metastatic recurrence. In summary, LAMC2 is a novel biomarker of bladder cancer metastasis that reflects the propensity of cells to metastasize via either lymphatic or hematogenous routes.

An array of insights: application of DNA chip technology in the study of cell biology.

The advent of DNA microarray technology has ushered in an era of systems biology whereby researchers can study the transcriptional behavior of thousands of genes in parallel. Advances in manufacturing techniques and informatics, and the availability of several genome sequences have furthered these capabilities to the point where whole-transcriptome studies can be accomplished in yeast, flies and plants, and soon will be possible in mammals. Concomitant with the expanding ability of the technology has been the development of novel techniques and their application towards the study of cellular biology.

An integrated clinical-genomics approach identifies a candidate multi-analyte blood test for serous ovarian carcinoma.

PURPOSE: Cancer of the ovary confers the worst prognosis among women with gynecologic malignancies, underscoring the need to develop new biomarkers for detection of early disease, particularly those that can be readily monitored in the blood. EXPERIMENTAL DESIGN: We developed an algorithm to identify secreted proteins encoded among approximately 22,500 genes on commercial oligonucleotide arrays and applied it to gene expression profiles of 67 stage I to IV serous papillary carcinomas and 9 crudely enriched normal ovarian tissues, to identify putative diagnostic markers. ELISAs were used to validate increased levels of secreted proteins in patient sera encoded by genes with differentially high expression. RESULTS: We identified 275 genes predicted to encode secreted proteins with increased/decreased expression in ovarian cancers (<0.5- or >2-fold, P < 0.001). The serum levels of four of these proteins (matrix metalloproteinase-7, osteopontin, secretory leukoprotease inhibitor, and kallikrein 10) were significantly elevated in a series of 67 independent patients with serous ovarian carcinomas compared with 67 healthy controls (P < 0.001, Wilcoxon rank sum test). Optimized support vector machine classifiers with as few as two of these markers (osteopontin or kallikrein 10/matrix metalloproteinase-7) in combination with CA-125 yielded sensitivity and specificity values ranging from 96% to 98.7% and 99.7% to 100%, respectively, with the ability to discern early-stage disease from normal, healthy controls. CONCLUSIONS: Our data suggest that this assay combination warrants further investigation as a multi-analyte diagnostic test for serous ovarian adenocarcinoma.

The metastasis-associated gene CD24 is regulated by Ral GTPase and is a mediator of cell proliferation and survival in human cancer.

Ral GTPases are important mediators of transformation, tumorigenesis, and cancer progression. We recently identified the metastasis-associated protein CD24, a glycosyl phosphatidyl inositol-linked surface protein, as a downstream target of Ral signaling by profiling the expression of RalA/B-depleted bladder carcinoma cells. Because CD24 is highly expressed in bladder and many other tumor types, we sought to determine if this protein plays an essential role in maintaining the malignant phenotype. Here, we show that loss of CD24 function in cell lines derived from common tumor types is associated with decreased rates of cell proliferation, clonogenicity in soft agar, changes in the actin cytoskeleton, and induction of apoptosis. Given these phenotypes, we evaluated a human bladder cancer tissue microarray by immunohistochemistry for CD24 to determine if CD24 is a prognostic cancer biomarker. Multivariate analysis showed that increased CD24 expression correlated with shorter patient disease-free survival (P = 0.07). In conclusion, we show that CD24 is a novel and functionally relevant Ral-regulated target and a potentially important prognostic marker. We suggest that these insights may lead to future therapeutic approaches that seek to eliminate CD24 function in cancer cells.

Prediction of drug combination chemosensitivity in human bladder cancer.

The choice of therapy for metastatic cancer is largely empirical because of a lack of chemosensitivity prediction for available combination chemotherapeutic regimens. Here, we identify molecular models of bladder carcinoma chemosensitivity based on gene expression for three widely used chemotherapeutic agents: cisplatin, paclitaxel, and gemcitabine. We measured the growth inhibition elicited by these three agents in a series of 40 human urothelial cancer cell lines and correlated the GI(50) (50% of growth inhibition) values with quantitative measures of global gene expression to derive models of chemosensitivity using a misclassification-penalized posterior approach. The misclassification-penalized posterior-derived models predicted the growth response of human bladder cancer cell lines to each of the three agents with sensitivities of between 0.93 and 0.96. We then developed an in silico approach to predict the cellular growth responses for each of these agents in the clinically relevant two-agent combinations. These predictions were prospectively evaluated on a series of 15 randomly chosen bladder carcinoma cell lines. Overall, 80% of the predicted combinations were correct (P = 0.0002). Together, our results suggest that chemosensitivity to drug combinations can be predicted based on molecular models and provide the framework for evaluation of such models in patients undergoing combination chemotherapy for cancer. If validated in vivo, such predictive models have the potential to guide therapeutic choice at the level of an individual's tumor.

