Clare Morgan

BCL-2 system analysis identifies high-risk colorectal cancer patients

Objective The mitochondrial apoptosis pathway is controlled by an interaction of multiple BCL-2 family proteins, and plays a key role in tumour progression and therapy responses. We assessed the prognostic potential of an experimentally validated, mathematical model of BCL-2 protein interactions (DR_MOMP) in patients with stage III colorectal cancer (CRC). Design Absolute protein levels of BCL-2 family proteins were determined in primary CRC tumours collected from n=128 resected and chemotherapy-treated patients with stage III CRC. We applied DR_MOMP to categorise patients as high or low risk based on model outputs, and compared model outputs with known prognostic factors (T-stage, N-stage, lymphovascular invasion). DR_MOMP signatures were validated on protein of n=156 patients with CRC from the Cancer Genome Atlas (TCGA) project. Results High-risk stage III patients identified by DR_MOMP had an approximately fivefold increased risk of death compared with patients identified as low risk (HR 5.2, 95% CI 1.4 to 17.9, p=0.02). The DR_MOMP signature ranked highest among all molecular and pathological features analysed. The prognostic signature was validated in the TCGA colon adenocarcinoma (COAD) cohort (HR 4.2, 95% CI 1.1 to 15.6, p=0.04). DR_MOMP also further stratified patients identified by supervised gene expression risk scores into low-risk and high-risk categories. BCL-2-dependent signalling critically contributed to treatment responses in consensus molecular subtypes 1 and 3, linking for the first time specific molecular subtypes to apoptosis signalling. Conclusions DR_MOMP delivers a system-based biomarker with significant potential as a prognostic tool for stage III CRC that significantly improves established histopathological risk factors.

A Stepwise Integrated Approach to Personalized Risk Predictions in Stage III Colorectal Cancer

Purpose: Apoptosis is essential for chemotherapy responses. In this discovery and validation study, we evaluated the suitability of a mathematical model of apoptosis execution (APOPTO-CELL) as a stand-alone signature and as a constituent of further refined prognostic stratification tools. Experimental Design: Apoptosis competency of primary tumor samples from patients with stage III colorectal cancer (n = 120) was calculated by APOPTO-CELL from measured protein concentrations of Procaspase-3, Procaspase-9, SMAC, and XIAP. An enriched APOPTO-CELL signature (APOPTO-CELL-PC3) was synthesized to capture apoptosome-independent effects of Caspase-3. Furthermore, a machine learning Random Forest approach was applied to APOPTO-CELL-PC3 and available molecular and clinicopathologic data to identify a further enhanced signature. Association of the signature with prognosis was evaluated in an independent colon adenocarcinoma cohort (TCGA COAD, n = 136). Results: We identified 3 prognostic biomarkers (P = 0.04, P = 0.006, and P = 0.0004 for APOPTO-CELL, APOPTO-CELL-PC3, and Random Forest signatures, respectively) with increasing stratification accuracy for patients with stage III colorectal cancer. The APOPTO-CELL-PC3 signature ranked highest among all features. The prognostic value of the signatures was independently validated in stage III TCGA COAD patients (P = 0.01, P = 0.04, and P = 0.02 for APOPTO-CELL, APOPTO-CELL-PC3, and Random Forest signatures, respectively). The signatures provided further stratification for patients with CMS1-3 molecular subtype. Conclusions: The integration of a systems-biology–based biomarker for apoptosis competency with machine learning approaches is an appealing and innovative strategy toward refined patient stratification. The prognostic value of apoptosis competency is independent of other available clinicopathologic and molecular factors, with tangible potential of being introduced in the clinical management of patients with stage III colorectal cancer. Clin Cancer Res; 23(5); 1200–12. ©2016 AACR.

A preclinical evaluation of the MEK inhibitor refametinib in HER2-positive breast cancer cell lines including those with acquired resistance to trastuzumab or lapatinib

