Douglas Robinson

Phase I/II and pharmacodynamic study of dovitinib (TKI258), an inhibitor of fibroblast growth factor receptors and VEGF receptors, in patients with advanced melanoma.

Purpose: Dovitinib (TKI258) is an orally available inhibitor of fibroblast growth factor (FGF), VEGF, and platelet-derived growth factor receptors. This phase I/II dose–escalation study was conducted to evaluate the safety, pharmacodynamics, and preliminary efficacy of dovitinib in the treatment of advanced melanoma. Experimental Design: Patients with advanced melanoma resistant or refractory to standard therapies or for whom no standard therapy was available were enrolled. Dovitinib was administered at doses ranging from 200 to 500 mg/d. Results: Forty-seven patients were enrolled. The most frequently reported adverse events were fatigue (77%; grade ≥3, 28%), diarrhea (77%; grade ≥3, 11%), and nausea (77%; grade ≥3, 9%). Six dose-limiting toxicities were observed in the 400-mg and 500-mg dose cohorts, which consisted of grade 3 nausea, fatigue, and diarrhea and grade 4 fatigue events. The maximum tolerated dose was 400 mg/d. The best tumor response was stable disease, which was observed in 12 patients. Increases in plasma FGF23, VEGF, and placental growth factor and decreases in soluble VEGF receptor 2 were noted during the first cycle of treatment, consistent with FGF receptor (FGFR) and VEGF receptor (VEGFR) inhibition. Dynamic contrast-enhanced MRI analysis showed a dose-dependent decrease in tumor blood flow and vascular permeability with dovitinib therapy. A decrease in FGFR phosphorylation was observed in paired tumor biopsy samples from a patient treated with dovitinib at a dose of 400 mg/d. Conclusions: At a dose of 400 mg/d, dovitinib showed an acceptable safety profile and limited clinical benefit and inhibited FGFR and VEGFR. Clin Cancer Res; 17(23); 7451–61. ©2011 AACR.

Correlative Analysis of Genetic Alterations and Everolimus Benefit in Hormone Receptor–Positive, Human Epidermal Growth Factor Receptor 2–Negative Advanced Breast Cancer: Results From BOLERO-2

PURPOSE To explore the genetic landscape of tumors from patients enrolled on the BOLERO-2 trial to identify potential correlations between genetic alterations and efficacy of everolimus treatment. The BOLERO-2 trial has previously demonstrated that the addition of everolimus to exemestane prolonged progression-free survival by more than twofold in patients with hormone receptor-positive, human epidermal growth factor receptor 2-negative, advanced breast cancer previously treated with nonsteroidal aromatase inhibitors. PATIENTS AND METHODS Next-generation sequencing was used to analyze genetic status of cancer-related genes in 302 archival tumor specimens from patients representative of the BOLERO-2 study population. Correlations between the most common somatic alterations and degree of chromosomal instability, and treatment effect of everolimus were investigated. RESULTS Progression-free survival benefit with everolimus was maintained regardless of alteration status of PIK3CA, FGFR1, and CCND1 or the pathways of which they are components. However, quantitative differences in everolimus benefit were observed between patient subgroups defined by the exon-specific mutations in PIK3CA (exon 20 v 9) or by different degrees of chromosomal instability in the tumor tissues. CONCLUSION The data from this exploratory analysis suggest that the efficacy of everolimus was largely independent of the most commonly altered genes or pathways in hormone receptor-positive, human epidermal growth factor receptor 2-negative breast cancer. The potential impact of chromosomal instabilities and low-frequency genetic alterations on everolimus efficacy warrants further investigation.

Molecular Alterations and Everolimus Efficacy in Human Epidermal Growth Factor Receptor 2–Overexpressing Metastatic Breast Cancers: Combined Exploratory Biomarker Analysis From BOLERO-1 and BOLERO-3

