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Supplementary Figure 5. A) Receiver Operator Curve (ROC), for the accuracy of the epigenetic lymph node prediction signature in cross validation. B) Examples of FLI1 and IRX4 immunohistochemical staining of a PeCa TMA, in samples showing either methylation.

Supplementary Figure 1. Proportion of MVPs in penile cancers.

Other than an association with HPV infection, little is known about the genetic alterations determining the development of penile cancer. Although penile cancer is rare in the developed world, it presents a significant burden in developing countries. Here, we report the findings of whole-exome sequencing (WES) to determine the somatic mutational landscape of penile cancer. WES was performed on penile cancer and matched germline DNA from 27 patients undergoing surgical resection. Targeted resequencing of candidate genes was performed in an independent 70 patient cohort. Mutation data were also integrated with DNA methylation and copy-number information from the same patients. We identified an HPV-associated APOBEC mutation signature and an NpCpG signature in HPV-negative disease. We also identified recurrent mutations in the novel penile cancer tumor suppressor genes CSN1(GPS1) and FAT1. Expression of CSN1 mutants in cells resulted in colocalization with AGO2 in cytoplasmic P-bodies, ultimately leading to the loss of miRNA-mediated gene silencing, which may contribute to disease etiology. Our findings represent the first comprehensive analysis of somatic alterations in penile cancer, highlighting the complex landscape of alterations in this malignancy. Cancer Res; 76(16); 4720–7. ©2016 AACR.

Supplementary Table 1. Patient details for A) Fresh Frozen test cohort and B) archival validation cohort

Supplementary Figure 6. Heatmap of 6 HNSCC cell lines (in duplicate) showing the methylation of the 30 probe set HPV classifier

Supplementary methods

Supplementary Figure 2. Comparison of gene expression between PeCa and matched normal tissue for PRC2 complex members

Purpose: Penile cancer is a rare malignancy in the developed world with just more than 1,600 new cases diagnosed in the United States per year; however, the incidence is much higher in developing countries. Although HPV is known to contribute to tumorigenesis, little is known about the genetic or epigenetic alterations defining penile cancer.Experimental Design: Using high-density genome-wide methylation arrays, we have identified epigenetic alterations associated with penile cancer. Q-MSP was used to validate lymph node metastasis markers in 50 cases. A total of 446 head and neck squamous cell carcinoma (HNSCC) and cervical squamous cell carcinoma (CESCC) samples were used to validate HPV-associated epigenetic alterations.Results: We defined 6,933 methylation variable positions (MVP) between normal and tumor tissue, which includes 997 hypermethylated differentially methylated regions associated with tumor supressor genes, including CDO1, AR1, and WT1. Analysis of penile cancer tumors identified a 4 gene epi-signature which accurately predicted lymph node metastasis in an independent cohort (AUC of 89%). Finally, we explored the epigenetic alterations associated with penile cancer HPV infection and defined a 30 loci lineage-independent HPV specific epi-signature which predicts HPV status and survival in independent HNSCC, CESC cohorts. Epi-signature–negative patients have a significantly worse overall survival [HNSCC P = 0.00073; 95% confidence interval (CI), 0.021–0.78; CESC P = 0.0094; HR = 3.91, 95% CI = 0.13–0.78], HPV epi-signature is a better predictor of survival than HPV status alone.Conclusions: These data demonstrate for the first time genome-wide epigenetic events involved in an aggressive penile cancer phenotype and define the epigenetic alterations common across multiple HPV-driven malignancies. Clin Cancer Res; 21(5); 1196–206. ©2014 AACR.

Supplementary Table 1. A)Patient details for Fresh Frozen test cohort, B) Patient details for validation cohort. Supplementary Figure 1. APOBEC mutation fraction of across multiple cancer types. Supplementary Figure 2. DNA methylation. Supplementary Figure 3. Candidate PeCa driver mutations. Supplementary Figure 4. Schematic of FAT1 is shown with locations of mutations and ethylation states of PeCa and normal penile tissue across the canonical FAT1 gene. Supplementary Figure 5. A) Example plot of Copy Number Alterations (CNA) in penile cancers, 450K methylation profiles for penile cancer and normal squamous epithelium, and output from GISTIC showing the frequency and significance of CNAs.

