Formalin-fixed paraffin-embedded (FFPE) tissue specimens comprise a potentially valuable resource for retrospective biomarker discovery studies, and recent work indicates the feasibility of using shotgun proteomics to characterize FFPE tissue proteins. A critical question in the field is whether proteomes characterized in FFPE specimens are equivalent to proteomes in corresponding fresh or frozen tissue specimens. Here we compared shotgun proteomic analyses of frozen and FFPE specimens prepared from the same colon adenoma tissues. Following deparaffinization, rehydration, and tryptic digestion under mild conditions, FFPE specimens corresponding to 200 μg of protein yielded ∼400 confident protein identifications in a one-dimensional reverse phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The major difference between frozen and FFPE proteomes was a decrease in the proportions of lysine C-terminal to arginine C-terminal peptides observed, but these differences had little effect on the proteins identified. No covalent peptide modifications attributable to formaldehyde chemistry were detected by analyses of the MS/MS datasets, which suggests that undetected, cross-linked peptides comprise the major class of modifications in FFPE tissues. Fixation of tissue for up to 2 days in neutral buffered formalin did not adversely impact protein identifications. Analysis of archival colon adenoma FFPE specimens indicated equivalent numbers of MS/MS spectral counts and protein group identifications from specimens stored for 1, 3, 5, and 10 years. Combination of peptide isoelectric focusing-based separation with reverse phase LC-MS/MS identified 2554 protein groups in 600 ng of protein from frozen tissue and 2302 protein groups from FFPE tissue with at least two distinct peptide identifications per protein. Analysis of the combined frozen and FFPE data showed a 92% overlap in the protein groups identified. Comparison of gene ontology categories of identified proteins revealed no bias in protein identification based on subcellular localization. Although the status of posttranslational modifications was not examined in this study, archival samples displayed a modest increase in methionine oxidation, from ∼17% after one year of storage to ∼25% after 10 years. These data demonstrate the equivalence of proteome inventories obtained from FFPE and frozen tissue specimens and provide support for retrospective proteomic analysis of FFPE tissues for biomarker discovery.

OBJECTIVE-Ménétrier's disease (MD) is a rare hypertrophic gastropathy characterised by giant rugal folds, hypochlorhydria, protein loss and a classic constellation of symptoms -nausea, vomiting, abdominal pain and peripheral oedema. It is considered a clinical diagnosis that, at times, may be difficult to establish. We propose firm diagnostic criteria for MD by delineating the clinicopathological features that best differentiate MD from its mimics. DESIGN-Forty-eight patients referred to Vanderbilt University Medical Center for consideration of enrolment in a clinical trial to treat MD patients with cetuximab were evaluated for a definitive diagnosis by assessment of clinical presentation, pertinent laboratory values and histopathological features.RESULTS-MD was confirmed in 25/48 (52%) of the patients. We designated the remaining 23 patients as mimics of MD; the most common diagnoses among the MD mimics were gastric polyps or polyposis syndromes (13/23, 57%). Gastric slides were available from 40 of 48 cases for detailed histological analysis (22/25 MD cases and 18/23 non-MD cases). Foveolar hyperplasia, glandular tortuosity and dilatation, and a marked reduction in parietal cell number were present in all 22 MD cases; lamina propria smooth muscle hyperplasia and oedema characterised most cases (18/22 and 19/22, respectively); more than half had prominent eosinophils (11/22) and/or plasma cells (12/22) in the lamina propria. The clinical presentation of MD patients was characterised by significantly younger age at onset, male predominance and increased vomiting compared to non-MD, and lower prevalence of anaemia compared to non-MD with polyps; there was trend towards increased frequency of peripheral oedema in MD versus non-MD.CONCLUSIONS-MD is most accurately diagnosed by clinicohistopathological analysis including esophagogastroduodenoscopy with gastric pH, appropriate laboratory tests (complete blood count, serum albumin, serum gastrin, H. pylori and cytomegalovirus serology) and full thickness mucosal biopsy of the involved gastric mucosa.Corresponding Author: Robert Coffey, robert.coffey@vanderbilt.edu, Tel: 615-343-6228, Fax: 615-343-1591 however, each has yielded inconsistent benefits, and none have been evaluated in a clinical trial. [9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26] The only definitive treatment is total gastrectomy. A recently published clinical trial from our group reported that MD patients treated with cetuximab, a monoclonal antibody to the epidermal growth factor receptor (EGFR), showed significant clinical and biochemical improvement in all 7 patients that completed the one-month trial with subsequent histological reversion to normal or near normal in 4 of these patients. [27][28][29] The pathogenesis of MD is related to increased EGFR signalling in the stomach. In vitro, administration of transforming growth factor-α (TGF-α), one of seven mammalian EGFR ligands, stimulates gastric epithelial growth, inhibits acid production and increases mucin levels...

Ménétrier’s disease is a rare, premalignant disorder of the stomach with no proven effective medical therapy. Increased epidermal growth factor (EGF) receptor signaling has been implicated in the pathogenesis of Ménétrier’s disease. We conducted a single-arm clinical trial with cetuximab, a monoclonal antibody that blocks EGF receptor signaling, in 9 individuals with clinically and histologically documented severe Ménétrier’s disease that impaired quality-of-life to the extent that gastrectomy was being considered. Of the 7 patients who completed the one-month course of treatment, all showed statistically significant improvement both clinically (quality-of-life indices) and biochemically (increased parietal cell mass and gastric acidity). Furthermore, all seven patients who completed the one-month trial elected to continue treatment, and four subsequently showed near complete histological remission. Cetuximab should be considered first line therapy for Ménétrier’s disease.

