1542 Background: For patients with advanced non-small cell lung cancer, tumour genotyping identifies actionable variants that inform targeted therapeutic choices, that improve outcomes. Liquid biopsy genotyping (LBG) is a non-invasive approach to tissue biopsy genotyping (TBG) that reduces turnaround, avoids repeat tissue biopsy, and can identify additional actionable variants. However, despite these benefits, patient access to LBG is not universal in a range of healthcare systems. While others have developed models evaluating the cost-effectiveness of LBG, these have are limited by assumptions regarding frequency of oncogenic variants and treatment utilisation. We utilised a micro-costing model (MCM) to quantify the cost/resources of LBG and TBG in a prospective trial ( PLAN; ClinicalTrials.gov Identifier: NCT05542485) aimed at investigating the feasibility of LBG in a tertiary cancer centre. Methods: A deterministic MCM was developed to enumerate the cost to generate a genomics report for both LBG and TBG in NSCLC. Capital costs were calculated based on up-front investment and annual depreciation/maintenance. Costs of consumables and staff time associated with each procedure was sourced from relevant hospital departments (e.g. Medical Physics) and evaluated for accuracy by a health economist and medical oncologist. We calculated the cost of sample acquisition (endobronchial ultrasound-guided biopsy or phlebotomy), processing, and genotyping for both LBG and TBG, from patients enrolled on the PLAN study (n = 100) between 08/2023-07/2024. Finally, we performed an exploratory analysis investigating potential reduction in staff time associated with automated library preparation, using currently available technology. Results: We identified that TBG requires more staff time (€534 vs €330), capital investment (€326 vs €16), and consumables (€1544 vs €788), resulting in an overall increased cost, compared with LBG (€2404 vs €1135). Automation of library preparation would reduce staff time required for LBG (Reduced to €191; 33% reduction) with less of an impact on TBG (Reduced to €485; 10% reduction). This difference was due to the increased wet-lab time with LBG and greater staff time for sample acquisition in TBG vs LBG (€298 vs €8). Finally, in the PLAN study, LBG resulted in cancellation of 12 repeat tissue biopsies, resulting in further savings. Conclusions: LBG is a cheaper alternative to TBG. Our data indicates LBG saves cost in the areas of healthcare staffing and capital infrastructure with further savings made through avoidance of repeat tissue biopsies. Thus, the resources required for LBG and TBG are different and should be considered in service planning for tumour types such as NSCLC in which genotyping is standard-of-care. Clinical trial information: NCT05542485 .

Supplementary Table 2: Differentially expressed gene list contrasting tumour region of origin between Central Tumor and Invasive Front. Supplementary Table 3: Differentially expressed gene list contrasting tumour region of origin between CT-LN. Supplementary Table 4: Differentially expressed gene list contrasting tumour region of origin between LN-IF.

Supplementary Figures S1-S2 and Supplementary Tables S1-S3.