Supplementary Figure 3. Methylation profiles of PeCa (green) and normal penile tissue (red) across canonical gene structure

Supplementary Figure 4. Comparison of DMR profiles across canonical features for PeCa (green) and normal squamous epithelium (red), for three candidate epigenetically regulated genes involved in the development of penile cancer (AR &CDO1).

Background: KRAS mutations (mt) are found in ~30% of non-small cell lung cancers (NSCLC). Despite the approval and development of KRAS G12C and KRAS G12D inhibitors respectively, mechanisms of resistance to MAPK pathway inhibitors are emerging and combination strategies are needed for patients with KRAS mt NSCLC . Avutometinib (VS-6766) is a unique RAF/MEK clamp that inhibits MEK kinase activity and blocks RAF-mediated phosphorylation of MEK. We studied the preclinical and clinical activity of the combination of avutometinib with the mTOR inhibitor everolimus in KRAS mt NSCLC. Methods: A panel of KRAS mt NSCLC cell lines were treated for 1 hour with clinically relevant concentrations of avutometinib and everolimus and changes in phosphoproteins were measured using an antibody array. We then tested for synergy of avutometinib and everolimus in 3D proliferation assays and in the H441 NSCLC xenograft model. A clinical trial is ongoing (NCT02407509) testing the combination of 3.2 mg avutometinib with 5 mg of everolimus administered twice weekly 3 weeks on/1 week off in 28-day cycles (previously defined as the recommended phase 2 dose) in a cohort of patients with KRAS mt NSCLC. Results: Avutometinib inhibited the MAPK pathway (p-MEK, p-ERK, p-90RSK) with an increase in p-PRAS40, suggesting activation of the PI3K pathway as an adaptive resistance mechanism. Everolimus inhibited the PI3K pathway (p-p70S6K and p-RPS6). Among a panel of KRAS mt NSCLC cell lines, avutometinib + everolimus showed synergistic anti-proliferative activity across KRAS G12C, G12V and G12D variants (mean synergy score of ~18). In the H441 KRAS G12V NSCLC xenograft model, there was a significant reduction in tumor volume and increase in survival with the combination of avutometinib and everolimus (87% TGI; 66 days vs 36.5 days in control group) compared to control. In the clinical trial expansion, 16 patients with KRAS mt NSCLC have been treated so far with avutometinib and everolimus (5 G12V, 3 Q61H, 2 G12C, 2 G12A, 2 G12D, 1 G13A, 1 G13D; median prior lines = 2). The current objective response rate (ORR) among the 14 patients who have at least one post assessment scan is 3/14 (21%; 1 G12V, 1 G12A, 1 G13A) with 11/14 showing a reduction in tumor size as best response. The current progression free survival (PFS) is 5.3 months (95% CI 2.8-7.4 months) with 4 patients still on study. Updated data on the planned cohort size of 20 patients will be presented. Conclusion: The combination of avutometinib and everolimus overcomes the activation of the PI3K/AKT/mTOR pathway which is an adaptive resistance mechanism to MAPK pathway inhibition. We have shown that avutometinib and everolimus induce synergistic anti-tumor effects preclinically, and preliminary data suggest clinically meaningful ORR and PFS in patients with KRAS mt NSCLC including non-G12C variants. Citation Format: Simon Rodney, Adam R. Stewart, Victoria Sanchez Perez, Cienne Morton, Lisa A. Pickard, Taleen Shakouri, Toby Prout, Mona Parmar, Alison J. Turner, Silvia Coma, Jonathan Pachter, Laura Finneran, Emma Hall, James Spicer, Anna Minchom, Udai Banerji. Preclinical and clinical evaluation of the RAF/MEK clamp avutometinib (VS-6766) in combination with the mTOR inhibitor everolimus for the treatment of KRAS mutated non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3461.