A transcriptional MAPK Pathway Activity Score (MPAS) is a clinically relevant biomarker in multiple cancer types.

KRAS- and BRAF-mutant tumors are often dependent on MAPK signaling for proliferation and survival and thus sensitive to MAPK pathway inhibitors. However, clinical studies have shown that MEK inhibitors are not uniformly effective in these cancers indicating that mutational status of these oncogenes does not accurately capture MAPK pathway activity. A number of transcripts are regulated by this pathway and are recurrently identified in genome-based MAPK transcriptional signatures. To test whether the transcriptional output of only 10 of these targets could quantify MAPK pathway activity with potential predictive or prognostic clinical utility, we created a MAPK Pathway Activity Score (MPAS) derived from aggregated gene expression. In vitro, MPAS predicted sensitivity to MAPK inhibitors in multiple cell lines, comparable to or better than larger genome-based statistical models. Bridging in vitro studies and clinical samples, median MPAS from a given tumor type correlated with cobimetinib (MEK inhibitor) sensitivity of cancer cell lines originating from the same tissue type. Retrospective analyses of clinical datasets showed that MPAS was associated with the sensitivity of melanomas to vemurafenib (HR: 0.596) and negatively prognostic of overall or progression-free survival in both adjuvant and metastatic CRC (HR: 1.5 and 1.4), adrenal cancer (HR: 1.7), and HER2+ breast cancer (HR: 1.6). MPAS thus demonstrates potential clinical utility that warrants further exploration.

Aromatase inhibitors--gene discovery.

Microarray analysis of tumour RNA is an extremely powerful tool which allows global gene expression to be measured. When used in combination with neoadjuvant treatment protocols in which therapy is given with the primary tumour within the breast, sequential biopsies may be analysed and results correlated with clinical and pathological response. In the present study, a neoadjuvant protocol has been used, administering the third generation inhibitor, letrozole, for 3 months and subjecting RNA extracted from biopsies taken before and after 10-14 days of treatment to microarray analysis. The objectives were to discover: (i) genes that change with estrogen deprivation (the only known biological effect of letrozole is to inhibit aromatase activity and reduce endogenous estrogens in postmenopausal women) and (ii) genes whose basal, on treatment or change in expression differ between tumours which are either responsive or resistant to treatment (so that predictive indices of response/resistance may be developed). Early changes in gene expression were identified by comparing paired tumour core biopsies taken before and after 14 days treatment in 58 patients using three different approaches based on frequency of changes, magnitude of changes and SAM analysis. All three approaches showed a greater number of genes were down-regulated than up-regulated. Merging of the data produced a total of 143 genes which were subject to gene ontology and cluster analysis. The ontology of the 91 down-regulated genes showed that they were functionally associated with cell cycle progression, particularly mitosis. In contrast, up-regulated genes were associated with organ development and extra-cellular matrix turnover and regulation. Clinical response was assessable in 52 patients; 37 (71%) tumours were classified as clinical responders (>50% reduction in volume at 3 months). Microarray analysis of pre- and 14-day biopsies identified 291 covariates (84 baselines, 72 14-day and 135 changes) highly predictive of response status. A similarity matrix using the covariates showed responding tumours have a similar genetic profile which was dissimilar to non-responding cancers whereas non-responsive cases were distinctive from each other. Changed genes predicting for response showed no concordance with those changed significantly by treatment in the overall group.

Measurement of serum levels of macrophage inhibitory cytokine 1 combined with prostate-specific antigen improves prostate cancer diagnosis.

