Molecular Profiling Results Research Articles

Abstract PR03: Molecular profiling of advanced solid tumors at Princess Margaret Cancer Centre and patient outcomes with genotype-matched clinical trials

Abstract Background: IMPACT and COMPACT are ongoing clinical trials at Princess Margaret (PM) to match advanced solid tumor patients with actionable somatic mutations to clinical trials with investigational therapies [NCT01505400]. We compared the outcome of patients profiled at PM treated on genotype-matched versus genotype-unmatched clinical trials. Methods: Patients with advanced solid tumors treated at PM or collaborating local institutions with available formalin fixed paraffin-embedded (FFPE) tumor tissue were prospectively consented for molecular profiling during standard treatment. Only patients with ECOG performance status ≤1 who were considered therapeutic trial candidates by their treating oncologist were eligible. Following pathology review, tumor DNA from FFPE blocks or unstained slides was extracted and genotyped using a customized Sequenom SNP genotyping panel (23 genes, 279 mutations) or a targeted next generation sequencing (NGS) panel, either the Illumina MiSeq TruSeq Amplicon Cancer Panel (48 genes, 212 amplicons) or the Ion Proton Ampliseq Cancer Hotspot Panel version 2 (50 genes, 207 amplicons) with ≤500x coverage in a Clinical Laboratory Improvement Amendments (CLIA) certified laboratory. An annotated molecular profiling report with somatic variants classified according to clinical actionability was included in the patient's electronic medical record. Oncologists were provided with regular summary tables of their patients' molecular profiling results and mutation-specific clinical trial listings to facilitate genotype-matched trial enrolment. Results: From March 2012 to July 2014, 1893 patients were enrolled with gynaecological (22%), breast (18%), lung (18%) colorectal (17%), pancreatobiliary (8%), upper aerodigestive (6%), genitourinary (5%), and other (5%) cancers. Patients had received a median of 4 prior systemic treatments (range 1-23). Of 253 (13%) screen failures, 10% were for insufficient tissue and 3% for clinical deterioration or other reasons. Patients were more likely to have one or more somatic mutations identified by NGS testing [597/813 (73%); average 1.23 mutations/patient; range 0-9 mutations/patient] compared with SNP genotyping [341/827 (41%); average 0.46 mutations/patient; range 0-2 mutations/patient; p<0.0001]. After a median follow-up of 18 months, a total of 244 patients (13%) were treated on 287 therapeutic clinical trials, including 84 patients (4%) on 92 genotype-matched trials. There was no difference in the proportion of trial enrolment onto genotype-matched therapies between patients profiled on Sequenom compared with targeted NGS [63/176 (36%) vs 29/101 (29%); p=0.23). Patients with pancreatobiliary and upper aerodigestive tract cancers were least likely to be treated on genotype-matched trials. A higher proportion of patients enrolled in genotype-matched trials were treated in phase I studies (80%) compared with genotype-unmatched trials (45%; p<0.001). The overall response rate by RECIST version 1.1 was higher in patients treated on genotype-matched trials (20%) compared with genotype-unmatched trials (11%; p<0.04). Patients treated on genotype-matched trials were more likely to achieve a best response of any shrinkage in the sum of their target lesions (61%) compared with patients treated on genotype-unmatched trials (32%; p<0.001). Conclusions: Few advanced solid tumor patients enrolled in a prospective institutional molecular profiling program were subsequently treated on genotype-matched therapeutic trials. Compared with SNP genotyping, profiling with a broader NGS panel did not increase the likelihood of receiving treatment on a genotype-matched trial. In this non-randomized comparison, genotype-enrichment of early phase clinical trials was associated with an increased objective tumor response rate. Greater efforts should be made to expand opportunities for genotype-trial matching and further studies are needed to evaluate the clinical utility of targeted NGS profiling. This abstract is also presented as Poster 18. Citation Format: Philippe L. Bedard, Amit Oza, Blaise Clarke, Ming-Sound Tsao, Natasha B. Leighl, Eric X. Chen, Albiruni Razak, Hal K. Berman, Stefano Serra, Michael Roehrl, Nadia Califaretti, Mateya Trinkaus, Tong Zhang, Mahadeo A. Sukhai, Anca Milea, Aaron R. Hansen, Trevor J. Pugh, Tracy Stockley, Suzanne Kamel-Reid, Lillian L. Siu. Molecular profiling of advanced solid tumors at Princess Margaret Cancer Centre and patient outcomes with genotype-matched clinical trials. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr PR03.