Background: The global COVID-19 pandemic has placed unprecedented burden on individual oncology patients, oncology departments, hospitals and national health systems. In order to protect individual patients from the risk of COVID-19 infection as well as improve capacity for COVID-19 patient management, a series of national and internationally agreed measures were proposed. During the first wave of infections the virulence and effect on cancer patients was not known. It is vitally important that the measures implemented to combat these risks are assessed and evaluated in order to learn best how to manage potential future waves of known and unknown viral infections.Methods: The international ESMO COVID-19 guidelines and national (NICE) guidelines were implemented across Kent, UK. All changes to treatment regimens were audited to assess what were the most frequent changes and in which patient groups could these be implemented. Data was subdivided for both early and advanced breast cancer as well as ER+, HER2+ or triple negative disease.Results: We collected full treatment history from 1,718 breast cancer patients currently receiving active oncology treatments. We were able to change treatment regimens due to COVID19 for 32.8% of patients. Of these 27.1% were early breast cancer patients compared with 43.7% were those with advanced metastatic disease. The most common changes for neoadjuvant changes were proceeding to surgery before completion of planned chemotherapy (10.2%), switch to 3 weekly Paclitaxel (10%) and chemotherapy break (8%). For adjuvant patients the most common changes included postponement of bisphosphonates (70.8%), chemotherapy break (13.5%), and curtailment to 6 months of adjuvant Trastuzumab (10.4%). For our palliative patients the most common changes included delay CDK4/6 inhibitor treatment (79.2%), postponement of bisphosphonates (24.8%), break in HER2 antibody (9.6%) and break in chemotherapy (8%).Conclusions: A large proportion of breast cancer oncology patients were deemed suitable to have a change in original planned treatment. We are fortunate to have comparatively large number of treatment options that can be customised on a patient basis to individual reduce risk of COVID-19. Further analysis is needed over time to compare the oncological outcomes of those in whom treatment was changes from the current gold standards of care. Citation Format: Simon N Rodney, Charlotte Abson, Russell Burcombe, Rema Jyothirmayi, Catherine Harper-Wynne. Changes in management of breast cancer patients during first wave of COVID19, throughout the area of Kent, United Kingdom. An audit of ESMO guideline implementation [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS14-06.

Coronavirus disease 2019 (COVID-19) antiviral response in a pan-tumor immune monitoring (CAPTURE) (NCT03226886) is a prospective cohort study of COVID-19 immunity in patients with cancer. Here we evaluated 585 patients following administration of two doses of BNT162b2 or AZD1222 vaccines, administered 12 weeks apart. Seroconversion rates after two doses were 85% and 59% in patients with solid and hematological malignancies, respectively. A lower proportion of patients had detectable titers of neutralizing antibodies (NAbT) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOC) versus wild-type (WT) SARS-CoV-2. Patients with hematological malignancies were more likely to have undetectable NAbT and had lower median NAbT than those with solid cancers against both SARS-CoV-2 WT and VOC. By comparison with individuals without cancer, patients with hematological, but not solid, malignancies had reduced neutralizing antibody (NAb) responses. Seroconversion showed poor concordance with NAbT against VOC. Previous SARS-CoV-2 infection boosted the NAb response including against VOC, and anti-CD20 treatment was associated with undetectable NAbT. Vaccine-induced T cell responses were detected in 80% of patients and were comparable between vaccines or cancer types. Our results have implications for the management of patients with cancer during the ongoing COVID-19 pandemic.

DNA methylation is a key epigenetic modification in the regulation of cell fate and differentiation, and its analysis is gaining increasing importance in both basic and clinical research. Targeted Bisulfite Sequencing (TBS) has become the method of choice for the cost-effective, targeted analysis of the human methylome at base-pair resolution. Here we benchmarked five commercially available TBS platforms, including three hybridization capture-based (Agilent, Roche, and Illumina) and two RRBS-based (Diagenode and NuGen), across 11 samples. A subset of these were also compared to whole-genome DNA methylation sequencing with the Illumina and Oxford Nanopore platforms. We assessed performance with respect to workflow complexity, on/off-target performance, coverage, accuracy, and reproducibility. We find all platforms able to produce usable data but with major differences for some performance criteria, especially in the number and identity of the CpG sites covered, which affects the interoperability of datasets generated on these different platforms. To overcome this limitation, we used imputation and show that it improves the interoperability from an average of 10.35% (0.8M CpG sites) to 97% (7.6M CpG sites). Our study provides cross-validated guidance on which TBS platform to use for different features of the methylome and offers an imputation-based harmonization solution for improved interoperability between platforms, allowing comparative and integrative analysis.

