MALDI Imaging Mass Spectrometry (MALDI-IMS)—Application of Spatial Proteomics for Ovarian Cancer Classification and Diagnosis

MALDI imaging mass spectrometry (MALDI-IMS) allows acquisition of mass data for metabolites, lipids, peptides and proteins directly from tissue sections. IMS is typically performed either as a multiple spot profiling experiment to generate tissue specific mass profiles, or a high resolution imaging experiment where relative spatial abundance for potentially hundreds of analytes across virtually any tissue section can be measured. Crucially, imaging can be achieved without prior knowledge of tissue composition and without the use of antibodies. In effect MALDI-IMS allows generation of molecular data which complement and expand upon the information provided by histology including immuno-histochemistry, making its application valuable to both cancer biomarker research and diagnostics. The current state of MALDI-IMS, key biological applications to ovarian cancer research and practical considerations for analysis of peptides and proteins on ovarian tissue are presented in this review

Can we accurately report PTEN status in advanced colorectal cancer?

BACKGROUND: Loss of phosphatase and tensin homologue (PTEN) function evaluated by loss of PTEN protein expression on immunohistochemistry (IHC) has been reported as both prognostic in metastatic colorectal cancer and predictive of response to anti-EGFR monoclonal antibodies although results remain uncertain. Difficulties in the methodological assessment of PTEN are likely to be a major contributor to recent conflicting results. METHODS: We assessed loss of PTEN function in 51 colorectal cancer specimens using Taqman® copy number variation (CNV) and IHC. Two blinded pathologists performed independent IHC assessment on each specimen and inter-observer variability of IHC assessment and concordance of IHC versus Taqman® CNV was assessed. RESULTS: Concordance between pathologists (PTEN loss vs no loss) on IHC assessment was 37/51 (73%). In specimens with concordant IHC assessment, concordance between IHC and Taqman® copy number in PTEN loss assessment was 25/37 (68%). CONCLUSION: Assessment PTEN loss in colorectal cancer is limited by the inter-observer variability of IHC, and discordance of CNV with loss of protein expression. An understanding of the genetic mechanisms of PTEN loss and implementation of improved and standardized methodologies of PTEN assessment are required to clarify the role of PTEN as a biomarker in colorectal cancer

Metallothionien 3 expression is frequently down-regulated in oesophageal squamous cell carcinoma by DNA methylation

BACKGROUND: Metallothionein 3 (MT3) inhibits growth in a variety of cell types. We measured MT3 gene expression by RT-PCR, and DNA methylation in the MT3 promoter by combined bisulphite restriction analysis, in four oesophageal cancer cell lines and the resected oesophagus from 64 patients with oesophageal squamous cell carcinoma (SCC). RESULTS: MT3 expression was not detected in one of the four oesophageal cell lines. The MT3 promoter was methylated in all of the oesophageal cell lines, but the degree of methylation was greater in the non-expressing cell line. After treatment with 5-aza-2'-deoxycytidine there was a reduction in the degree of methylation, and an increase in MT3 expression, in each of the cell lines (p < 0.01). Methylation was detected in 52% (33 of 64) of primary SCC and 3% (2 of 62) of histologically normal resection margins. MT3 expression was measured in 29 tumours, 17 of which had methylation of MT3. The expression of MT3 was significantly less in the methylated tumours compared to either the unmethylated tumours (p = 0.03), or the matched margin (p = 0.0005). There was not a significant difference in MT3 expression between the tumour and the margin from patients with unmethylated tumour. No correlations were observed between methylation of MT3 and survival time, patient age, gender, smoking or drinking history, tumour stage, volume, or lymph node involvement. CONCLUSION: We conclude that MT3 expression is frequently down-regulated in oesophageal SCC, by DNA methylation, but that this is not a prognostic indicator

Similarity of aberrant DNA methylation in Barrett's esophagus and esophageal adenocarcinoma