This Supplementary Figure indicates the project plan

This Supplementary Table indicates the sample source for the study

Multiple myeloma (MM) is an incurable malignancy characterised by clonal proliferation of malignant plasma cells in bone marrow (BM). Currently, there are a lack of standardised approaches to determine patient prognosis and response to therapy. The MMProfiler is a prognostic test based on the well validated SKY92 gene signature which can accurately identify high-risk (HR) patients at diagnosis with survival of less than 2 years. The achievement of minimal residual disease (MRD) negativity after therapy is considered a key goal of treatment. Recent evidence indicates that achievement of MRD negativity may improve the PFS of MM patients with HR cytogenetics but data supporting this in SKY92-defined HR are lacking. NGS approaches have aimed to standardise MRD testing by monitoring clonal, disease-driving IGH rearrangements. Through an ongoing national study, for the first time in Ireland we are combining two genomic approaches with the aim of improving baseline genetic risk-stratification as well as subsequent assessment of response to treatment based on MRD. Ultimately, we aim to determine how SKY92 risk impacts the ability to achieve MRD negativity in transplant eligible (TE) patients. Diagnostic and follow-up BM samples were provided from TE MM patients across Ireland. Both a mononuclear cell (MNC) fraction and CD138+ fraction were isolated using SepMate tubes and the EasySep CD138+ selection kit respectively. Briefly, RNA from CD138+ plasma cells at diagnosis was used for SKY92 classification using MMProfiler (Affymetrix arrays). Invivoscribe LymphoTrack PCR-based IGH-FR1 assay and sequencing using the Illumina MiSeq platform were used to identify clonal DNA rearrangements at diagnosis. Clonality is defined as ≥2.5% of the total reads of that sample and greater than two times the third-ranked top sequence. This clonal sequence identified will be used for MRD monitoring using DNA from MNCs post-ASCT. Preliminary results show that 11/33 (33.3%) patients were classified as SKY92 HR. In addition, t(4;14) was detected in 27.2% patients, t(11;14) in 12.1%, t(14;16)/t(14;20) in 6.1% and gain(1q) in 45.5%. There was an association between SKY92 HR signature and t(4;14) (63% HR vs 9.1% SR, p<0.01), and a higher proportion also had gain(1q) (63.6% HR vs 36.4% SR). Both t(4;14) and gain(1q) are often considered as independent HR markers. Interestingly, the prevalence of SR cytogenetic marker t(11;14) was similar (9.1% HR vs 13.6% SR). LymphoTrack IGH-FR1 assay detected clonality in 16/19 patients (84.2%). Remaining patients will be sequenced using additional LymphoTrack IGH assays until a clonal sequence is determined. At follow-up, we will determine if these clonal sequences remain present post-ASCT using the appropriate targeted assay. Optimisation of DNA input and sequencing depth will define the sensitivity of MRD negativity. We successfully identified clonal IGH rearrangements in the majority of newly diagnosed MM patients, and expect this to reach >95% in combination with additional IGH-targeting assays. These clonal sequences will be used to monitor MRD. A significant proportion of MM patients in Ireland present with HR disease as defined by both SKY92 and cytogenetic profile. Future work will aim to evaluate the achievement of MRD negativity based on SKY92 status, and whether this impacts survival. We will continue to profile Irish patients and ultimately, we hope this could lead to a more risk-stratified treatment approach.

Introduction: Small diagnostic tissue samples can be inadequate in testing an expanding list of validated oncogenic driver alterations and fail to reflect intratumour heterogeneity (ITGH) in lung cancer. Liquid biopsies are non-invasive and may better reflect ITGH. Most liquid biopsies are performed in the context of circulating tumour DNA (ctDNA) in plasma but Exhaled Breath Condensate (EBC) shows promise as a lung-specific liquid biopsy. Methods: In this prospective, proof-of-concept study we carried out targeted Next Generation Sequencing (NGS) on diagnostic tissue samples from 125 patients with lung cancer and compared results to plasma and EBC for 5 oncogenic driver mutations (EGFR, KRAS, PIK3CA, ERBB2, BRAF) using an ultrasensitive PCR technique (UltraSEEK T Lung Panel on the MassARRAY (R) System, Agena Bioscience, San Diego, CA, USA). Results: There was a significantly higher failure rate due to unamplifiable DNA in tissue NGS (57/125, 45.6%) compared to plasma (27/125, 21.6%, p <= 0.001 and EBC (26/125,20.8%, p = 0.001. Consequently, both plasma and EBC identified higher number of mutations compared to tissue NGS. Specifically, there were significantly higher numbers of mutations detected in EGFR, KRAS and PIK3CA in plasma (p = 9.82 x 10(-3), p = 3.14 x 10(-5), p = 1.95 x 10(-3)) and EBC (p = 2.18 x 10(-3), p = 2.28 x 10(-4),p = 0.016) compared to tissue NGS. There was considerable divergence in mutation profiles between plasma and EBC with 34/76 (44%) mutations detected in plasma and 37/74 (41.89%) in EBC unique to their respective liquid biopsy. Conclusions: The results suggest that EBC is effective in identifying clinically relevant alterations in patients with lung cancer using UltraSEEK T and has a potential role as an adjunct to plasma testing.