PURPOSE: Current serum testing for the detection of prostate cancer (PCa) lacks specificity. On diagnosis, the optimal therapeutic pathway is not clear and tools for adequate risk assessment of localized PCa progression are not available. This leads to a significant number of men having unnecessary diagnostic biopsies and surgery. A search for novel tumor markers identified macrophage inhibitory cytokine 1 (MIC-1) as a potentially useful marker. Follow-up studies revealed MIC-1 overexpression in local and metastatic PCa whereas peritumoral interstitial staining for MIC-1 identified lower-grade tumors destined for recurrence. Consequently, we sought to assess serum MIC-1 measurement as a diagnostic tool. EXPERIMENTAL DESIGN: Using immunoassay determination of serum MIC-1 concentration in 1,000 men, 538 of whom had PCa, we defined the relationship of MIC-1 to disease variables. A diagnostic algorithm (MIC-PSA score) based on serum levels of MIC-1, total serum prostate-specific antigen, and percentage of free prostate-specific antigen was developed. RESULTS: Serum MIC-1 was found to be an independent predictor of the presence of PCa and tumors with a Gleason sum > or =7. We validated the MIC-PSA score in a separate population and showed an improved specificity for diagnostic blood testing for PCa over percentage of free prostate-specific antigen, potentially reducing unnecessary biopsies by 27%. CONCLUSIONS: Serum MIC-1 is an independent marker of the presence of PCa and tumors with a Gleason sum of > or =7. The use of serum MIC-1 significantly increases diagnostic specificity and may be a future tool in the management of PCa.

Endothelin axis is a target of the lung metastasis suppressor gene RhoGDI2.

Half of patients treated for locally advanced bladder cancer relapse with often fatal metastatic disease to the lung. We have recently shown that reduced expression of the GDP dissociation inhibitor, RhoGDI2, is associated with decreased survival of patients with advanced bladder cancer. However, the effectors by which RhoGDI2 affects metastasis are unknown. Here we use DNA microarrays to identify genes suppressed by RhoGDI2 reconstitution in lung metastatic bladder cancer cell lines. We identify such RNAs and focus only on those that also increase with tumor stage in human bladder cancer samples to discover only clinically relevant targets of RhoGDI2. Levels of endothelin-1 (ET-1), a potent vasoconstrictor, were affected by both RhoGDI2 reconstitution and tumor stage. To test the hypothesis that the endothelin axis is important in lung metastasis, lung metastatic bladder carcinoma cells were injected in mice treated with the endothelin receptor-specific antagonist, atrasentan, thereby blocking engagement of the up-regulated ET-1 ligand with its cognate receptor. Endothelin antagonism resulted in a dramatic reduction of lung metastases, similar to the effect of reexpressing RhoGDI2 in these metastatic cells. Taken together, these experiments show a novel approach of identifying therapeutic targets downstream of metastasis suppressor genes. The data also suggest that blockade of the ET-1 axis may prevent lung metastasis, a new therapeutic concept that warrants clinical evaluation.

Molecular and prognostic distinction between serous ovarian carcinomas of varying grade and malignant potential.

Profiles of gene transcription have begun to delineate the molecular basis of ovarian cancer, including distinctions between carcinomas of differing histology, tumor progression and patient outcome. However, the similarities and differences among the most commonly diagnosed noninvasive borderline (low malignant potential, LMP) lesions and invasive serous carcinomas of varying grade (G1, G2 and G3) have not yet been explored. Here, we used oligonucleotide arrays to profile the expression of 12,500 genes in a series of 57 predominantly stage III serous ovarian adenocarcinomas from 52 patients, eight with borderline tumors and 44 with adenocarcinomas of varying grade. Unsupervised and supervised analyses showed that LMP lesions were distinct from high-grade serous adenocarcinomas, as might be expected; however, well-differentiated (G1) invasive adenocarcinomas showed a strikingly similar profile to LMP tumors as compared to cancers with moderate (G2) or poor (G3) cellular differentiation, which were also highly similar. Comparative genomic hybridization of an independent cohort of five LMP and 63 invasive carcinomas of varying grade demonstrated LMP and G1 were again similar, exhibiting significantly less chromosomal aberration than G2/G3 carcinomas. A majority of LMP and G1 tumors were characterized by high levels of p21/WAF1, with concomitant expression of cell growth suppressors, gadd34 and BTG-2. In contrast, G2/G3 cancers were characterized by the expression of genes associated with the cell cycle and by STAT-1-, STAT-3/JAK-1/2-induced gene expression. The distinction between the LMP-G1 and G2-G3 groups of tumors was highly correlated to patient outcome (chi(2) for equivalence of death rates=7.681189; P=0.0056, log-rank test). Our results are consistent with the recent demonstration of a poor differentiation molecular 'meta-signature' in human cancer, and underscore a number of cell-cycle- and STAT-associated targets that may prove useful as points of therapeutic intervention for those patients with aggressive disease.