Purpose The MEK/MAPK pathway is commonly activated in HER2-positive breast cancer, but little investigation of targeting this pathway has been undertaken. Here we present the results of an in vitro preclinical evaluation of refametinib, an allosteric MEK1/2 inhibitor, in HER2-positive breast cancer cell lines including models of acquired resistance to trastuzumab or lapatinib. Methods A panel of HER2-positive breast cancer cells were profiled for mutational status and also for anti-proliferative response to refametinib alone and in combination with the PI3K inhibitor (PI3Ki) copanlisib and the HER2-targeted therapies trastuzumab and lapatinib. Reverse phase protein array (RPPA) was used to determine the effect of refametinib alone and in combination with PI3Ki and HER2-inhibitors on expression and phosphorylation of proteins in the PI3K/AKT and MEK/MAPK pathways. We validated our proteomic in vitro findings by utilising RPPA analysis of patients who received either trastuzumab, lapatinib or the combination of both drugs in the NCT00524303/LPT109096 clinical trial. Results Refametinib has anti-proliferative effects when used alone in 2/3 parental HER2-positive breast cancer cell lines (HCC1954, BT474), along with 3 models of these 2 cell lines with acquired trastuzumab or lapatinib resistance (6 cell lines tested). Refametinib treatment led to complete inhibition of MAPK signalling. In HCC1954, the most refametinib-sensitive cell line (IC50 = 397 nM), lapatinib treatment inhibits phosphorylation of MEK and MAPK but activates AKT phosphorylation, in contrast to the other 2 parental cell lines tested (BT474-P, SKBR3-P), suggesting that HER2 may directly activate MEK/MAPK and not PI3K/AKT in HCC1954 cells but not in the other 2 cell lines, perhaps explaining the refametinib-sensitivity of this cell line. Using RPPA data from patients who received either trastuzumab, lapatinib or the combination of both drugs together with chemotherapy in the NCT00524303 clinical trial, we found that 18% (n=38) of tumours had decreased MAPK and increased AKT phosphorylation 14 days after treatment with HER2-targeted therapies. The combination of MEK inhibition (MEKi) with refametinib and copanlisib led to synergistic inhibition of growth in 4/6 cell lines tested (CI @ED75 = 0.39-0.75), whilst the combinations of lapatinib and refametinib led to synergistic inhibition of growth in 3/6 cell lines (CI @ED75 = 0.39-0.80). Conclusion Refametinib alone or in combination with copanlisib or lapatinib could represent an improved treatment strategy for some patients with HER2-positive breast cancer, and should be considered for clinical trial evaluation. The direct down-regulation of MEK/MAPK but not AKT signalling by HER2 inhibition (e.g. by lapatinib or trastuzumab), which we demonstrate occurs in 18% of HER2-positive breast cancers may serve as a potential biomarker of responsiveness to the MEK inhibitor refametinib.

Development of a personalized therapeutic strategy for ERBB-gene-mutated cancers

Background: The application of genomic technologies to patient tumor samples identified groups of signaling pathways which acquire activating mutations. Some cancers are dependent on these mutations and the aberrant proteins resulting from these mutations can be targeted by novel drugs which can eradicate the cancer. Methods: We used www.cbioportal.org to determine the frequency of ERBB mutations in solid tumors. We then determined the sensitivity of a panel of cell lines to clinically available PI3K inhibitors. Using proliferation and apoptosis assays as well as functional interrogation with reverse phase protein arrays we demonstrated the impact of targeting ERBB-mutant cancers with the combination of a PI3K inhibitor and the pan-HER family inhibitor afatinib. Results: In over 14,000 patients we found that 12% of their tumors have an ERBB family gene mutation (EGFR, ERBB2, ERBB3 and ERBB4). In cancers not commonly associated with HER family protein overexpression, such as ovarian, endometrial, melanoma and head and neck cancers (n = 2116), we found that ERBB family mutations are enriched, occurring at rates from 14% to 34% and commonly co-occur with PIK3CA mutations. Importantly, we demonstrate that ERBB family mutant cancers are sensitive to treatment with PI3K inhibitors. Finally we show that the combination of afatinib and copanlisib represents a novel therapeutic strategy for patients whose cancers harbor both ERBB family and PIK3CA mutation. Conclusions: We demonstrate that ERBB family mutations are common in cancers not associated with overexpression or amplification of HER family proteins. These ERBB family mutant cancers are sensitive to treatment with PI3K inhibitors, and when combined with pan-HER inhibitors have synergistic antiproliferative effects.

