Irene Chong

Identification of novel determinants of resistance to lapatinib in ERBB2-amplified cancers

The gene encoding the receptor tyrosine kinase ERBB2, also known as HER2, is amplified and/or overexpressed in up to 15% of breast cancers. These tumours are characterised by an aggressive phenotype and poor clinical outcome. Although therapies targeted at ERBB2 have proven effective, many patients fail to respond to treatment or become resistant and the reasons for this are still largely unknown. Using a high-throughput functional screen we assessed whether genes found to be recurrently amplified and overexpressed in ERBB2+ve breast cancers mediate resistance to the ERBB2-targeted agent lapatinib. Lapatinib-resistant ERBB2-amplified breast cancer cell lines were screened, in the presence or absence of lapatinib, with an RNA interference library targeting 369 genes recurrently amplified and overexpressed in both ERBB2-amplified breast cancer tumours and cell lines. Small interfering RNAs targeting a number of genes caused sensitivity to lapatinib in this context. The mechanisms of resistance conferred by the identified genes were further investigated and in the case of NIBP (TRAPPC9), lapatinib resistance was found to be mediated through NF-κB signalling. Our results indicate that specific amplified and/ or overexpressed genes found in ERBB2-amplified breast cancer may mediate response to ERBB2-targeting agents.

Quantification of organ motion during chemoradiotherapy of rectal cancer using cone-beam computed tomography.

PURPOSE There has been no previously published data related to the quantification of rectal motion using cone-beam computed tomography (CBCT) during standard conformal long-course chemoradiotherapy. The purpose of the present study was to quantify the interfractional changes in rectal movement and dimensions and rectal and bladder volume using CBCT and to quantify the bony anatomy displacements to calculate the margins required to account for systematic (Σ) and random (σ) setup errors. METHODS AND MATERIALS CBCT images were acquired from 16 patients on the first 3 days of treatment and weekly thereafter. The rectum and bladder were outlined on all CBCT images. The interfraction movement was measured using fixed bony landmarks as references to define the rectal location (upper, mid, and low), The maximal rectal diameter at the three rectal locations was also measured. The bony anatomy displacements were quantified, allowing the calculation of systematic (Σ) and random (σ) setup errors. RESULTS A total of 123 CBCT data sets were analyzed. Analysis of variance for standard deviation from planning scans showed that rectal anterior and lateral wall movement differed significantly by rectal location. Anterior and lateral rectal wall movements were larger in the mid and upper rectum compared with the low rectum. The posterior rectal wall movement did not change significantly with the rectal location. The rectal diameter changed more in the mid and upper than in the low rectum. No consistent relationship was found between the rectal and bladder volume and time, nor was a significant relationship found between the rectal volume and bladder volume. CONCLUSIONS In the present study, the anterior and lateral rectal movement and rectal diameter were found to change most in the upper rectum, followed by the mid rectum, with the smallest changes seen in the low rectum. Asymmetric margins are warranted to ensure phase 2 coverage.

Vaginal vault brachytherapy as sole postoperative treatment for low-risk endometrial cancer

PURPOSE To evaluate the efficacy and side effect profile of adjuvant vaginal vault brachytherapy alone after hysterectomy in Stage I endometrial carcinoma. METHODS AND MATERIALS Between 23/11/1992 and 16/05/2005, a total of 173 patients with early endometrial carcinoma treated with vaginal vault brachytherapy alone postoperatively were identified. Patients were treated using a single-line source vaginal stump applicator (Varian Medical Systems) to deliver a dose of 5.5 Gy per fraction at a depth of 5mm from the applicator surface. A total of four fractions were delivered treating twice a week giving an overall treatment time of 10 days to deliver the total dose of 22 Gy in four fractions. RESULTS There were 19 deaths in this series, 6 (3.5%) from disseminated endometrial cancer and 13 (7.5%) from unrelated causes. High-risk features of Stage 1C, Grade G3, or clear cell histology were present in all 6 patients who developed metastatic disease. One patient developed a local recurrence alone, which was salvaged with external beam pelvic radiotherapy. The low-risk group defined by Stage 1B and G1 or G2 did not develop distant relapse in this series. Late morbidity was rare except for vaginal stenosis seen in 13%. CONCLUSIONS This series confirms that vaginal vault brachytherapy is associated with a high rate of pelvic control and survival after simple hysterectomy for low- and intermediate-risk endometrial cancer with minimal toxicity.

Gefitinib and EGFR Gene Copy Number Aberrations in Esophageal Cancer.