PURPOSE Two recent phase III trials, BOLERO-1 and BOLERO-3 (Breast Cancer Trials of Oral Everolimus), evaluated the addition of everolimus to trastuzumab and chemotherapy in human epidermal growth factor receptor 2-overexpressing advanced breast cancer. The current analysis aimed to identify biomarkers to predict the clinical efficacy of everolimus treatment. METHODS Archival tumor samples from patients in BOLERO-1 and BOLERO-3 were analyzed using next-generation sequencing, immunohistochemistry, and Sanger sequencing. RESULTS Biomarker data were available for 549 patients. PIK3CA activating mutations and PTEN loss were reported in 30% and 16% of BOLERO-1 samples and in 32% and 12% of BOLERO-3 samples, respectively. PI3K pathway was hyperactive (PIK3CA mutations and/or PTEN loss and/or AKT1 mutation) in 47% of BOLERO-1 and 41% of BOLERO-3 samples. In both studies, differential progression-free survival (PFS) benefits of everolimus were consistently observed in patient subgroups defined by their PI3K pathway status. When analyzing combined data sets of both studies, everolimus was associated with a decreased hazard of progression in patients with PIK3CA mutations (hazard ratio [HR], 0.67; 95% CI, 0.45 to 1.00), PTEN loss (HR, 0.54; 95% CI, 0.31 to 0.96), or hyperactive PI3K pathway (HR, 0.67; 95% CI, 0.48 to 0.93). Patients with wild-type PIK3CA (HR, 1.10; 95% CI, 0.83 to 1.46), normal PTEN (HR, 1.00; 95% CI, 0.80 to 1.26), or normal PI3K pathway activity (HR, 1.19; 95% CI, 0.87 to 1.62) did not derive PFS benefit from everolimus. CONCLUSION This analysis, although exploratory, suggests that patients with human epidermal growth factor receptor 2-positive advanced breast cancer having tumors with PIK3CA mutations, PTEN loss, or hyperactive PI3K pathway could derive PFS benefit from everolimus.

A Five-Gene Hedgehog Signature Developed as a Patient Preselection Tool for Hedgehog Inhibitor Therapy in Medulloblastoma

Purpose: Distinct molecular subgroups of medulloblastoma, including hedgehog (Hh) pathway–activated disease, have been reported. We identified and clinically validated a five-gene Hh signature assay that can be used to preselect patients with Hh pathway–activated medulloblastoma. Experimental Design: Gene characteristics of the Hh medulloblastoma subgroup were identified through published bioinformatic analyses. Thirty-two genes shown to be differentially expressed in fresh-frozen and formalin-fixed paraffin-embedded tumor samples and reproducibly analyzed by RT-PCR were measured in matched samples. These data formed the basis for building a multi-gene logistic regression model derived through elastic net methods from which the five-gene Hh signature emerged after multiple iterations. On the basis of signature gene expression levels, the model computed a propensity score to determine Hh activation using a threshold set a priori. The association between Hh activation status and tumor response to the Hh pathway inhibitor sonidegib (LDE225) was analyzed. Results: Five differentially expressed genes in medulloblastoma (GLI1, SPHK1, SHROOM2, PDLIM3, and OTX2) were found to associate with Hh pathway activation status. In an independent validation study, Hh activation status of 25 medulloblastoma samples showed 100% concordance between the five-gene signature and Affymetrix profiling. Further, in medulloblastoma samples from 50 patients treated with sonidegib, all 6 patients who responded were found to have Hh-activated tumors. Three patients with Hh-activated tumors had stable or progressive disease. No patients with Hh-nonactivated tumors responded. Conclusions: This five-gene Hh signature can robustly identify Hh-activated medulloblastoma and may be used to preselect patients who might benefit from sonidegib treatment. Clin Cancer Res; 21(3); 585–93. ©2014 AACR.

Abstract 4818: The predictive value of a 5-gene signature as a patient pre-selection tool in medulloblastoma for Hedgehog pathway inhibitor therapy