Clinical Outcomes of Patients with Rare and Heavily Pretreated Solid Tumors Treated according to the Results of Tumor Molecular Profiling.

Patients with heavily pretreated advanced cancer or with rare tumors are difficult to treat. Molecular profiling (MP) of tumors to identify biomarkers that predict potential outcomes with individual therapies is an emerging strategy to guide treatment decisions. Patients with rare tumors for which standard-of-care therapy was unavailable or more common tumors for which standard-of-care options had been exhausted underwent MP at a single Australian center. Data regarding treating physicians' choice of therapy, MP results and recommendations, and patient outcomes were collected. Seven patients had received prior standard first-line therapy (PST), 16 had rare tumors, and 31 had been heavily pretreated (HPT; ≥2 prior lines). Most treatments suggested by MP (541/594; 91.1%) were common chemotherapy drugs available in generic formulations. MP-guided therapy recommendations differed from physician's recommendations in 48 patients (88.9%). MP-guided therapy produced clinical benefit (improved QOL and/or performance status, symptoms, bodyweight, or RECIST) in 19/31 (61.3%), 11/16 (68.8%), and 3/7 (42.9%) patients with HPTs, rare tumors, and PSTs, respectively, and had a PFS ratio ≥1.3 in 22/37 evaluable patients (59.5%; 95% confidence interval 44–76%). The null hypothesis that ≤15% of these patients would have a PFS ratio ≥1.3 was rejected (one-sided p < 0.0001). In conclusion, using MP to guide therapy selection is feasible in clinical practice and may improve patient outcomes.

Effects of Long-Term Odanacatib Treatment on Bone Gene Expression in Ovariectomized Adult Rhesus Monkeys: Differentiation From Alendronate.

Similar efficacy of the cathepsin K inhibitor odanacatib (ODN) and the bisphosphonate alendronate (ALN) in reducing bone turnover markers and increasing bone mineral density in spine and hip were previously demonstrated in ovariectomized (OVX)-monkeys treated for 20 months in prevention mode. Here, we profiled RNA from tibial metaphysis and diaphysis of the same study using Affymetrix microarrays, and selected 204 probe sets (p < 0.001, three-group ANOVA) that were differentially regulated by ODN or ALN versus vehicle. Both drugs produced strikingly different effects on known bone-related genes and pathways at the transcriptional level. Although ALN either reduced or had neutral effects on bone resorption-related genes, ODN significantly increased the expression of osteoclast genes (eg, APC5, TNFRSF11A, CTSK, ITGB3, and CALCR), consistent with previous findings on the effects of this agent in enhancing the number of nonresorbing osteoclasts. Conversely, ALN reduced the expression of known bone formation-related genes (eg, TGFBR1, SPP1, RUNX2, and PTH1R), whereas ODN either increased or had neutral effects on their expression. These differential effects of ODN versus ALN on bone resorption and formation were highly correlative to the changes in bone turnover markers, cathepsin K (Catk) target engagement marker serum C-terminal cross-linked telopeptide (1-CTP) and osteoclast marker tartrate resistant acid phosphatase isoform 5b (TRAP5b) in the same monkeys. Overall, the molecular profiling results are consistent with the known pharmacological actions of these agents on bone remodeling and clearly differentiate the molecular mechanisms of ODN from the bisphosphonates.