A phosphoproteomic analysis of the ErbB2 receptor tyrosine kinase signaling pathways.

Overexpression of the ErbB2 receptor tyrosine kinase is common in human cancers and is associated with an increased level of metastasis. To better understand the cellular signaling networks activated by ErbB2, a phosphoproteomic analysis of tyrosine-phosphorylated proteins was carried out in ErbB2-overexpressing breast and ovarian cancer cell lines. A total of 153 phosphorylation sites were assigned on 78 proteins. Treatment of cells with Herceptin, a monoclonal antibody that inhibits ErbB2 activity, significantly reduced the number of detectable protein phosphorylation sites, suggesting that many of these proteins participate in ErbB2-driven cell signaling. Of the 71 proteins that were differentially phosphorylated, only 13 were previously reported to directly associate with ErbB2. The differentially phosphorylated proteins included kinases, adaptor/docking proteins, proteins involved in cell proliferation and migration, and several uncharacterized RNA binding proteins. Selective depletion of some of these proteins, including RNA binding proteins SRRM2, SFRS1, SFRS9, and SFRS10, by siRNAs reduced the rate of migration of ErbB2-overexpressing ovarian cancer cells.

RhoGDI2 is an invasion and metastasis suppressor gene in human cancer.

To discover novel metastasis suppressor genes that are clinically relevant in common human cancers, we used isogenic human bladder cancer cell lines and used DNA microarray technology to identify genes whose expression diminishes as a function of invasive and metastatic competence. We then evaluated the expression profile of such genes in 105 pathologically characterized tumors from seven common organ sites, and we identified one gene, RhoGDI2, whose expression was diminished as a function of primary tumor stage and grade. When RhoGDI2 was transferred back into cells with metastatic ability that lacked its expression, it suppressed experimental lung metastasis but did not affect in vitro growth, colony formation, or in vivo tumorigenicity. In addition, RhoGDI2 reconstitution in these cells blocked invasion in an organotypic assay and led to a reduction of in vitro motility. These results indicate that RhoGDI2 is a metastasis suppressor gene, a marker of aggressive human cancer, and a promising target for therapy.

Profiling the evolution of human metastatic bladder cancer.

Pulmonary metastases frequently develop in patients with aggressive bladder cancer, yet investigation of this process at the molecular level suffers from the poor availability of human metastatic tumor tissue and the absence of suitable animal models. To address this, we developed progressively more metastatic human bladder cancer cell lines and an in vivo bladder-cancer lung-metastasis model, and we successfully used these to identify genes of which the expression levels change according to the degree of pulmonary metastatic potential. By initially intravenously injecting the poorly metastatic T24T human urothelial cancer cells into nude mice, and then serially reintroducing and reisolating the human tumor cells from the resultant mouse lung tumors, three derivative human lines with increasingly metastatic phenotypes, designated FL1, FL2, and FL3, were sequentially isolated. To identify the genes associated with the most lung-metastatic phenotype, the RNA complement from the parental and derivative cells was evaluated with oligonucleotide microarrays. In doing so, we found 121 genes to be progressively up-regulated during the transition from T24T to FL3, whereas 43 genes were progressively down-regulated. As expected, many of the genes identified in these groups could, according to the ascribed functions of their protein product, theoretically participate in tissue invasion and metastasis. In addition, the magnitude of gene expression changes observed during the metastatic transition correlated with the in vivo propensity for earlier lung colonization and decreased host survival. To additionally define which genes found in the experimental system were of relevance to human bladder cancer lung metastasis, we evaluated gene expression profiles of 23 primary human bladder tumors of various stages and grades, and then we compared these gene expression profiles to the altered profiles in our model cell lines. Here we found that the expression of epiregulin, urokinase-type plasminogen activator (uPA), matrix metalloproteinase (MMP)14, and tissue inhibitor of metalloproteinase (TIMP-2) were consistently and progressively up-regulated when viewed as a function of tumor stage in tissues of patients versus the metastatic potential seen in the mouse lung model. The strong correlation of these four markers between the experimental and clinical situations helps validate this system as a useful tool for the study of lung metastasis and defines targets of therapy that may reduce the incidence of this process in patients.