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Medulloblastoma (MB), an invasive primitive neuroectodermal tumor of the posterior fossa, is the most common brain tumor in children, comprising ∼20% of childhood and <2% of adult brain tumors. Current standard of care treatment, surgery followed by craniospinal radiation and chemotherapy, can lead to significant long term toxicities, especially in very young patients. At the time of relapse, no standard salvage therapy exists. Therefore, targeted therapies are needed. Several studies have used gene expression profiling to identify distinct molecular subgroups of MB, including one characterized by activated Hedgehog (Hh) signaling. Using available gene expression data, a 5-gene Hh signature that can be assayed in formalin-fixed paraffin-embedded (FFPE) samples by standard RT-PCR was identified. Two sets of matched fresh frozen and FFPE MB specimens were used; one for development of the 5-gene signature and one for its independent validation. Hh activation status was determined in fresh frozen samples by gene expression profiling using the GeneChip human genome U133 Plus 2.0 array (Affymetrix, Santa Clara, CA) and in FFPE samples by RT-PCR analysis. The 5-gene Hh signature was selected from a larger panel of 73 genes that were associated with the Hh subgroup classification, as determined by standard Affymetrix gene expression profiling. Eighteen of these genes shown to be differentially expressed in FFPE were chosen for the RT-PCR gene card that formed the basis of the Elastic Net model building exercise. Based on the expression levels of the 5-gene signature, a predictive model was used to compute a propensity score (0-100%) representative of the Hh activation status of each tumor sample. The median propensity scores for the 17 non-Hh-activated tumors was 0.7% (range: 0.1-3.0%) compared to 87.9% (range: 69.1-97.6%) in the eight Hh-activated tumors. Hh activation status of 25 independent MB samples defined by the 5-gene signature and assayed by RT-PCR were in 100% agreement with the Hh activation status determined by gene expression profiling. In order to determine the predictive value of this assay as a tool to identify patients who might benefit from treatment with a Hh pathway inhibitor, MB samples from patients (n=13) enrolled in recent phase I trials of the Smoothened inhibitor LDE225 were analyzed and correlated with the respective tumor responses. Using the 5-gene signature, all patients (n=4) who responded to LDE225 treatment (PR or CR) were found to have Hh-pathway activated tumors, whereas all patients who did not respond (n=9) were found to have Hh non-activated tumors. These results suggest an association between Hh activation status determined by the 5-gene Hh signature and tumor response to LDE225 treatment. Data from an ongoing phase I/II trial in pediatric patients will enable determination of the predictive value of this patient pre-selection assay. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4818. doi:1538-7445.AM2012-4818

HER2 status in advanced or metastatic gastric, esophageal, or gastroesophageal adenocarcinoma for entry to the TRIO-013/LOGiC trial of lapatinib

HER2/ERBB2 status is used to select patients for HER2-targeted therapy. HER2/ERBB2 amplification/overexpression of upper gastrointestinal (UGI) adenocarcinomas was determined locally or in two central laboratories to select patients for the TRIO-013/LOGiC trial of chemotherapy with or without lapatinib. Patients selected locally had central laboratory confirmation of HER2 amplification for inclusion in the primary efficacy population. HER2 was assessed with PathVysion or IQ PharmDx FISH and HercepTest immunohistochemistry assays. Associations with outcomes were retrospectively evaluated. Overall, HER2 status was determined in UGI cancers from 4,674 patients in a central laboratory for eligibility (1,995 cases) and for confirmation of local HER2 results (333 cases). Of 1,995 adenocarcinomas screened centrally, 322 (16.1%) had HER2-amplified disease with 29 (1.5%) showing HER2 genomic heterogeneity. Men and older patients had higher rates of amplification. Of 545 patients accrued to the trial (gastric, 87.3%; GEJ, 8.3% and esophageal cancer, 4.4%), 487 patients (89%) were centrally confirmed as having HER2-amplified disease. Concordance between central and local HER2 testing was 83%. Concordance between PathVysion and IQ PharmDx FISH assays was 99% and FISH in the two central laboratories was 95%. Lapatinib-treated Asian participants and those less than 60 years had significant improvement in progression-free survival (PFS), particularly among those whose cancers had 5.01–10.0 and >10.0-fold amplification of HER2. In conclusion, HER2 is commonly amplified in UGI adenocarcinomas with amplification highly correlated to overexpression, and HER2 amplification levels correlated with PFS. While HER2 genomic heterogeneity occurs, its prevalence is low. Mol Cancer Ther; 16(1); 228–38. ©2016 AACR.

Abstract 4564: Assessment of genetic alterations using next-generation sequencing in postmenopausal women with hormone receptor-positive, HER2-negative advanced breast cancer: results from the BOLERO-2 phase III trial.