BACKGROUND:An increasing number of anti-cancer therapeutic agents target specific mutant proteins that are expressed by many different tumor types. Successful use of these therapies is dependent on the presence or absence of somatic mutations within the patient's tumor that can confer clinical efficacy or drug resistance. METHODS:The aim of our study was to determine the type, frequency, overlap and functional proteomic effects of potentially targetable recurrent somatic hotspot mutations in 47 cancer-related genes in multiple disease sites that could be potential therapeutic targets using currently available agents or agents in clinical development. RESULTS:Using MassArray technology, of the 1300 patient tumors analysed 571 (43.9%) had at least one somatic mutation. Mutations were identified in 30 different genes. KRAS (16.5%), PIK3CA (13.6%) and BRAF (3.8%) were the most frequently mutated genes. Prostate (10.8%) had the lowest number of somatic mutations identified, while no mutations were identified in sarcoma. Ocular melanoma (90.6%), endometrial (72.4%) and colorectal (66.4%) tumors had the highest number of mutations. We noted high concordance between mutations in different parts of the tumor (94%) and matched primary and metastatic samples (90%). KRAS and BRAF mutations were mutually exclusive. Mutation co-occurrence involved mainly PIK3CA and PTPN11, and PTPN11 and APC. Reverse Phase Protein Array (RPPA) analysis demonstrated that PI3K and MAPK signalling pathways were more altered in tumors with mutations compared to wild type tumors. CONCLUSIONS:Hotspot mutational profiling is a sensitive, high-throughput approach for identifying mutations of clinical relevance to molecular based therapeutics for treatment of cancer, and could potentially be of use in identifying novel opportunities for genotype-driven clinical trials.

Additional file 4: Table S3. Significant Spearman’s Rho correlations from RPPA measurements of stromal cells.

Stromal gene expression patterns predict patient outcomes in colorectal cancer. TRIM28 is a transcriptional co-repressor that regulates an abundance of genes through the KRAB domain family of transcription factors. We have previously shown that stromal expression of TRIM28 is a marker of disease relapse and poor survival in colorectal cancer. Here, we perform differential epithelium-stroma proteomic network analyses to characterize signaling pathways associated with TRIM28 within the tumor microenvironment.

In this study, we developed an image analysis algorithm for quantification of two potential apoptotic biomarkers in non-small-cell lung cancer (NSCLC): FLIP and procaspase-8. Immunohistochemical expression of FLIP and procaspase-8 in 184 NSCLC tumors were assessed. Individual patient cores were segmented and classified as tumor and stroma using the Definiens Tissue Studio. Subsequently, chromogenic expression of each biomarker was measured separately in the nucleus and cytoplasm and reported as a quantitative histological score. The software package pROC was applied to define biomarker thresholds. Cox proportional hazards analysis was applied to generate hazard ratios (HRs) and associated 95% CI for survival. High cytoplasmic expression of tumoral (but not stromal) FLIP was associated with a 2.5-fold increased risk of death in lung adenocarcinoma patients, even when adjusted for known confounders (HR 2.47, 95% CI 1.14-5.35). Neither nuclear nor cytoplasmic tumoral procaspase-8 expression was associated with overall survival in lung adenocarcinoma patients; however, there was a significant trend (P for trend = 0.03) for patients with adenocarcinomas with both high cytoplasmic FLIP and high cytoplasmic procaspase-8 to have a multiplicative increased risk of death. Notably, high stromal nuclear procaspase-8 expression was associated with a reduced risk of death in lung adenocarcinoma patients (adjusted HR 0.31, 95% CI 0.15-0.66). On further examination, the cells with high nuclear procaspase-8 were found to be of lymphoid origin, suggesting that the better prognosis of patients with tumors with high stromal nuclear procaspase-8 is related to immune infiltration, a known favorable prognostic factor. No significant associations were detected in analysis of lung squamous cell carcinoma patients. Our results suggest that cytoplasmic expression of FLIP in the tumor and nuclear expression of procaspase-8 in the stroma are prognostically relevant in non-small-cell adenocarcinomas but not in squamous cell carcinomas of the lung.