The NEXT-1 (Next generation pErsonalized tX with mulTi-omics and preclinical model) trial: prospective molecular screening trial of metastatic solid cancer patients, a feasibility analysis

We conducted a prospective genomic screening trial with high throughput sequencing and copy number variation (CNV) assay, and immunohistochemistry array in metastatic solid cancer patients. We used Ion AmpliSeq Cancer Hotspot Panel v2 and nCounter Copy Number Variation Assay (21 genes) to identify molecular targets for potential matched therapy. Metastatic solid tumor patients were prospectively consented for molecular profiling tests. The primary outcome for this trial was the feasibility of molecular tests and response rate (matched vs non-matched treatment). Between November 2013 and August 2014, a total of 428 metastatic solid tumor patients were enrolled on to this study. The mutational profiles were obtained for 407 (95.1%) patients. CNV 21-gene assays were successfully performed in 281 (65.7%) of 428 patients. Of the 407 patients with molecular profiling results, 342 (84.0%) patients had one or more aberrations detected. Of the 342 patients, 103 patients were matched to molecularly targeted agents in the context of clinical trials or clinical practice. The response rate was significantly higher in the genome-matched treated group for gastrointestinal/hepatobiliary/rare tumors (matched vs non-matched treatment, 42.6% vs 24.3%, P = .009) and lung cancer cohort (matched vs non-matched treatment, 61.2% vs 28.6% < P = .001) when compared with the non-matched group. In this trial, we demonstrate that genome-matched treatment based on molecular profiling result in better treatment outcome in terms of response rate.

Results of comprehensive molecular profiling of gliomas and the potential therapeutic implications.

2013 Background: Gliomas are the most common type of primary brain tumors with underlying molecular heterogeneity contributing to differential treatment response. Our retrospective study was design...

Genome-wide multi-omics profiling of colorectal cancer identifies immune determinants strongly associated with relapse.

The use and benefit of adjuvant chemotherapy to treat stage II colorectal cancer (CRC) patients is not well understood since the majority of these patients are cured by surgery alone. Identification of biological markers of relapse is a critical challenge to effectively target treatments to the ~20% of patients destined to relapse. We have integrated molecular profiling results of several “omics” data types to determine the most reliable prognostic biomarkers for relapse in CRC using data from 40 stage I and II CRC patients. We identified 31 multi-omics features that highly correlate with relapse. The data types were integrated using multi-step analytical approach with consecutive elimination of redundant molecular features. For each data type a systems biology analysis was performed to identify pathways biological processes and disease categories most affected in relapse. The biomarkers detected in tumors urine and blood of patients indicated a strong association with immune processes including aberrant regulation of T-cell and B-cell activation that could lead to overall differences in lymphocyte recruitment for tumor infiltration and markers indicating likelihood of future relapse. The immune response was the biologically most coherent signature that emerged from our analyses among several other biological processes and corroborates other studies showing a strong immune response in patients less likely to relapse.

DNA mismatch repair deficiency and benefit from adjuvant bevacizumab in NSABP C-08: Molecular profiling results.

55 Background: The purpose of this study was to identify biomarkers that define a subset of patients who received benefit from bevacizumab (bev) in NSABP trial C-08, even though bev did not improve outcomes over standard adjuvant chemotherapy (CT) in the treatment of stage II and III colon cancer. Methods: A randomly selected cohort of C-08 cases (N=500) were profiled for whole genome expression (N=445) and for mutations (N=463) in KRAS, NRAS, PIK3CA, and MET. BRAF mutations and mismatch repair (MMR) status were profiled on the available cases (N=1,764 and 1,993, respectively). Cox proportional hazard models were used to assess prognosis and prediction for the value of bev using overall survival (0S) and time to recurrence (TTR) as end points. Results: The effect of bev was different for MMR deficient (MMR-d) and proficient tumors for OS (interaction p=.035) but not TTR (interaction p=.08). Patients with MMR-d (N=252) tumors showed a significant benefit from the addition of bev to CT for OS (hazard ratio =0.52 (95% CI: 0.29-0.94, p=0.028). KRAS, NRAS, PIK3CA, and MET were not significant for interaction with bev in the discovery cohort. BRAF mutations were associated with MMR status (p&lt;.0001) and the prognostic value of MMR depended on BRAF for TTR (interaction p=.027) but not OS (interaction p=.31). The effect of bev was independent of BRAF (interaction p=.28 TTR and .37 OS). Three-factor interaction tests for bev, MMR, and BRAF were not significant for either endpoint. Gene expression analysis with BRB array tools identified 5 BioCarta pathways (p&lt;0.05) which differentially expressed in 4 statistical tests; 4 of these pathways were directly or indirectly involved in T cell activation and one was involved in the activation of VEGF. Conclusions: Patients in C-08 with MMR-d tumors received benefit from bev treatment but these results need to be validated in a separate study. Gene expression data suggest that T-cells may be differentially expressed based on MMR status. Activation of VEGF has been shown to suppress T-cell development (Ohm et al. Blood. 2003:10;4878). A speculative possibility for the benefit of bev in MMR-d tumors may be due to blocking of VEGF, releasing T cells from VEGF suppression.

