Quality control in microarray assessment of gene expression in human airway epithelium

<p>Abstract</p> <p>Background</p> <p>Microarray technology provides a powerful tool for defining gene expression profiles of airway epithelium that lend insight into the pathogenesis of human airway disorders. The focus of this study was to establish rigorous quality control parameters to ensure that microarray assessment of the airway epithelium is not confounded by experimental artifact. Samples (total n = 223) of trachea, large and small airway epithelium were collected by fiberoptic bronchoscopy of 144 individuals and hybridized to Affymetrix microarrays. The pre- and post-chip quality control (QC) criteria established, included: (1) RNA quality, assessed by RNA Integrity Number (RIN) ≥ 7.0; (2) cRNA transcript integrity, assessed by signal intensity ratio of GAPDH 3' to 5' probe sets ≤ 3.0; and (3) the multi-chip normalization scaling factor ≤ 10.0.</p> <p>Results</p> <p>Of the 223 samples, all three criteria were assessed in 191; of these 184 (96.3%) passed all three criteria. For the remaining 32 samples, the RIN was not available, and only the other two criteria were used; of these 29 (90.6%) passed these two criteria. Correlation coefficients for pairwise comparisons of expression levels for 100 maintenance genes in which at least one array failed the QC criteria (average Pearson r = 0.90 ± 0.04) were significantly lower (p < 0.0001) than correlation coefficients for pairwise comparisons between arrays that passed the QC criteria (average Pearson r = 0.97 ± 0.01). Inter-array variability was significantly decreased (p < 0.0001) among samples passing the QC criteria compared with samples failing the QC criteria.</p> <p>Conclusion</p> <p>Based on the aberrant maintenance gene data generated from samples failing the established QC criteria, we propose that the QC criteria outlined in this study can accurately distinguish high quality from low quality data, and can be used to delete poor quality microarray samples before proceeding to higher-order biological analyses and interpretation.</p

Limited heterogeneity of known driver gene mutations among the metastases of individual patients with pancreatic cancer

The extent of heterogeneity among driver gene mutations present in naturally occurring metastases - that is, treatment-naive metastatic disease - is largely unknown. To address this issue, we carried out 60× whole-genome sequencing of 26 metastases from four patients with pancreatic cancer. We found that identical mutations in known driver genes were present in every metastatic lesion for each patient studied. Passenger gene mutations, which do not have known or predicted functional consequences, accounted for all intratumoral heterogeneity. Even with respect to these passenger mutations, our analysis suggests that the genetic similarity among the founding cells of metastases was higher than that expected for any two cells randomly taken from a normal tissue. The uniformity of known driver gene mutations among metastases in the same patient has critical and encouraging implications for the success of future targeted therapies in advanced-stage disease

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

GSTP1 hypermethylation is associated with reduced protein expression, aggressive disease and prognosis in neuroblastoma

Epigenetic modifications such as methylation of CpG islands in tumor-suppressor gene promoter regions have been associated with tumor development in many human cancers. Using methylation specific multiplex ligation-dependent probe amplification method, we analyzed the methylation status of 35 different genes in 16 neuroblastoma (NB) cell lines and 50 NB tumor samples (NBs), and investigated whether specific hypermethylation was associated with biological and/or clinical parameters. Among the genes found hypermethylated, the effect of GSTP1 hypermethylation on mRNA and protein expression was also explored. The median number of hypermethylated genes was higher in cell lines compared to NBs (5.5 vs. 2). For eight genes, aberrant methylation of CpG-islands in NB was not (ESR1, PAX5, WT1, CADM1, MSH6, and CDKN2B) or very rarely (CDH13 and GSTP1) reported in literature. GSTP1 was found hypermethylated in 44% of the NB cell lines and in 33% of the stage 4-11qLOH -non MYCN-amplified high risk NBs. Hypermethylation was correlated with reduced mRNA and protein expression. In the whole NBs cohort, GSTP1 hypermethylation was less frequently detected (8%), but found to be associated with lower event-free (EFS) and overall survival. Hypermethylation of GSTP1 showed also association with lower EFS in high risk subgroups as stage 4 and older patients (≥547 days). Our results suggest that, as in several adult cancers, aberrant methylation of GSTP1 may contribute to the carcinogenetic process in NB and could be potentially used as a new marker leading to define an ultra-high risk subgroup

Patterns of Failure in Patients With Borderline Resectable/Locally Advanced Pancreatic Cancer After Preoperative Chemotherapy and Stereotactic Body Radiation Therapy.

PURPOSE: The role of preoperative stereotactic body radiation therapy (SBRT) in pancreatic cancer is controversial, and questions regarding the optimal dose and radiation treatment field remain. To better inform future investigations of SBRT dose and radiation fields, we evaluated the patterns of failure in patients with borderline resectable/locally advanced pancreatic cancer (BR/LAPC) after preoperative chemotherapy and SBRT in patients who underwent surgical resection. METHODS AND MATERIALS: We performed a single-institution retrospective review of consecutive patients treated from September 2017 to January 2022 with BR/LAPC. Patients who underwent preoperative chemotherapy and SBRT followed by surgical resection were reviewed. SBRT was delivered to a dose of 33 Gy in 5 fractions. Kaplan-Meier overall survival and progression-free survival estimates were calculated. RESULTS: In total, 18 patients (12 BRPC, 6 LAPC) were included. Median age was 69 years (range 41-84 years). Median follow-up was 30 months (range 13-59 months). Seventeen patients (94%) had a R0 resection and 13 (72%) underwent vascular reconstruction. Median overall survival and progression-free survival was 42 months (range 13-59 months) and 23 months (range 1-45 months), respectively. In total, 61% (11/18) patients experienced progression at any point during follow-up. Of the patients who experienced recurrence, 27% (3/11) experienced local progression as component of their first recurrence, whereas 100% (11/11) experienced distant progression as a component of their first recurrence. When examining all recurrences that occurred at any point in follow-up, 28% (5/18) of patients experienced local or locoregional recurrence and 61% (11/18) experienced distant progression. CONCLUSIONS: Local control and margin negative resection rates were excellent with preoperative chemotherapy and nondose-escalated SBRT in surgically resected patients with BR/LAPC. Distant recurrence was the predominant site of failure with lower incidences of isolated locoregional recurrences. Additional research is needed to determine the ideal treatment volume and patients who may benefit from dose escalation

Additional file 1: Table S1. of Melanoma genome evolution across species

ZMEL1 whole-genome sequencing. This file contains all of the mutations called by MuTect and Shimmer, along with their associated quality scores and overlapping mutations. (XLSX 6276 kb

Additional file 4: Table S4. of Melanoma genome evolution across species

ZMELR1 vs. ZMEL1 mRNA differential expression. This file contains the differentially expressed genes from RNA-seq of the ZMEL1 line vs. the ZMELR1 line. (XLSX 3569 kb

Additional file 2: Table S2. of Melanoma genome evolution across species

ZMEL1 validation primers for MiSeq run. This file contains all of the PCR primer sequences used to validate the subset of 384 called mutations that were validated in the ZMEL1 line via MiSeq. (XLSX 58 kb