There remains a lack of consensus among guideline relating to which patients require investigation for haematuria. We determined the incidence of urinary tract cancer in a prospective observational study of 3556 patients referred for investigation of haematuria across 40 hospitals between March 2016 and June 2017 (DETECT 1; ClinicalTrials.gov: NCT02676180) and the appropriateness of age at presentation in cases with visible (VH) and nonvisible (NVH) haematuria. The overall incidence of urinary tract cancer was 10.0% (bladder cancer 8.0%, renal parenchymal cancer 1.0%, upper tract transitional cell carcinoma 0.7%, and prostate cancer 0.3%). Patients with VH were more likely to have a diagnosis of urinary tract cancer compared with NVH patients (13.8% vs 3.1%). Older patients, male gender, and smoking history were independently associated with urinary tract cancer diagnosis. Of bladder cancers diagnosed following NVH, 59.4% were high-risk cancers, with 31.3% being muscle invasive. The incidence of cancer in VH patients45 yr of age was 3.5% (n=7) and 1.0% (n=4) in NVH patients60 yr old. Our results suggest that patients with VH should be investigated regardless of age. Although the risk of urinary tract cancer in NVH patients is low, clinically significant cancers are detected below the age threshold for referral for investigation.This study highlights the requirement to investigate all patients with visible blood in the urine and an age threshold of ≥60 yr, as recommended in some guidelines, as the investigation of nonvisible blood in the urine will miss a significant number of urinary tract cancers. Patient preference is important, and evidence that patients are willing to submit to investigation should be considered in reaching a consensus recommendation for the investigation of haematuria. International consensus to guide that patients will benefit from investigation should be developed.

In Europe and North America penile cancer is a rare disease, with an incidence of approximately 1.5 per 100,000 males, although this increases to 4.4 per 100,000 men in South America and Africa. Penile cancer represents a significant global health problem due to the often devastating consequences of treatment at this site of malignancy, and the mortality associated with metastatic disease. The primary lymphatic drainage of penile cancer is to the inguinal lymph nodes, and the presence of metastatic disease within the inguinal lymph nodes is the most important prognostic factor. The 5-year survival for men with lymph node metastasis is 57%, compared with 90% for those without. Of those patients who present with clinically node negative (cN0) disease, 20%-25% will have occult metastases. Therefore if all patients with ZT1G2 disease undergo inguinal lymphadenectomy, potential overtreatment will occur in 75%-80% of cases where the inguinal lymph nodes are pathologically clear. Furthermore, open inguinal lymphadenectomy is associated with significant morbidity, with up to 70% of patients developing complications related to wound healing, or long-term genital or lower limb lymphoedema. Therefore, there is a clear clinical need to accurately predict the presence of lymph node micro-metastasis and to determine prognosis so as to select only those patients who would benefit from radical inguinal lymphadenectomy. This information would be invaluable when discussing and determining the extent of surgical resections and further treatment required. This review will evaluate the evidence for the use of molecular biomarkers to predict lymph node status as well as prognosis in penile carcinoma. It will also discuss the next generation of biomarkers, which have the potential to change the diagnostic landscape in penile cancer. The pathways and logic behind biomarkers that have been studied thus far will also be considered.