Activating Mutations in PIK3CB Confer Resistance to PI3K Inhibition and Define a Novel Oncogenic Role for p110ß.

Activation of the PI3K pathway occurs commonly in a wide variety of cancers. Experience with other successful targeted agents suggests that clinical resistance is likely to arise and may reduce the durability of clinical benefit. Here, we sought to understand mechanisms underlying resistance to PI3K inhibition in PTEN-deficient cancers. We generated cell lines resistant to the pan-PI3K inhibitor GDC-0941 from parental PTEN-null breast cancer cell lines and identified a novel PIK3CB D1067Y mutation in both cell lines that was recurrent in cancer patients. Stable expression of mutant PIK3CB variants conferred resistance to PI3K inhibition that could be overcome by downstream AKT or mTORC1/2 inhibitors. Furthermore, we show that the p110ß D1067Y mutant was highly activated and induced PIP3 levels at the cell membrane, subsequently promoting the localization and activation of AKT and PDK1 at the membrane and driving PI3K signaling to a level that could withstand treatment with proximal inhibitors. Finally, we demonstrate that the PIK3CB D1067Y mutant behaved as an oncogene and transformed normal cells, an activity that was enhanced by PTEN depletion. Collectively, these novel preclinical and clinical findings implicate the acquisition of activating PIK3CB D1067 mutations as an important event underlying the resistance of cancer cells to selective PI3K inhibitors.

Development and Application of a Microfluidics-Based Panel in the Basal/Luminal Transcriptional Characterization of Archival Bladder Cancers.

In the age of personalized medicine stratifying tumors into molecularly defined subtypes associated with distinctive clinical behaviors and predictable responses to therapies holds tremendous value. Towards this end, we developed a custom microfluidics-based bladder cancer gene expression panel for characterization of archival clinical samples. In silico analysis indicated that the content of our panel was capable of accurately segregating bladder cancers from several public datasets into the clinically relevant basal and luminal subtypes. On a technical level, our bladder cancer panel yielded robust and reproducible results when analyzing formalin-fixed, paraffin-embedded (FFPE) tissues. We applied our panel in the analysis of a novel set of 204 FFPE samples that included non-muscle invasive bladder cancers (NMIBCs), muscle invasive disease (MIBCs), and bladder cancer metastases (METs). We found NMIBCs to be mostly luminal-like, MIBCs to include both luminal- and basal-like types, and METs to be predominantly of a basal-like transcriptional profile. Mutational analysis confirmed the expected enrichment of FGFR3 mutations in luminal samples, and, consistently, FGFR3 IHC showed high protein expression levels of the receptor in these tumors. Our bladder cancer panel enables basal/luminal characterization of FFPE tissues and with further development could be used for stratification of bladder cancer samples in the clinic.

Heterogeneity and clinical significance of ESR1 mutations in ER-positive metastatic breast cancer patients receiving fulvestrant.

Mutations in ESR1 have been associated with resistance to aromatase inhibitor (AI) therapy in patients with ER+ metastatic breast cancer. Little is known of the impact of these mutations in patients receiving selective oestrogen receptor degrader (SERD) therapy. In this study, hotspot mutations in ESR1 and PIK3CA from ctDNA were assayed in clinical trial samples from ER+ metastatic breast cancer patients randomized either to the SERD fulvestrant or fulvestrant plus a pan-PI3K inhibitor. ESR1 mutations are present in 37% of baseline samples and are enriched in patients with luminal A and PIK3CA-mutated tumours. ESR1 mutations are often polyclonal and longitudinal analysis shows distinct clones exhibiting divergent behaviour over time. ESR1 mutation allele frequency does not show a consistent pattern of increases during fulvestrant treatment, and progression-free survival is not different in patients with ESR1 mutations compared with wild-type patients. ESR1 mutations are not associated with clinical resistance to fulvestrant in this study.