Purpose The Cancer Esophagus Gefitinib trial demonstrated improved progression-free survival with the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor gefitinib relative to placebo in patients with advanced esophageal cancer who had disease progression after chemotherapy. Rapid and durable responses were observed in a minority of patients. We hypothesized that genetic aberration of the EGFR pathway would identify patients benefitting from gefitinib. Methods A prespecified, blinded molecular analysis of Cancer Esophagus Gefitinib trial tumors was conducted to compare efficacy of gefitinib with that of placebo according to EGFR copy number gain (CNG) and EGFR, KRAS, BRAF, and PIK3CA mutation status. EGFR CNG was determined by fluorescent in situ hybridization (FISH) using prespecified criteria and EGFR FISH-positive status was defined as high polysomy or amplification. Results Biomarker data were available for 340 patients. In EGFR FISH-positive tumors (20.2%), overall survival was improved with gefitinib compared with placebo (hazard ratio [HR] for death, 0.59; 95% CI, 0.35 to 1.00; P = .05). In EGFR FISH-negative tumors, there was no difference in overall survival with gefitinib compared with placebo (HR for death, 0.90; 95% CI, 0.69 to 1.18; P = .46). Patients with EGFR amplification (7.2%) gained greatest benefit from gefitinib (HR for death, 0.21; 95% CI, 0.07 to 0.64; P = .006). There was no difference in overall survival for gefitinib versus placebo for patients with EGFR, KRAS, BRAF, and PIK3CA mutations, or for any mutation versus none. Conclusion EGFR CNG assessed by FISH appears to identify a subgroup of patients with esophageal cancer who may benefit from gefitinib as a second-line treatment. Results of this study suggest that anti-EGFR therapies should be investigated in prospective clinical trials in different settings in EGFR FISH-positive and, in particular, EGFR-amplified esophageal cancer.

Optimal treatment of metastatic pancreatic cancer

Approximately 60% of patients with pancreatic cancer have metastatic disease at the time of diagnosis.1 Gemcitabine, which has been shown to improve overall survival (OS) and clinical response compared with 5-fluorouracil (5-FU), is standard treatment with a median survival of 6 months and a 1 year survival rate of only 20%.2 The addition of other drugs to gemcitabine to improve outcome has been generally unsuccessful with the exception of erlotinib, an oral human epidermal growth factor 1 receptor/epidermal growth factor receptor ( HER1/EGFR ) tyrosine kinase inhibitor. The combination of erlotinib with gemcitabine resulted in an improved 1 year survival compared with gemcitabine alone (23% vs 17%, p=0.023).3 Data from a phase III trial evaluating gemcitabine with capecitabine (GEM-CAP) showed an improvement in response and progression-free survival with GEM-CAP over single-agent gemcitabine, and pooling of these results with two other randomised controlled trials in a meta-analysis resulted in a survival advantage with GEM-CAP.4 At this years American Society of Clinical Oncology annual meeting, the interim analysis from the randomised phase III PRODIGE 4/ACCORD 11 trial comparing FOLFIRINOX (5-FU/leucovorin, irinotecan and oxaliplatin) with gemcitabine as first-line treatment for fit patients with metastatic pancreatic adenocarcinoma was presented.5 These preliminary results were encouraging, with a significantly longer OS, progression-free survival and improved response rate associated with the experimental arm compared with gemcitabine alone. Although …

Session 3: Boosting primary and recurrent rectal cancer: how far can we push the radiotherapy envelope?

Neoadjuvant pelvic radiotherapy is widely used for patients with advanced rectal cancer. The trade‐off between dose and response is well‐established, yet little consensus remains on the precise methods of delivery and doses given in different scenarios. Professor Vuong reviews the evidence base and trial evidence on the escalation of radiotherapy dose and the methods of achieving this.

Session 3: Intra-operative radiotherapy - creating new surgical boundaries

In patients with advanced and recurrent colorectal cancer, surgical resection with clear margins is the greatest challenge and is limited by known anatomical constraints. Preoperative or intra‐operative assessment of the limits of surgical dissection may help to explore the possibility of improving resectability through either targeted external beam radiotherapy or intra‐operative radiotherapy. Professor Chang reviews the evidence base and potential advantages and disadvantages of this approach, whilst the expert panel agree a consensus on the evidence for assessment and therapy of such patients.

Abstract 69: Identification of new therapeutic targets and molecular predictors of response in oesophageal cancer