Purpose: Penile cancer is a rare malignancy in the developed world with just more than 1,600 new cases diagnosed in the United States per year; however, the incidence is much higher in developing countries. Although HPV is known to contribute to tumorigenesis, little is known about the genetic or epigenetic alterations defining penile cancer. Experimental Design: Using high-density genome-wide methylation arrays, we have identified epigenetic alterations associated with penile cancer. Q-MSP was used to validate lymph node metastasis markers in 50 cases. A total of 446 head and neck squamous cell carcinoma (HNSCC) and cervical squamous cell carcinoma (CESCC) samples were used to validate HPV-associated epigenetic alterations. Results: We defined 6,933 methylation variable positions (MVP) between normal and tumor tissue, which includes 997 hypermethylated differentially methylated regions associated with tumor supressor genes, including CDO1, AR1, and WT1. Analysis of penile cancer tumors identified a 4 gene epi-signature which accurately predicted lymph node metastasis in an independent cohort (AUC of 89%). Finally, we explored the epigenetic alterations associated with penile cancer HPV infection and defined a 30 loci lineage-independent HPV specific epi-signature which predicts HPV status and survival in independent HNSCC, CESC cohorts. Epi-signature–negative patients have a significantly worse overall survival [HNSCC P = 0.00073; 95% confidence interval (CI), 0.021–0.78; CESC P = 0.0094; HR = 3.91, 95% CI = 0.13–0.78], HPV epi-signature is a better predictor of survival than HPV status alone. Conclusions: These data demonstrate for the first time genome-wide epigenetic events involved in an aggressive penile cancer phenotype and define the epigenetic alterations common across multiple HPV-driven malignancies. Clin Cancer Res; 21(5); 1196–206. ©2014 AACR.

To assess the prevalence of preoperative anemia and the impact of preoperative anemia and blood transfusion requirement on 30- and 90-day complications in a cohort of patients undergoing robot-assisted radical cystectomy with intracorporeal urinary diversion (iRARC).IRARC was performed on 166 patients between June 2011 and March 2016. Prospective data were collected for patient demographics, clinical and pathologic characteristics, perioperative variables, transfusion requirements, and hospital length of stay. Thirty- and 90-day complications were classified according to the modified Memorial Sloan Kettering Cancer Center Clavien-Dindo system.Preoperative anemia was common (43.4%) and greatest in patients receiving neoadjuvant chemotherapy (48.6%) (p 0.001). Patients with preoperative anemia were significantly more likely to have an Ileal conduit (p = 0.033), higher cystectomy stage (≥pT3) (p = 0.028), and a lower lymph node yield (p = 0.031). Preoperative anemia was not associated with increased perioperative morbidity but was associated with the requirement for blood transfusion (p = 0.001). Blood transfusion required in 20.4% of patients with intraoperative and postoperative blood transfusion rate was 10.2% and 13.9%, respectively. The 30-day all complication rate and 30-day major complication rate were 55.4% and 15.7%, respectively, while 90-day all complication rate and 90-day major complication rate were 65.7% and 19.3%, respectively. Intraoperative blood transfusion was not associated with increased complications, but postoperative blood transfusion requirement was independently associated with perioperative morbidity: all 30-day complications (p = 0.003), all 90-day complications (p = 0.009), and 90-day major complications (p = 0.004).The presence of preoperative anemia in patients undergoing iRARC is not associated with increased surgical risk, although preoperative anemic patients were significantly more likely to require blood transfusion. Blood transfusion requirement and specifically postoperative blood transfusion are independently associated with perioperative morbidity and are an important factor for the optimization of postoperative outcomes.

Bladder cancer is the 8th most common cancer with 74,000 new cases in the United States in 2015. Non-muscle invasive bladder cancer (NMIBC) accounts for 75% of all bladder cancer cases. Transurethral resection and intravesical treatments remain the main treatment modality. Up to 31-78% of cases recur, hence the need for intensive treatment and surveillance protocols which makes bladder cancer one of the most expensive cancers to manage. The purpose of this review is to compare contemporary guidelines from Europe, (European Association of Urology), the United States (National Comprehensive Cancer Network), the United Kingdom (National Institute for Health and Care Excellence), Japan (Japanese Urological Association) and the International Consultation on Bladder Cancer (ICUD). We compare and contrast the different guidelines and the evidence on which their recommendations are based.