Somatic mutations in patient tumor DNA samples can be readily detected based on mass spectrometry. The MassARRAY system is a high-throughput matrix-assisted laser desorption time-of-flight (MALDI) mass spectrometer for detection of nucleic acids. The technique is based on single-nucleotide base extension. A series of PCR assays amplify specific DNA regions of interest harboring mutations. A third primer is then introduced into the reaction which corresponds to the DNA template immediately in front of the mutation site. A final round of PCR is then performed using mass-modified nucleotides. These nucleotides are designed so that no additional bases can be added to the extension primer (terminating bases) after a single-base extension and are mass modified to exaggerate mass differences between nucleotides allowing easier identification by mass spectrometry.The sequences of the extension primer and possible extension products (wild type and mutations) are known; therefore, it is possible to calculate their mass. The mass spectrometer can identify the mass peaks for each assay and identify those with mutations (multiple peaks). The technique was originally designed to screen multiple single-nucleotide polymorphisms (SNPs) in a large number of specimens. A SNP in the coding region of DNA that alters the gene and subsequent protein expression is considered a mutation. Mutations often occur in genes whose protein product is in a key signaling pathway and/or drug target. Rationale treatment options can be designed based upon the presence or absence of these mutations. In this chapter, we describe the process for detection of somatic mutations in DNA extracted from formalin-fixed paraffin-embedded (FFPE) material.

Abstract Purpose: A number of independent gene expression profiling studies have identified transcriptional subtypes in colorectal cancer with potential diagnostic utility, culminating in publication of a colorectal cancer Consensus Molecular Subtype classification. The worst prognostic subtype has been defined by genes associated with stem-like biology. Recently, it has been shown that the majority of genes associated with this poor prognostic group are stromal derived. We investigated the potential for tumor misclassification into multiple diagnostic subgroups based on tumoral region sampled. Experimental Design: We performed multiregion tissue RNA extraction/transcriptomic analysis using colorectal-specific arrays on invasive front, central tumor, and lymph node regions selected from tissue samples from 25 colorectal cancer patients. Results: We identified a consensus 30-gene list, which represents the intratumoral heterogeneity within a cohort of primary colorectal cancer tumors. Using a series of online datasets, we showed that this gene list displays prognostic potential HR = 2.914 (confidence interval 0.9286–9.162) in stage II/III colorectal cancer patients, but in addition, we demonstrated that these genes are stromal derived, challenging the assumption that poor prognosis tumors with stem-like biology have undergone a widespread epithelial–mesenchymal transition. Most importantly, we showed that patients can be simultaneously classified into multiple diagnostically relevant subgroups based purely on the tumoral region analyzed. Conclusions: Gene expression profiles derived from the nonmalignant stromal region can influence assignment of colorectal cancer transcriptional subtypes, questioning the current molecular classification dogma and highlighting the need to consider pathology sampling region and degree of stromal infiltration when employing transcription-based classifiers to underpin clinical decision making in colorectal cancer. Clin Cancer Res; 22(16); 4095–104. ©2016 AACR. See related commentary by Morris and Kopetz, p. 3989

Ceramide synthase 5 is involved in the de novo synthesis of ceramide, a sphingolipid involved in cell death and proliferation. In this study, we investigated the role of ceramide synthase 5 in colorectal cancer by examining ceramide synthase 5 expression, clinico-pathological parameters and association with survival/death signalling pathways in cancer. Immunohistochemical analysis of CerS5 was performed on 102 colorectal cancer samples using tissue microarrays constructed from formalin-fixed and paraffin-embedded tissues. We found strong membranous ceramide synthase 5 staining in 57 of 102 (56%) colorectal cancers. A multivariate Cox regression analysis of ceramide synthase 5 expression adjusted for disease stage, differentiation and lympho-vascular invasion revealed reduced 5-year overall survival (p = 0.001) and 5-year recurrence-free survival (p = 0.002), with hazard ratios of 4.712 and 4.322, respectively. The effect of ceramide synthase 5 expression on tumourigenic processes was further characterised by reverse phase protein array analysis. Reverse phase protein arrays were generated from laser capture microdissection-enriched carcinoma cells from 19 fresh-frozen colorectal cancer tissues. Measurements of phosphorylation and total levels of signalling proteins involved in apoptosis, autophagy and other cancer-related pathways revealed two distinct signalling networks; weak membranous ceramide synthase 5 intensity was associated with a proteomic network dominated by signalling proteins linked to apoptosis, whereas strong ceramide synthase 5 intensity was associated with a proteomic sub-network mostly composed of proteins linked to autophagy. In conclusion, high ceramide synthase 5 expression was found in colorectal cancer tissue and was associated with poorer patient outcomes. Our findings suggest that this may be mediated by a transition from apoptotic to autophagy signalling pathways in ceramide synthase 5 High expressing tumours, thus implicating ceramide synthase 5 in the progression of colorectal cancer.