The NEDD4 family of E3 ubiquitin ligases includes nine members. Each is a modular protein, containing an N-terminal C2 domain for cell localization, two-to-four central WW domains for substrate recognition, and a C-terminal, catalytic HECT domain, which is responsible for catalyzing the ubiquitylation reaction. Members of this family are known to affect pathways central to the pathogenesis of colorectal cancer, including the WNT, TGFβ, EGFR, and p53 pathways. Recently, NEDD4 mRNA was reported to be overexpressed in colorectal cancer, but tumor stage was not considered in the analysis. Expression of the other family members has not been studied in colorectal cancer. Herein, we determined the expression patterns of all nine NEDD4 family members in 256 patients who presented with disease ranging from premalignant adenoma to stage IV colorectal cancer. NEDD4 mRNA was significantly increased in all stages of colorectal cancer. In contrast, NEDD4L mRNA, the closest homolog to NEDD4, was the most highly downregulated family member, and was significantly downregulated in all tumor stages. We also found NEDD4L protein was significantly decreased by western blotting in colorectal cancer samples compared to adjacent normal mucosa. In addition, NEDD4L, but not catalytically inactive NEDD4L, inhibited canonical WNT signaling at or below the level of β-catenin in vitro. These findings suggest that NEDD4L may play a tumor suppressive role in colorectal cancer, possibly through inhibition of canonical WNT signaling.

Effective spatio-temporal control of transcription and replication during S-phase is paramount to maintaining genomic integrity and cell survival. Dysregulation of these systems can lead to conflicts between the transcription and replication machinery, causing DNA damage and cell death. BRD4 allows efficient transcriptional elongation by stimulating phosphorylation of RNA polymerase II (RNAPII). We report that bromodomain and extra-terminal domain (BET) protein loss of function (LOF) causes RNAPII pausing on the chromatin and DNA damage affecting cells in S-phase. This persistent RNAPII-dependent pausing leads to an accumulation of RNA:DNA hybrids (R-loops) at sites of BRD4 occupancy, leading to transcription-replication conflicts (TRCs), DNA damage, and cell death. Finally, our data show that the BRD4 C-terminal domain, which interacts with P-TEFb, is required to prevent R-loop formation and DNA damage caused by BET protein LOF.

The NCI Common Terminology Criteria for Adverse Events (CTCAE) v5.0 is the standard for oncology toxicity encoding and grading despite limited validation. We assessed inter-rater reliability (IRR) in multi-reviewer toxicity identification. Methods and Materials: Two reviewers independently reviewed 100 randomly selected notes for weekly on-treatment visits during radiotherapy from the electronic health record (EHR). Discrepancies were adjudicated by a third reviewer for consensus. Term harmonization was performed to account for overlapping symptoms in CTCAE. IRR was assessed based on unweighted and weighted Cohen's kappa coefficients. Results: Between reviewers, unweighted kappa was 0.68 (95% CI 0.65-0.71) and weighted kappa 0.59 (0.22-1.00). IRR was consistent between noted present or absent symptoms with kappa of 0.6 (0.66-0.71) and 0.6 (0.65-0.69), respectively. Conclusions: Significant discordance suggests toxicity identification, particularly retrospectively, is a complex and error prone task. Strategies to minimize IRR, including training and simplification of the CTCAE criteria, should be considered in trial design and future terminologies.

Objectives Expert abstraction of acute toxicities is critical in oncology research but is labor-intensive and variable. We assessed the accuracy of a natural language processing (NLP) pipeline to extract symptoms from clinical notes compared to physicians. Materials and Methods Two independent reviewers identified present and negated National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE) v5.0 symptoms from 100 randomly selected notes for on-treatment visits during radiation therapy with adjudication by a third reviewer. A NLP pipeline based on Apache clinical Text Analysis Knowledge Extraction System was developed and used to extract CTCAE terms. Accuracy was assessed by precision, recall, and F1. Results The NLP pipeline demonstrated high accuracy for common physician-abstracted symptoms, such as radiation dermatitis (F1 0.88), fatigue (0.85), and nausea (0.88). NLP had poor sensitivity for negated symptoms. Conclusion NLP accurately detects a subset of documented present CTCAE symptoms, though is limited for negated symptoms. It may facilitate strategies to more consistently identify toxicities during cancer therapy.

Prolonged treatment with adjuvant valganciclovir has been shown in one retrospective study to exert a significant effect on overall survival (OS) in newly diagnosed patients with glioblastoma multiforme (GBM). However, studies evaluating the effectiveness of valganciclovir in the treatment of recurrent GBM have not been performed. We evaluated the effect of valganciclovir in the recurrent setting in combination with bevacizumab therapy. A retrospective analysis was performed on patients treated for recurrent GBM with off-label valganciclovir and bevacizumab at Vanderbilt University. We identified 13 patients who received valganciclovir plus bevacizumab at some point during their treatment, 8 of whom were started on valganciclovir and bevacizumab concurrently upon first recurrence, whereas 5 had valganciclovir added to their bevacizumab regimen prior to a second recurrence. of these patients, 12 were pathologically confirmed to have GBM, and 1 patient was diagnosed with gliosarcoma. We also identified an institutional cohort of 50 patients who had not been exposed to valganciclovir, but were treated with bevacizumab for first recurrence. The progression-free survival (PFS) at 6 months (PF6) and median OS (mOS) in the valganciclovir plus bevacizumab group was 62% and 13.1 months, respectively, for all 13 patients, and 50% and 11.3 months, respectively, for the 8 concurrently treated patients. In the institutional bevacizumab cohort, the PF6 and mOS were 34% and 8.7 months, respectively. In this retrospective analysis, valganciclovir in combination with bevacizumab exhibited a trend toward improved survival in patients with recurrent GBM. However, given the small sample size and the retrospective nature of this study, a larger prospective study is required to confirm these results.