Other than an association with HPV infection, little is known about the genetic alterations determining the development of penile cancer. Although penile cancer is rare in the developed world, it presents a significant burden in developing countries. Here, we report the findings of whole-exome sequencing (WES) to determine the somatic mutational landscape of penile cancer. WES was performed on penile cancer and matched germline DNA from 27 patients undergoing surgical resection. Targeted resequencing of candidate genes was performed in an independent 70 patient cohort. Mutation data were also integrated with DNA methylation and copy-number information from the same patients. We identified an HPV-associated APOBEC mutation signature and an NpCpG signature in HPV-negative disease. We also identified recurrent mutations in the novel penile cancer tumor suppressor genes CSN1(GPS1) and FAT1. Expression of CSN1 mutants in cells resulted in colocalization with AGO2 in cytoplasmic P-bodies, ultimately leading to the loss of miRNA-mediated gene silencing, which may contribute to disease etiology. Our findings represent the first comprehensive analysis of somatic alterations in penile cancer, highlighting the complex landscape of alterations in this malignancy. Cancer Res; 76(16); 4720–7. ©2016 AACR.

Bladder cancer (BC) is the 5th most common cancer in Western societies with a rising global incidence. The major challenge to improving patient outcomes is to better identify patients at risk for recurrence, progression, and metastasis, and to monitor treatment response. A non-invasive monitoring assay is needed in order to interrogate the molecular features and clinical tumor heterogeneity of bladder cancers, which will potentially lead to better stratified patient management. We developed a targeted amplicon-based re-sequencing workflow for detecting and tracking genomic mutations in cfDNA using dual molecular indexed primers for specific amplification of a panel of 20 of known (TERT, FGFR3, PIK3CA, HRAS) and novel BC mutations. Only 3hr time is needed for the total workflow for the library generation and followed by next generation sequencing (NGS) performed on Illumina MiSeq sequencing platform. Data analysis and variants calling was performed. After a thorough test, optimization, and validation of the targeted panel and the workflow on selected samples of matched bladder tumor-blood pairs and controls, 45 samples were included for testing the workflow, which includes samples of 16 matched bladder tumor-blood pairs, technical duplicates of cfDNA DNA Reference Standards (Horizon Discovery) with mutations at known allelic frequencies. Technical duplicates of cfDNA extracted from blood from patients with metastatic BC (pre and post chemotherapy) were also included in this study. Somatic variants from matched tumor-blood pairs were detected. We showed that detection sensitivity at 1.0% allelic frequency was achieved. Our preliminary results demonstrated that we were able to detect mutation burden from cfDNA samples and track mutation burden change in response to chemotherapy for patients with metastatic BC. Here we developed a sensitive, non-invasive targeted NGS workflow to detect somatic mutations for bladder cancers from both tumor DNA and cfDNA samples. Our early results demonstrated that we can detect and track mutational changes from patients with metastatic BC. Citation Format: Liqin Dong, Pramit Khetrapal, Simon Rodney, Wei Shen Tan, Sheida Rezaee, Patricia de Winter, John Kelly, Andrew Feber. A targeted re-sequencing assay for molecular profiling of somatic mutations from plasma cell-free dna (cfdna) for bladder cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5702. doi:10.1158/1538-7445.AM2017-5702

e20652 Background: Detection of EGFR mutations in ctDNA can help determine appropriateness of TKI therapy for patients with NSCLC. We investigated whether longitudinal monitoring of ctDNA levels can be used to assess response to therapy and disease progression, with a focus on EGFR mutation-positive patients treated with immunotherapy. Methods: Serially collected blood from patients with EGFR mutation-positive NSCLC treated with TKIs and/or immunotherapy was analyzed using an ultrasensitive 24-gene next-generation sequencing assay. Clinical characteristics and outcomes were analyzed retrospectively by chart review. Results: We studied quantitative changes in ctDNA levels during treatment by analyzing somatic mutations in 91 plasma samples from 8 patients with EGFR-mutant NSCLC, including samples collected around the time of disease progression for a subset of patients. Two patients treated with PD-1 inhibitor monotherapy experienced a rise in ctDNA harboring EGFR-sensitizing mutations prior to radiographic progression. A third patient was started on anti-PD-1 monotherapy following disease progression on erlotinib. Plasma levels of L858R, T790M, and TP53 mutations were detectable on treatment initiation and decreased with radiographic response. The levels of these mutations rose at progression,fell with response to EGFR-directed therapy, and increased again before disease progression. Another patient was found to have mutations in EGFR, T790M, and TP53 that fell upon treatment with combination TKI therapy. The remaining four patients studied were treated with concurrent TKI and immunotherapy. In all of these cases, sensitizing EGFR mutations were present in plasma at low levels during response to treatment. Two of the four patients had a rise in ctDNA level at the time of radiographic progression; the other two patients had durable responses with persistently low ctDNA levels. Analysis of additional cases is ongoing. Conclusions: Monitoring quantitative changes in ctDNA may enable assessment of response or disease progression in immunotherapy- and TKI-treated EGFR-mutant NSCLC patients.