The BOLERO-2 phase III trial compared the combination of everolimus and exemestane to placebo and exemestane in 724 postmenopausal women with hormone receptor-positive, HER2-negative advanced breast cancer. Results showed significant improvement in progression-free survival, response rate, and clinical benefit rate. Although significant benefit has been observed in all prospectively defined subgroups, variations were seen among patients. As the first step to delineate the molecular determinants of sensitivity to everolimus and interactions between estrogen receptor and mTOR pathways, we used next-generation sequencing technology to comprehensively assess the genetic alterations in archival tumor specimens. Formalin-fixed, paraffin-embedded archival tumor samples were obtained from 496 patients. DNA was extracted from 348 samples of sufficient quantity and 230 samples (representing approximately 1/3 of all patients) qualified for the analysis. No indication of sampling bias was observed when the data were assessed and compared against several covariates as well as to the full trial data. The coding regions of 182 cancer-related genes were analyzed using an Illumina HiSeq 2000. Mutations and copy number variations were evaluated in 230 samples for which DNA extraction, library construction, and hybrid capture were successful and sequencing depth was sufficient (250-1500×). Alterations predicted to be germline events were discarded. One hundred seventy-three genes were found to be altered in at least one tumor sample. The number of alterations per sample varied from 1 to 24 (average 9 ± 4). Sequence variations (N = 1565) consisted primarily of point mutations (85%), followed by deletions (9%) and insertions (6%). 225 of 230 (98%) patients had at least two sequence variations. The most frequently mutated genes included PIK3CA (49%), TP53 (24%), and ARID1A (16%). ESR1 and IGF1R mutations were detected in 9% and 4% of the cohort, respectively. PIK3CA and AKT1 (6%) mutations were found to be mutually exclusive. Copy-number alterations comprised of 524 amplifications and 27 bi-allelic deletions. The most frequently amplified genes included CCND1 (32%) and FGFR1 (18%). Specific rearrangements assayed in 14 genes occurred infrequently (9% of the samples). These results demonstrated the feasibility of performing large-scale next-generation sequencing in a global phase III clinical trial. High alteration frequencies were observed in specific genes, resulting in activated PI3K, FGFR1, and estrogen receptor pathways, corroborating the rationale of concomitant targeting of the mTOR and estrogen receptor pathways. The results generate testable hypotheses for the development of combinations of new targeted therapies in this patient population. Citation Format: Jose Baselga, Martine Piccart, Hope Rugo, David Chen, Howard A. Burris, Mario Campone, Shinzaburo Noguchi, Alejandra Perez, Inas Deleu, Mikhail Shtivelband, Louise Provencher, Adnan Derti, Alan Huang, Rob McDonald, Creton Kalfoglou, Douglas Robinson, Tetiana Taran, Tarek Sahmoud, David Lebwohl, Gabriel N. Hortobagyi. Assessment of genetic alterations using next-generation sequencing in postmenopausal women with hormone receptor-positive, HER2-negative advanced breast cancer: results from the BOLERO-2 phase III trial. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4564. doi:10.1158/1538-7445.AM2013-4564

Abstract A26: Detecting PIK3CA mutations in circulating cell-free DNA from patients with metastatic cancer: An exploratory analysis in patients with endometrial and lung cancer

Background: Personalized therapy relies on the ability to characterize the tumor at the time of treatment. Circulating cell-free DNA (cfDNA) may offer the potential for representative mutation analysis in patients with cancer irrespective of tumor tissue availability. Recent publications have started to establish the feasibility of this approach. For example, 72.5% concordance of PIK3CA mutations was reported between temporally unmatched cfDNA and archival tumor tissue from patients with metastatic breast cancer (Higgins et al. Clin Cancer Res 2012). Here, we present a retrospective analysis evaluating the reliability of detecting PIK3CA mutations in plasma samples from patients with endometrial and lung cancer – two malignancies known to harbor alterations in the PI3K pathway. Methods: Baseline plasma DNA samples were available from 61 patients with advanced endometrial cancer (NCT01289041) and 37 patients with advanced non-small cell lung cancer (NSCLC; NCT01297491). BEAMing technology (Richardson & Iglehart. Clin Cancer Res 2012) and Sanger analysis were used in all samples. Sanger analysis was able to detect any mutation in exons 1, 5, 7, 9, and 20, whereas only selected mutations (14 in total) known to affect PI3K function in exons 1, 4, 7, 9, and 20 could be detected in cfDNA. Plasma samples were all temporally unmatched to the archival tissue specimen. Concordance analysis was performed by comparing the mutation status of samples (i.e. the proportion of samples that were detected as either wildtype or mutant consistently) between Sanger and cfDNA sequencing. Results: 54 of 61 patients with endometrial cancer had interpretable mutation results by both Sanger and cfDNA analysis. Concordance of PIK3CA mutation was 74% between plasma and tissue. The PIK3CA mutations detected in cfDNA were distributed over exons 1, 7, 9, and 20 (29%, 19%, 33%, and 19%, respectively). Among the 37 patients with NSCLC, overall concordance was 54%. Variant distribution of PIK3CA mutations in this small number of lung tumors appeared to differ from the usual “hotspots” as two-thirds of mutations detected by Sanger analysis were not available on the BEAMing panel. Conclusion: Concordance for PIK3CA mutation between temporally unmatched archival specimens and blood samples in this endometrial cancer patient population was in line with the published rate for metastatic breast cancer. The different mutations identified in samples from patients with NSCLC indicate a need for better understanding of the potential role of the PI3K pathway in this tumor type. Overall these results support the feasibility of assessing PIK3CA mutations in plasma samples. The outcomes show a similar trend to multiple recent publications, thus warranting rapid further exploration of the clinical utility of cfDNA in metastatic cancer. Citation Format: Emmanuelle di Tomaso, Bradley Monk, Grace Dy, Douglas Robinson, Paola Aimone, Lucia Trandafir, Cristian Massacesi, Samit Hirawat. Detecting PIK3CA mutations in circulating cell-free DNA from patients with metastatic cancer: An exploratory analysis in patients with endometrial and lung cancer. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A26.