Because of advances in targeted therapies, the clinical evaluation of cutaneous melanoma is increasingly based on a combination of traditional histopathology and molecular pathology. Therefore, it is necessary to expand our knowledge of the molecular events that accompany the development and progression of melanoma to optimize clinical management. The central objective of this study was to increase our knowledge of the mutational events that complement melanoma progression. High-throughput genotyping was adapted to query 159 known single nucleotide mutations in 33 cancer-related genes across two melanoma cohorts from Ireland (n=94) and Belgium (n=60). Results were correlated with various clinicopathological characteristics. A total of 23 mutations in 12 genes were identified, that is--BRAF, NRAS, MET, PHLPP2, PIK3R1, IDH1, KIT, STK11, CTNNB1, JAK2, ALK, and GNAS. Unexpectedly, we discovered significant differences in BRAF, MET, and PIK3R1 mutations between the cohorts. That is, cases from Ireland showed significantly lower (P<0.001) BRAF(V600E) mutation rates (19%) compared with the mutation frequency observed in Belgian patients (43%). Moreover, MET mutations were detected in 12% of Irish cases, whereas none of the Belgian patients harbored these mutations, and Irish patients significantly more often (P=0.027) had PIK3R1-mutant (33%) melanoma versus 17% of Belgian cases. The low incidence of BRAF(V600E)(-) mutant melanoma among Irish patients was confirmed in five independent Irish cohorts, and in total, only 165 of 689 (24%) Irish cases carried mutant BRAF(V600E). Together, our data show that melanoma-driving mutations vary by demographic area, which has important implications for the clinical management of this disease.

Colorectal cancer is a leading cause of cancer related deaths worldwide. Early diagnosis and treatment is the key to improving survival rates and as such a need exists to identify patients who may benefit from adjuvant chemotherapy. The dysregulation of the ubiquitin-proteasome system (UPS) has been implicated in oncogenesis and cancer cell survival, and proteasome inhibitors are in clinical use for a number of malignancies including multiple myeloma. In this study we examined the protein expression of several key components of the ubiquitin-proteasome system in colorectal cancer using immunohistochemistry to determine expression levels of ubiquitinylated proteins and the proteasomal subunits, 20S core and Rpt4 in a cohort of 228 colon cancer patients. Multivariate Cox analysis revealed that neither the intensity of either ubiquitinylated proteins or the 20S core were predictive in either Stage II or III colon cancer for disease free survival or overall survival. In contrast, in Stage II patients increased Rpt4 staining was significantly associated with better disease free survival (Cox proportional hazard ratio 0.605; p=0.0217). Our data suggest that Rpt4 is an independent prognostic variable for Stage II colorectal cancer and may aid in the decision of which patients undergo adjuvant chemotherapy.