P3-16-01: Safety Profile and Clinical Activity of Single-Agent BKM120, a Pan-Class I PI3K Inhibitor, for the Treatment of Patients with Metastatic Breast Carcinoma.

Abstract Background Phosphatidylinositol 3-kinase (PI3K) is critical to cancer cell growth, survival, and metabolism. BKM120 is an oral pan-class I (α, β, γ, δ) PI3K inhibitor that has demonstrated in vitro and in vivo tumor cell growth inhibition in a range of cancer types including breast cancer. Materials and methods: The Phase I study CBKM120X2101 investigating single-agent daily BKM120 in patients (pts) with advanced solid tumors has been recently completed with the maximum tolerated dose established at 100 mg/day. Here, we report the analysis of metastatic breast carcinoma (MBC) pts enrolled in this study. Results: Overall, 83 pts have enrolled, 21 of whom have MBC. At the cut-off date of 25th February 2011, 20 MBC pts were evaluable: 1 pt at 80 mg, 1 pt at 150 mg and 18 pts at 100 mg. Patient characteristics were as follows: median age 55 years (range 37–71); performance status ECOG 0/1/2 for 7/12/1 pts, respectively; visceral disease was reported in 16 pts, including liver, 10 pts (50%); lung, 9 pts (45%); and pleura, 5 pts (25%); all pts had &amp;gt;3 lines of systemic therapy (3-12). The median time from last treatment and study entry was 46 days (29-235). The median duration of BKM120 treatment administered was 7.5 weeks (1.0−96.4). The most frequent grade 3 drug-related adverse events (AEs) were: transaminases increase, 4 pts; psychiatric disorders, 3 pts, consisting of anxiety, affective disorder, and mood alteration (1 pt each); diarrhea, 2 pts; fatigue, 2 pts; and hyperglycemia, 1 pt. The only grade 4 drug-related AE was hyperglycemia, reported in 1 pt at 150 mg. Most AEs were manageable with treatment interruption and dose reductions. Eighteen pts were evaluable for objective tumor response by RECIST. Two pts (11%) exhibited partial responses, which were confirmed in a triple-negative MBC pt, and unconfirmed in an ER+ HER2− MBC pt. For these 2 pts, the treatment duration was 29+ (ongoing) and 6 months, respectively. An additional 9 pts (50%) had stable disease, lasting &amp;gt;4 months in 7 pts (35%). Conclusions: This preliminary analysis showed that BKM120 has single-agent activity in heavily pretreated pts with MBC, and an acceptable safety profile. Molecular profiling and updated pharmacokinetic results will be presented at the meeting. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-16-01.

Vascular regrowth following photodynamic therapy in the chicken embryo chorioallantoic membrane