4518 Background: Penile cancer is a rare but mutilating disease. Due to its rarity few studies have investigated the molecular oncogenic changes. There is a huge need to improve the poor outcome of metastatic disease by advancing the standard practice of platinum based therapies. Treatment based on actionable mutations and the potential of immunotherapies may improve clinical outcomes. We present the first analysis of intra-tumour heterogeneity within penile cancer. Methods: Tissue samples, greater than 80% purity, were collected from four spatially distinct tumor regions together with matched normal and metastatic tissue. Deep whole exome sequencing was performed on the resulting 48 samples from eight patients. Mutations were called by using Mutect2 and annotated with Annovar. Copy number changes were estimated using Sequenza and phylogenetic trees created using pyclone. Results: A mean of 650 single nucleotide variants and insertion/deletion events were discovered per tumor sample. Extensive intra-tumor heterogeneity was found with a wide range of different predicted subclonal architectures. Two distinct patterns were noted: either the acquisition of mutations by the lymph node metastasis was an early event with a lower mutational load, or it was a later event with a high mutational load. In the two instances where patients were negative for human papillomavirus infection, the lymph node metastasis evolved at a later date. However in 5/6 cases of HPV infection, an early subclone developed which potentially led to the lymph node metastasis. Driver mutations in p53, cMET or FAT1 were found to be truncal early events in 80% of cases. Actionable mutations in PIK3CA and EGFR were found to be subclonal in two further samples. Conclusions: There is extensive intra- and inter-tumour heterogeneity within penile cancer. The main truncal drivers in penile cancer are cMET, FAT1 and p53. Samples infected with human papillomavirus appeared to have a distinct signature in terms of both copy number changes and clonal architecture. Actionable mutations PIK3CA and EGFR were found to be subclonal in origin. This may have profound effects on the clinical utility of targetable treatments such as tyrosine kinase inhibitors.

Prostate cancer is the most common cancer and second leading cause of death in men. The evidence base for the diagnosis and treatment of prostate cancer is continually changing. We aim to review and discuss past and contemporary papers on these topics to provoke debate and highlight key dilemmas faced by the urological community. We review key papers on prostate-specific antigen screening, radical prostatectomy versus surveillance strategies, targeted therapies, timing of radiotherapy and alternative anti-androgen therapeutics. Previously, the majority of patients, irrespective of risk, underwent radical open surgical procedures associated with considerable morbidity and mortality. Evidence is emerging that not all prostate cancers are alike and that low-grade disease can be safely managed by surveillance strategies and localized treatment to the prostate. The question remains as to how to accurately stage the disease and ultimately choose which treatment pathway to follow.