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA Purpose of the study Oesophageal cancer is the seventh most common cause of cancer related death worldwide. Disease relapse is frequent and treatment options are limited. With the exception of HER-2 and EGFR amplification, there are currently no validated biomarkers to predict response to biological therapies in this disease. The purpose of this study was to identify new biomarker defined therapeutic approaches for patients with oesophageal cancer. Experimental approach We have undertaken a functional screening approach focused on the druggable kinome to generate a first map of the genetic dependencies found in oesophageal cancer. A panel of 19 oesophageal and 130 non-oesophageal cancer cell lines were screened with siRNA targeting 720 kinases to generate functional profiles. By integrating this dataset with exome sequencing and copy number data, we identified key genetic dependencies (KGDs) associated with oesophageal histology (adenocarcinoma or squamous cell carcinoma) or key driver genotypes including amplifications in c-MYC, CCND1, EGFR and ERBB2, as well as loss of functional mutations in SMAD4 and ARID1A. Results Interrogation of the oesophageal functional genomics dataset established oncogene addiction effects including c-MYC, which is amplified in 30% of oesophageal carcinomas. We confirmed that a novel isoform of Bruton agammaglobulinaemia tyrosine kinase gene (BTK-C) was expressed in oesophageal cancer models and that silencing of BTK resulted in selective lethality in c-MYC amplified oesophageal cell lines. To further assess the impact of BTK inhibition in oesophageal cancer, we treated a panel of oesophageal cell lines with increasing doses of ibrutinib, a small molecule tyrosine kinase inhibitor of BTK. Analysis of ibrutinib SF50 revealed that there was preferential sensitivity to ibrutinib in c-MYC amplified oesophageal cell lines. Furthermore, we observed a significant association between mutation of the chromatin remodelling factor gene SMARCA4 and dependency upon the bromodomain protein BRD4. We also identified SMAD4 kinase genetic dependencies including AKT1 and a number of its substrates (FGR, MAP3K3, CHEK1 and WEE1). We noted a densely connected group of kinases that regulate the mitotic cell cycle in the SMAD4 dependency network, and found that SMAD4 mutant cell lines exhibited an increased sensitivity to drugs, including paclitaxel, that target the mitotic checkpoint. Conclusions Our results support the potential of BTK small molecule inhibition as a novel therapeutic strategy in c-MYC amplified oesophageal carcinoma. Loss of function SMAD4 mutations represents a candidate biomarker of response to drugs that target the mitotic checkpoint in oesophageal cancer. Citation Format: Irene Chong, lauren Aronson, David Cunningham, James Campbell, Colm Ryan, Michael Davidson, Ian Chau, Alan Ashworth, Christopher J. Lord. Identification of new therapeutic targets and molecular predictors of response in oesophageal cancer. [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 69.

Abstract 4987: Identification of novel genes and pathways involved in resistance to HER2-targeting agents in breast cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The HER2 gene ERBB2 is amplified and/ or overexpressed in up to 15% of all invasive breast cancers. These cancers are characterised by an aggressive phenotype and poor clinical outcome. HER2 positive tumours often display multiple high level amplifications of genes in addition to HER2. Although targeted therapies to HER2 have proven clinical benefit, a substantial number of patients have tumours that are either de novo resistant or acquire resistance over time to these agents. Reasons for this are still largely unknown. By using a high throughput siRNA screen we sought to determine whether genes recurrently amplified and overexpressed in HER2-amplified breast cancer mediate resistance to HER2-targeting agents. We profiled 45 HER2-amplified primary breast cancers and 13 HER2-amplified cell lines with high resolution microarray-based comparative genomic hybridisation (aCGH) and Illumina WG6 v2 arrays. Overlaying of aCGH and gene expression data led to the identification of 369 genes recurrently amplified and overexpressed when amplified. Lapatinib-resistant HER2-amplified breast cancer cell lines were treated with lapatinib, a HER2 small molecule inhibitor, and screened with an RNA interference (RNAi) library targeting these 369 genes and controls. This RNAi screen and subsequent validation screens identified eight genes that when silenced lead to a significant sensitization to lapatinib. The mechanisms of resistance conferred by the identified genes were further investigated and pointed to the involvement of NF-kappaB-signalling, Wnt-signalling and reactive oxygen species scavenging. Our results indicate that specific amplified and overexpressed genes found in HER2 positive breast cancers mediate resistance to anti-HER2 agents. These findings might be used to identify patients likely to be de novo resistant to lapatinib and provide new targets for combined targeted therapies to overcome resistance to anti-HER2 therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4987. doi:10.1158/1538-7445.AM2011-4987

Abstract 3039: Identification of a novel biomarker of resistance to lapatinib common to both breast and oesophagogastric cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL HER2 is amplified and/or overexpressed in 15% and 20% of breast and oesophagogastric (OG) cancers, respectively. Results from the recently published ToGA trial reveal that trastuzumab plus standard chemotherapy improves survival in patients with advanced HER2-positive OG cancer compared with chemotherapy alone, which mirrors the survival benefit shown with trastuzumab plus chemotherapy in metastatic breast cancer. The clinical value of lapatinib, already confirmed in advanced trastuzumab-resistant breast cancer, is currently being evaluated in prospective phase III trials for HER2-positive OG cancer in the metastatic setting. Despite improvements in outcome with anti-HER2 therapy, less than half of patients respond to treatment, and predictive biomarkers to lapatinib for breast and OG cancer have not yet been identified. We sought to identify predictive biomarkers of resistance to lapatinib treatment, common to both breast and OG cancer, by using a siRNA screen of genes identified in a primary siRNA screen to sensitise lapatinib-resistant breast cancer cell lines to lapatinib. These genes are found recurrently amplified and overexpressed in 45 HER2-amplified primary breast cancers and 13 HER2-amplified breast cancer cell lines. OG junction cell lines were treated with lapatinib and screened using the siRNA library. We found that silencing of TP53INP1, a gene target of TP53 which is known to be a reactive oxygen species scavenger, resulted in significant sensitisation to lapatinib in two of the most lapatinib-resistant HER2-positive cell lines derived from the OG junction. TP53INP1 was over-expressed, but not amplified, in the sensitised OG junction cell lines. Our results suggest that genes which mediate resistance to anti-HER2 therapy in breast cancer may be relevant to OG cancer. We are currently developing assays for TP53INP1 in tumours for validation within the context of prospective randomised controlled clinical trials for OG cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3039. doi:10.1158/1538-7445.AM2011-3039