Background Barrett's esophagus (BE) is the metaplastic replacement of squamous with columnar epithelium in the esophagus, as a result of reflux. It is the major risk factor for the development of esophageal adenocarcinoma (EAC). Methylation of CpG dinucleotides of normally unmethylated genes is associated with silencing of their expression, and is common in EAC. This study was designed to determine at what stage, in the progression from BE to EAC, methylation of key genes occurs. Results We examined nine genes (APC, CDKN2A, ID4, MGMT, RBP1, RUNX3, SFRP1, TIMP3, and TMEFF2), frequently methylated in multiple cancer types, in a panel of squamous (19 biopsies from patients without BE or EAC, 16 from patients with BE, 21 from patients with EAC), BE (40 metaplastic, seven high grade dysplastic) and 37 EAC tissues. The methylation frequency, the percentage of samples that had any extent of methylation, for each of the nine genes in the EAC (95%, 59%, 76%, 57%, 70%, 73%, 95%, 74% and 83% respectively) was significantly higher than in any of the squamous groups. The methylation frequency for each of the nine genes in the metaplastic BE (95%, 28%, 78%, 48%, 58%, 48%, 93%, 88% and 75% respectively) was significantly higher than in the squamous samples except for CDKN2A and RBP1. The methylation frequency did not differ between BE and EAC samples, except for CDKN2A and RUNX3 which were significantly higher in EAC. The methylation extent was an estimate of both the number of methylated alleles and the density of methylation on these alleles. This was significantly greater in EAC than in metaplastic BE for all genes except APC, MGMT and TIMP3. There was no significant difference in methylation extent for any gene between high grade dysplastic BE and EAC. Conclusion We found significant methylation in metaplastic BE, which for seven of the nine genes studied did not differ in frequency from that found in EAC. This is also the first report of gene silencing by methylation of ID4 in BE or EAC. This study suggests that metaplastic BE is a highly abnormal tissue, more similar to cancer tissue than to normal epithelium.Eric Smith, Neville J De Young, Sandra J Pavey, Nicholas K Hayward, Derek J Nancarrow, David C Whiteman, B Mark Smithers, Andrew R Ruszkiewicz, Andrew D Clouston, David C Gotley, Peter G Devitt, Glyn G Jamieson and Paul A Dre

Identification of unique neoantigen qualities in long-term survivors of pancreatic cancer

Pancreatic ductal adenocarcinoma is a lethal cancer with fewer than 7% of patients surviving past 5 years. T-cell immunity has been linked to the exceptional outcome of the few long-term survivors1,2, yet the relevant antigens remain unknown. Here we use genetic, immunohistochemical and transcriptional immunoprofiling, computational biophysics, and functional assays to identify T-cell antigens in long-term survivors of pancreatic cancer. Using whole-exome sequencing and in silico neoantigen prediction, we found that tumours with both the highest neoantigen number and the most abundant CD8+ T-cell infiltrates, but neither alone, stratified patients with the longest survival. Investigating the specific neoantigen qualities promoting T-cell activation in long-term survivors, we discovered that these individuals were enriched in neoantigen qualities defined by a fitness model, and neoantigens in the tumour antigen MUC16 (also known as CA125). A neoantigen quality fitness model conferring greater immunogenicity to neoantigens with differential presentation and homology to infectious disease-derived peptides identified long-term survivors in two independent datasets, whereas a neoantigen quantity model ascribing greater immunogenicity to increasing neoantigen number alone did not. We detected intratumoural and lasting circulating T-cell reactivity to both high-quality and MUC16 neoantigens in long-term survivors of pancreatic cancer, including clones with specificity to both high-quality neoantigens and predicted cross-reactive microbial epitopes, consistent with neoantigen molecular mimicry. Notably, we observed selective loss of high-quality and MUC16 neoantigenic clones on metastatic progression, suggesting neoantigen immunoediting. Our results identify neoantigens with unique qualities as T-cell targets in pancreatic ductal adenocarcinoma. More broadly, we identify neoantigen quality as a biomarker for immunogenic tumours that may guide the application of immunotherapies