Abstract A02: Biomarkers of drug response to buparlisib: Results of next-generation sequencing in a phase II trial of advanced endometrial carcinoma

Background: Genetic alterations in the phosphatidylinositol 3-kinase (PI3K) pathway are frequent in endometrial carcinoma (EC; 59% PIK3CA and 66% PTEN alterations; Kandoth C et al., Nature 2013), thus providing a rationale for testing PI3K inhibitors in this tumor type. A Phase II, single-arm clinical trial of buparlisib, an oral pan-PI3K inhibitor that inhibits all four class I PI3K isoforms (α, β, δ, γ), as second-line therapy, was conducted in advanced EC (N=70). All patients (pts) received buparlisib 100 mg/day until disease progression or discontinuation due to an adverse event. The primary objective was objective response rate (RECIST v1.1) in pts with PI3K-activated tumors (PIK3CA and/or PTEN alteration) or in all pts. The activity of single-agent buparlisib was marginal and PI3K pathway activation was not associated with a better outcome in this pt population (Teneriello MG et al., ICACT 2013). Retrospective molecular characterization was undertaken to assess a potential alternate contribution to PI3K pathway activation in EC and the impact on response to treatment. Methods: Next-generation sequencing data were generated for 51 pts (serous n=17, endometrioid n=28, unknown n=6) and targeted coding regions of approximately 400 genes. Using standard bioinformatics tools, short nucleotide variants (SNVs) were called. To generate signatures of PI3K pathway activation, data from the TCGA endometrial cohort (Kandoth C et al., Nature 2013) were first leveraged. Levels of pAKT (pT308 and pS473) and pS6 (pS235 and pS240), proxies for PI3K pathway activation, were modeled using SNVs in the TCGA endometrial cohort. Specifically, elastic net models predicting pAKT and pS6 were optimized using somatic SNVs for endometrioid (6-gene model) and serous (3-gene model) subtypes separately. The TCGA models were then used to create pathway activation scores for each pt based on their molecular profile. Results: No significant relationships were found for pts with the serous subtype, hence the results focus on the endometrioid subtype. Subgroup analysis was performed to assess trends in progression-free survival (PFS) using Cox proportional hazards regression. Pts with the lowest PI3K pathway activation scores, based on the pAKT TCGA model, including pts with PTEN wildtype (n=5), trended towards a lower risk of PFS event compared with pts with moderate or high activation (n=23). In addition, pts with the highest pathway activation scores, based on the pS6 TCGA model, including pts with alterations in mTOR (n=5), trended towards a higher risk of PFS event compared with pts with moderate or low activation (n=23). When combining the two signatures, pts with high pS6 activation (n=5) had a higher risk of PFS event followed by pts with low pS6 and high pAKT scores (n=19) compared with pts with low pS6 and low pAKT scores (n=4). PIK3CA mutations were unrelated to pAKT in the TCGA model for the endometrioid subtype, but PIK3CA-mutated tumors in the TCGA model had slightly higher levels of pS6. Conclusion: In both models, pts with the lowest levels of PI3K pathway activation trended towards a lower risk of PFS event compared with pts with moderate or high levels of PI3K activation. A possible explanation is that the high levels of PI3K pathway activation observed in EC may represent a challenge for single-agent inhibition. Alternatively, for this single-arm study in EC, we can only elucidate the prognostic value of the biomarker and not the contribution of the treatment per se. Further validation with larger sample sizes is warranted to support this hypothesis further. Citation Format: Nadia Solovieff, Angad Pal Singh, Hans Bitter, Markus Riester, Michael Teneriello, Amit Oza, Bradley Monk, Douglas Robinson, Lucia Trandafir, Cristian Massacesi, Emmanuelle di Tomaso, R. Wendel Naumann. Biomarkers of drug response to buparlisib: Results of next-generation sequencing in a phase II trial of advanced endometrial carcinoma. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A02.