Abstract Background: TRIM28 is a universal transcriptional co-repressor with pleotropic effects in both normal and tumor cells. We have previously shown that varying TRIM28 levels in epithelial cells and stromal fibroblasts have a prognostic value in colorectal cancer patients. The pathophysiological role of TRIM28 in carcinogenesis may therefore be associated with TRIM28 expression levels and cell type of expression within the tumor microenvironment. In this study we aim to dissect the molecular pathways associated with TRIM28 expression ratios within the tumor microenvironment by isolating individual populations of neoplastic epithelial and stromal cells and investigating their protein signaling networks. Methods: TRIM28 expression was analyzed by immunohistochemistry on FFPE tissue from 19 colorectal cancer patients. TRIM28 staining was evaluated in both epithelial and stromal compartments. IHC scoring of at least 2 units of difference in staining intensity between stromal fibroblasts and epithelial cells was defined as a high TRIM28 expression ratio and a low TRIM28 expression ratio was defined as 1 or 0 units of difference in staining intensity. Reverse-phase protein microarrays (RPMA) were constructed from laser capture micro-dissection (LCM) enriched tumor epithelium and stroma isolated from fresh-frozen tissue of the same patient cohort. The protein signaling networks were measured for 32 downstream signaling endpoints. Spearman rank analysis was used to assess the correlations between individual protein pairs. Correlation coefficient ρ ≥ 0.75 with P ≤ 0.01 was considered significant. Results: Immunohistochemical analysis of the FFPE tissue sections identified 10 TRIM28 high ratio cases and 9 TRIM28 low ratio cases. Proteomic networks were assessed in TRIM28 high and low ratio cases in fresh-frozen tissue using RPMA. Spearman ρ rank correlation analyses for the 32 signaling proteins revealed that 184 highly correlated protein pairs exclusive to the TRIM28 high ratio group, whereas 157 protein pairs were found exclusively in the TRIM28 low ratio group. In addition 191 protein pairs were shared across both TRIM28 high and low ratio groups. The caspases 3 and 7, as well as the cell surface receptor RAGE, were prominent in the high TRIM28 ratio group proteomic network, whereas the Metalloprotease MMP9 and the GTPase Ras-GRF1 were exclusive to the low TRIM28 ratio group. Furthermore we found Survivin and JNK to be prominent in the stromal compartment of the high TRIM28 cases. Conclusions: We characterized molecular pathways associated with TRIM28 expression ratios within the tumor microenvironment. Proteomic analysis revealed distinct protein signaling networks associated with varying TRIM28 expression levels in epithelial and stromal compartments. The study presents a novel way of deciphering molecular crosstalk between the epithelial and stromal compartments within the microenvironment. Citation Format: Seán Fitzgerald, Virginia Espina, Katherine M. Sheehan, Robert Cummins, Anthony O'Grady, Dermot Kenny, Richard O'Kennedy, Lance Liotta, Elaine W. Kay, Gregor Kijanka. Molecular characterization of epithelial and stromal crosstalk associated with TRIM28 expression levels in colorectal cancer. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B55. doi:10.1158/1538-7445.CHTME14-B55

The expression profiles of the epidermal growth factor receptor (EGFR) and insulin-like growth factor receptor 1 (IGF1R) were determined by immunohistochemical (IHC) analysis (n = 184) and Western blot analysis (n = 40) in patients with nonesmall-cell lung cancer (NSCLC) who had undergone surgical resection. High coexpression of both receptors (H-score >= 200) was more common in squamous cell carcinoma (SCC) and correlated with poor overall survival (OS) (P = .04). This subset of patients may respond favorably to combination targeted therapies. Background: Recent experimental and biomarker evidence indicates that the epidermal growth factor receptor (EGFR) and insulin-like growth factor receptor 1 (IGF1R) interact in the pathogenesis of malignant epithelial tumors, including lung cancer. This study examines the expression of both receptors and their prognostic significance in surgically resected nonesmall-cell lung cancer (NSCLC). Methods: EGFR and IGF1R expression were evaluated in 184 patients with NSCLC (83 squamous cell carcinomas [SCCs], 83 adenocarcinomas [ADCs], and 18 other types) using immunohistochemical (IHC) analysis. Expression of both receptors was examined in matched fresh frozen normal and tumor tissues from 40 patients with NSCLC (20 SCCs and 20 ADCs) by Western blot analysis. Results: High EGFR expression was detected in 51% of patients, and SCCs had higher EGFR expression than did non-SCCs (57.4% vs. 42.5%; P = .028). High IGF1R expression was observed in 53.8% of patients, with SCC having higher expression than non-SCC (62.6% vs. 37.3%; P = .0004). A significant association was shown between EGFR and IGF1R protein overexpression (P < .005). Patients with high expression of both receptors had a poorer overall survival (OS) (P = .04). Higher EGFR and IGF1R expression was detected in resected tumors relative to matched normal tissues (P = .0004 and P = .0009), with SCC having higher expression levels than ADC. Conclusion: Our findings indicate a close interrelationship between EGFR and IGF1R. Coexpression of both receptors correlates with poor survival. This subset of patients may benefit from treatments cotargeting EGFR and IGF1R. (C) 2014 Elsevier Inc. All rights reserved.