Targeting DNA Damage Response and Replication Stress in Pancreatic Cancer

Background and aims: Continuing recalcitrance to therapy cements pancreatic cancer (PC) as the most lethal malignancy, which is set to become the second leading cause of cancer death in our society. The study aim was to investigate the association between DNA damage response (DDR), replication stress and novel therapeutic response in PC to develop a biomarker driven therapeutic strategy targeting DDR and replication stress in PC. Methods: We interrogated the transcriptome, genome, proteome and functional characteristics of 61 novel PC patient-derived cell lines to define novel therapeutic strategies targeting DDR and replication stress. Validation was done in patient derived xenografts and human PC organoids. Results: Patient-derived cell lines faithfully recapitulate the epithelial component of pancreatic tumors including previously described molecular subtypes. Biomarkers of DDR deficiency, including a novel signature of homologous recombination deficiency, co-segregates with response to platinum (P &lt; 0.001) and PARP inhibitor therapy (P &lt; 0.001) in vitro and in vivo. We generated a novel signature of replication stress with which predicts response to ATR (P &lt; 0.018) and WEE1 inhibitor (P &lt; 0.029) treatment in both cell lines and human PC organoids. Replication stress was enriched in the squamous subtype of PC (P &lt; 0.001) but not associated with DDR deficiency. Conclusions: Replication stress and DDR deficiency are independent of each other, creating opportunities for therapy in DDR proficient PC, and post-platinum therapy

HER-2/neu gene amplification in esophageal adenocarcinoma and its influence on survival

The original publication is available at the Annals website at www.springerlink.com/content/1534-4681.Background: HER-2/neu (c-erbB-2, HER2) gene amplification and protein overexpression have been associated with poor prognosis in several solid tumors, including breast and gastric cancer. Its incidence and significance in esophageal adenocarcinoma is unknown. Materials and Methods: Tissue microarrays were successfully constructed from 89 paraffin-embedded archival specimens of esophageal adenocarcinomas for HER2 gene amplification by silver-enhanced in situ hybridization (SISH). No patients had undergone neoadjuvant therapy. Protein overexpression was tested with immunohistochemistry (IHC) using automated immunostaining (Ventana Benchmark). Incidence of HER2 positivity, correlation to clinicopathological variables in esophageal cancer patients, and concordance between SISH and IHC were determined. Results: True HER2 gene amplification was detected in 14 esophageal cancer specimens (16%), and 92% of those with high-level HER2 amplification showed positive HER2 protein overexpression. No significant associations were found among gene amplification and clinicopathological factors. The 5-year survival rates were 57% for esophageal cancer patients with HER2 amplification compared with 32% without, but the difference in overall survival was not significant (P = .37). The correlation between SISH and IHC was statistically significant (P < .0001). Conclusion: While molecular targeting may be possible for approximately 16% of esophageal adenocarcinoma patients, HER2 oncogene amplification did not influence survival in this study.Sarah K. Thompson, Thomas R. Sullivan, Ruth Davies and Andrew R. Ruszkiewic

DNA methylation patterns identify subgroups of pancreatic neuroendocrine tumors with clinical association

Here we report the DNA methylation profile of 84 sporadic pancreatic neuroendocrine tumors (PanNETs) with associated clinical and genomic information. We identified three subgroups of PanNETs, termed T1, T2 and T3, with distinct patterns of methylation. The T1 subgroup was enriched for functional tumors and ATRX, DAXX and MEN1 wild-type genotypes. The T2 subgroup contained tumors with mutations in ATRX, DAXX and MEN1 and recurrent patterns of chromosomal losses in half of the genome with no association between regions with recurrent loss and methylation levels. T2 tumors were larger and had lower methylation in the MGMT gene body, which showed positive correlation with gene expression. The T3 subgroup harboured mutations in MEN1 with recurrent loss of chromosome 11, was enriched for grade G1 tumors and showed histological parameters associated with better prognosis. Our results suggest a role for methylation in both driving tumorigenesis and potentially stratifying prognosis in PanNETs