Genomic DNA methylation is one of the most important epigenetic modifications in eukaryotes necessary for cellular differentiation and plays a crucial role in gene regulation of many oncogenes and tumor suppressor genes. Aberrant DNA methylation at the level of both individual gene promoters and on a genome-wide scale has been heavily implicated in cancer initiation and progression. Whole genome bisulfite sequencing (WGBS) is the gold standard method for studying alterations in DNA methylation at a single base pare resolution. However, the high cost of WGBS is still prohibitive for high-throughput observational studies on large sample cohorts. Moreover, the WGBS data analysis requires substantial IT resources and a complex bioinformatics analysis. Targeted bisulfite sequencing enables a cost-effective and focused analysis of genomic regions of interest where most of DNA methylation alterations occur, such as promoters, CpG islands and shores, and enhancer regions. Currently, there are 2 main approaches for targeted bisulfite sequencing of medium-to-large genomic regions, reduced representation bisulfite sequencing (RRBS) and hybridization-based enrichment. We have performed a comparison of 5 commercially available platforms for RRBS and in-solution hybridization-based enrichment panels. We analyzed a panel of two cancer cell lines (Hela and Coriell NA12878) and a normal blood reference gDNA sample, in duplicate on each platform using recommended sample inputs, and compared the results to WGBS data. Given that sample input requirements differ significantly between RRBS (∼100ng or less), and hybridization based methods (500ng - 1 μg), we have analyzed the normal blood reference gDNA sample at 500ng input in duplicate across all platforms. All samples were sequenced to a ∼30x depth on an Illumina HiSeq 2500 sequencer. We compared the genomic coverage, precision and off-target effects and overall concordance in methylome profiles for each method. In summary, here we provide a comprehensive comparison of the performance of 5 different commercially available methods for medium to large scale targeted bisulfite sequencing, facilitating information to researchers for selection of appropriate method depending on required application. Citation Format: Miljana Tanic, Simon Rodney, James Barrett, Hannah Parker, Andrew Feber, Stephan Beck. EpiCapture: Benchmarking commercially available targeted bisulfite-sequencing platforms to gold-standard whole genome bisulfite sequencing. [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 4530.

Background. Contact lenses may be optimised to correct for the aberrations of the eye and provide superior vision. However, due to various requirements, soft contact lenses (SCL ) are deliberately fitted to be different from the shape of the eye. As a consequence, SCL will distort when placed on the eye in the clinical phenomenon known as the Lens Flexure Effect (LFE). The defocus effect of LFE has only been studied previously but little is known about its effect on aberrations. With the current intentions to correct aberrations using SCL, the effect of LFE on higher order aberrations becomes important. Method. We used finite element analysis (ANSYS) to investigate the shape change of SCL placed on eye. We transferred the output as sixteenth order even polynomials into a ray-tracing program (Zemax) to evaluate the optical performance of the pre-flexed and post-LFE SCL. A range of SCL base-curves (BOZR, 7.0 mm to 9.0 mm in seven steps) and back vertex powers (BVP, -12D to +3D in seven steps) were studied to look at the impact of these parameters on LFE. Results. Effect of LFE of BVP increases with BVP and departure of BOZR from corneal shape. However, within clinically practical values, the impact is small (around 0.5D). LFE also has an impact on spherical aberrations although its impact is varied according to BVP and BOZR. Conclusion. The optical impact of SCL LFE can be predicted using computation modelling. LFE may limit the performance of some designs of aberration-corrected SCL. Keywords: contact lens, flexure, finite elements, ray tracing, aberrations

Purpose. Pseudo-accommodating intra-ocular lenses (P-IOL) have been available for some time and the availability of accommodating IOL (A-IOL) is imminent. While these types of devices have been tested empirically, few studies have addressed the fundamental parameters governing their performance limits. We modelled the amplitude of accommodation of A-IOLs and P-IOLs to analyse parameters controlling their performance. Methods. Two types of two-element A-IOLs (those with a mobile anterior optical element, or a mobile posterior element) were modelled. Paraxial models were developed to identify key controlling parameters and potential optimal configurations, followed by finite modelling using computer assisted ray-tracing employing equi-convex/concave optical elements. A range of configurations representing varying focal lengths of front and back optical elements were tested. Degenerate cases representing P-IOLs were also tested. Results. P-IOLs have limited rate of pseudo-accommodation with axial shift (approximately 1.2D/mm). For A-IOLs, configurations with positive power front elements returned best rate of accommodation (up to approximately 3.0D/mm when the front element focal length is 25 mm). Conclusions. Considering the maximum potential amounts of axial shifts available, P-IOLs were predicted to provide less than 1D of accommodation whereas A-IOLs may provide up to 3-4D of accommodation, depending on design configuration.