Photodynamic therapy (PDT) induces damage to the endothelium, which can lead to increased vascular permeability and, under intensive PDT conditions, even to platelet aggregation, vasoconstriction, and blood flow stasis. Eventually, ischemia, hypoxia, and inflammation can occur, resulting in angiogenesis. We studied the sequence of the vascular events after Visudyne®-PDT in the chicken chorioallantoic membrane (CAM) at day 11 of development. Using epi-fluorescence microscopy, we monitored the regrowth of capillaries in the PDT treated area. Immediately after irradiation, the treatment resulted in blood flow arrest. And 24 h post PDT, sprouting of new blood vessels was observed at the edge of the PDT zone. Neovessels looping out from the edge of the PDT zone gave rise to specialized endothelial tip structures guiding the vessels towards the center of the treated area. At 48 h almost all of the treated area was repopulated with functional but morphologically altered vasculature. These observations also showed reperfusion of some of the vessels that had been closed by the PDT treatment. CAM samples were immunohistochemically stained for Ki-67 showing proliferation of endothelial cells in the PDT area. Also, several markers of immature and angiogenic blood vessels, such as αVβ3-integrin, vimentin and galectin-1, were found to be enhanced in the PDT area, while the endothelial maturation marker intercellular adhesion molecule (ICAM)-1 was found to be suppressed. These results demonstrate that the new vascular bed is formed by both neo-angiogenesis and reperfusion of existing vessels. Both the quantitative real-time RT–PCR profile and the response to pharmacological treatment with Avastin®, an inhibitor of angiogenesis, suggest that angiogenesis occurs after PDT. The observed molecular profiling results and the kinetics of gene regulation may enable optimizing combination therapies involving PDT for treatment of cancer and other diseases.

Molecular profiling in the diagnosis of synchronous primary cancers of the endometrium and ovary

e16500 Background: Synchronous primary cancers of the endometrium and ovary (SYNCH) have a better prognosis than either primary endometrial or ovarian cancer with metastasis (MET). Distinguishing SYNCH from MET can be a challenge when the neoplasms share similar histologic features. To date, histologic characteristics are used to make this distinction and guide treatment decisions. The purpose of this study was to determine if objective discrimination using an integrated molecular approach can assist in making this distinction. Methods: Paired tumor samples from 16 SYNCH and 11 MET were sent for mutational profiling. 13 samples were sent as blinded duplicates for internal validation. The mutational profile for each neoplasm was determined using PCR and quantitative genotyping for a panel of LOH markers targeting 1p,3p,5q,9p,10q,17p,17q,21q,22q and k-ras-2 exon 1 DNA sequencing for point mutational change. The paired samples were then re-defined as SYNCH versus MET based on the profile results. Survival was compared using Kaplan-Meier. Results: The results are in the table. All of the duplicate samples had the same molecular profile results. Median survival based on pathologic diagnosis was 106 months in SYNCH versus 37 months in MET (p=0.07). The median survival was 110 months in both groups based on molecular profiling results. For patients with a diagnosis of SYNCH by both pathology and molecular profiling median survival was 117 months. For those with a pathologic diagnosis of SYNCH but metastasis on molecular profiling survival was 129 months. Conclusions: Molecular profiling to distinguish synchronous primary cancers from either primary endometrial or ovarian cancer with metastasis, is currently being evaluated. In this study, the pathologic diagnosis was a better indicator of survival than a diagnosis based on molecular profiling alone. Further studies are being performed to determine the role of molecular profiling in women who are thought to have synchronous primary cancers of the endometrium and ovary. [Table: see text] [Table: see text]

New molecular targets in malignant gliomas

The purpose of this review is to provide an update on the identification of novel molecular targets in neurooncology and their translation into clinical practice. Basic research is providing novel insights into the complex molecular pathways involved in the pathogenesis of malignant glioma transformation and progression. By unraveling the intricate signaling cascades responsible for sustained proliferation, angiogenesis, invasion and resistance to apoptosis in glioma, we are now confronted with an ever-expanding list of molecular targets. Clinical studies using single targeted therapies have been disappointing, therefore providing the impetus for novel combination drug trials. The potential for combination regimens brings the challenge of testing an exponentially growing number of treatments. Success will depend on an integration of novel treatment regimens and innovative trial designs combined with careful patient selection based on the results of molecular profiling of tumor tissue. Technologic advances in oncogenomics, proteomics and functional genomic screens (such as synthetic lethality) are providing mechanisms to rapidly identify the critical targets whose inactivation will lead to a substantive tumor growth arrest. Tumor tissue biomarkers that identify those tumors most likely to respond to a specific inhibitor are needed as a mechanism toward tailoring therapy to the individual patient with malignant glioma.