Frequency, impact and a preclinical study of novel ERBB gene family mutations in HER2-positive breast cancer:

Background: Somatic mutations in the ERBB genes (epidermal growth factor receptor: EGFR, ERBB2, ERBB3, ERBB4) promote oncogenesis and lapatinib resistance in metastatic HER2+ (human epidermal growth factor-like receptor 2) breast cancer in vitro. Our study aimed to determine the frequency of mutations in four genes: EGFR, ERBB2, ERBB3 and ERBB4 and to investigate whether these mutations affect cellular behaviour and therapy response in vitro and outcomes after adjuvant trastuzumab-based therapy in clinical samples. Methods: We performed Agena MassArray analysis of 227 HER2+ breast cancer samples to identify the type and frequency of ERBB family mutations. Of these, two mutations, the somatic mutations ERBB4-V721I and ERBB4-S303F, were stably transfected into HCC1954 (PIK3CA mutant), HCC1569 (PIK3CA wildtype) and BT474 (PIK3CA mutant, ER positive) HER2+ breast cancer cell lines for functional in vitro experiments. Results: A total of 12 somatic, likely deleterious mutations in the kinase and furin-like domains of the ERBB genes (3 EGFR, 1 ERBB2, 3 ERBB3, 5 ERBB4) were identified in 7% of HER2+ breast cancers, with ERBB4 the most frequently mutated gene. The ERBB4-V721I kinase domain mutation significantly increased 3D-colony formation in 3/3 cell lines, whereas ERBB4-S303F did not increase growth rate or 3D colony formation in vitro. ERBB4-V721I sensitized HCC1569 cells (PIK3CA wildtype) to the pan class I PI3K inhibitor copanlisib but increased resistance to the pan-HER family inhibitor afatinib. The combinations of copanlisib with trastuzumab, lapatinib, or afatinib remained synergistic regardless of ERBB4-V721I or ERBB4-S303F mutation status. Conclusions: ERBB gene family mutations, which are present in 7% of our HER2+ breast cancer cohort, may have the potential to alter cellular behaviour and the efficacy of HER- and PI3K-inhibition.

Abstract LB-354: Investigating potential molecular biomarkers for cetuximab response in metastatic colorectal cancer tissue using reverse phase protein array

Colorectal cancer (CRC) is the third and second most commonly diagnosed cancer in males and females, and the second most common cause of cancer-related deaths in the developed world. Identifying the importance of epidermal growth factor (EGF) signalling pathways for the survival of CRC cells (Van Cutsem, Kohne et al. 2011) resulted in development of therapies that target EGF-receptors (EGFR). Late stage CRC patients are mainly treated with monoclonal anti-EGFR antibodies such as cetuximab. Although anti-EGFR therapies have significantly improved survival in CRC patients (Van Cutsem, Kohne et al. 2009), they are not effective in patients with activating KRAS, BRAF, NRAS and PI3KCA mutations (De Roock, Claes et al. 2010). Nevertheless, between 50-60% of patients will not benefit from the additional anti-EGFR treatment, even when these have a quadruple wild-type status (De Roock, De Vriendt et al. 2011), suggesting that novel biomarkers and targeted therapies are required. In this study, we investigate potential biomarkers for cetuximab response in a panel of 93 quadruple wild-type, liver metastatic CRC samples from patient derived xenografts (PDX) with known responses to cetuximab. Matching PDXs were used to measure changes in tumour volumes post cetuximab treatment (Bertotti, Papp et al. 2015) and were later categorised as regressing, progressing or stabilised. Protein expression levels were measured in matched PDXs, using a reverse phase protein array (RPPA) with a panel of 72 antibodies targeting key cancer related proteins as previously described). Statistical analysis of measured values showed significant correlation between high protein expression levels, such as Chk-1, PARP, HIAP-2 (cIAP-1), and PDX progression post cetuximab treatment. These proteins were significantly expressed lower in both regressing and stable PDXs. Interestingly, we found a high cross correlation between expression levels of these proteins across all the samples, giving them a great potential to act as predictive biomarkers for cetuximab response. Bioinformatics pathway enrichment of these proteins showed a significant enrichment of intrinsic apoptotic and cell cycle signalling pathways. These results have also been investigated in publically available patient data with cetuximab response and together will be used to construct a deterministic mathematical cell cycle model that will help to predict the outcome of cetuximab therapy in future. Citation Format: Naser Monsefi, Robert O’Byrne, Steven Carberry, Eugenia R. Zanella, Ana Barat, Mattia Cremona, Clare Morgan, Bryan T. Hennessy, Andrea Bertotti, Livio Trusolino, Jochen H.M. Prehn. Investigating potential molecular biomarkers for cetuximab response in metastatic colorectal cancer tissue using reverse phase protein array. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-354.