Introduction: Human saliva contains a wealth of proteins that can be monitored for disease diagnosis and progression. Saliva, which is easy to collect, has been extensively studied for the diagnosis of numerous systemic and infectious diseases. However, the presence of amylase, the most abundant protein in saliva, can obscure the detection of low-abundance proteins by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-ToF MS), thus reducing its diagnostic utility. Objectives: In this study, we used a device to deplete salivary amylase from water-gargle samples by affinity adsorption. Following depletion, saliva proteome profiling was performed using MALDI-ToF MS on gargle samples from individuals confirmed to have COVID-19 based on nasopharyngeal (NP) swab reverse transcription quantitative polymerase chain reaction (RT-qPCR). Results: The depletion of amylase led to increased signal intensities of various peaks and the detection of previously unobserved peaks in the MALDI-ToF MS spectra. The overall specificity and sensitivity after amylase depletion were 100% and 85.17%, respectively, for detecting COVID-19. Conclusion: This simple, rapid, and inexpensive technique for depleting salivary amylase can reveal spectral diversity in saliva using MALDI-ToF MS, expose low-abundance proteins, and assist in establishing novel biomarkers for diseases.

Current artificial intelligence (AI) applications for the diagnosis of coronavirus disease 2019 (COVID-19) often lack a biological foundation in the decision-making process. In this study, we have employed AI for COVID-19 diagnosis using mass spectrometry (MS) data and leveraged explainable AI (X-AI) to explain the decision-making process on a local (per-sample) and global (all samples) basis. We first assessed eight machine learning models with five feature engineering techniques using a five-fold stratified cross-validation. The best accuracy was achieved by Random Forest (RF) classifier using the ratio of areas under the curve (AUC) from the MS data as features. These features were chosen on the basis of tentatively representing both human and viral proteins in human gargle samples. We evaluated the RF classifier on a 70%−30% train-test split strategy of 152 human gargle samples, yielding an accuracy of 94.12% on the test dataset. Employing X-AI, we further interpreted the RF model using shapely additive explanations (SHAP) and feature importance techniques, including permutation and impurity-based feature importances. With these interpretation models offering a local and global explanation for the machine learning model decisions, we devised a straightforward, three-stage X-AI framework that can enable medical practitioners to understand the mechanisms of a black-box AI model. To the medical practitioner, this instills trust in the AI model by providing the rationales for its decisions.

Human saliva contains a plethora of proteins whose presence and concentration can be monitored for diagnosis and progression of disease. Saliva has been extensively probed for the diagnosis of several systemic and infectious diseases because of the ease with which it can be collected. However, amylase, the most abundant protein found in saliva can obscure the detection of low-abundance proteins by MALDI-ToF MS (matrix-assisted laser desorption/ionization-time of flight mass spectrometry) and diminish the diagnostic utility of this specimen type. In the present study, we used a device to deplete salivary amylase from water-gargle samples through affinity adsorption. After depletion, profiling of the saliva proteome was performed by MALDI-ToF MS on gargle samples from subjects whose COVID-19 (coronavirus disease 2019) status was confirmed by NP (nasopharyngeal) swab RT-qPCR (reverse transcription polymerase chain reaction). Amylase depletion led to the enhancement of signal intensities of various peaks as well as the detection of previously unobserved peaks in the MALDI-ToF spectra. The overall specificity and sensitivity after amylase depletion was 100% and 85.17% respectively for detecting COVID-19. Our simple, rapid and inexpensive technique to deplete salivary amylase can be used to unmask spectral diversity in saliva by MALDI-ToF MS, reveal low-abundant proteins and aid in the establishment of novel biomarkers for diseases.

SARS-CoV-2 has infected more than 30 million persons throughout the world. A subset of patients suffer serious consequences that require hospitalization and ventilator support. Current tests for SARS-CoV-2 generate qualitative results and are vital to make a diagnosis of the infection. However, they are not helpful to follow changes in viral loads after diagnosis. The ability to quantitatively assess viral levels is necessary to determine the effectiveness of therapy with anti-viral or immune agents. Viral load analysis is also necessary to determine the replicative potential of strains with different mutations, emergence of resistance to anti-viral agents and the stability of viral nucleic acid and degree of RT-PCR inhibition in different types of collection media. Quantitative viral load analysis in body fluids, plasma and tissue may be helpful to determine the effects of the infection in various organ systems. To address these needs, we developed two assays to quantitate SARS-CoV-2. The assays target either the S or E genes in the virus, produce comparable viral load results, are highly sensitive and specific and have a wide range of quantitation. We believe that these assays will be helpful to manage the clinical course of infected patients and may also help to better understand the biology of infection with SARS-CoV-2.

Since December 2019, SARS‐CoV‐2 has spread extensively throughout the world, with more than 117 million reported cases and 2.6 million deaths (Johns Hopkins coronavirus resource center, https://coronavirus.jhu.edu/map.html). Detecting the virus is the first step in diagnosing the infection, followed by quarantine to prevent transmission. Nasopharyngeal/oropharyngeal swabs (NP/OP) and saliva are two specimen types that are most often analyzed to detect SARS‐CoV‐2 by molecular tests that detect viral RNA or by antigen/antibody tests that detect viral proteins and/or the host immune response against the virus. Compared to antigen/antibody tests, molecular tests are highly sensitive and specific for detecting the virus. A significant drawback is that specimen collection requirements are specific to each test and cannot be interchanged with another test. Some tests are qualified to be used on NP swabs or saliva, but not both specimen types. Even with NP swabs, a test may be qualified to detect the virus only with swabs collected in viral transport medium (VTM) but not in other media. These restrictive pre‐analytic steps are disadvantageous in that a lab would have to develop and validate different tests for SARS‐CoV‐2 depending on the specimen type and collection media, with added setup cost, infrastructure, and training requirements. To overcome these problems, we developed and validated a cost‐effective multiplex reverse‐transcription real‐time PCR assay that can be used to detect SARS‐CoV‐2 in different specimen types. The assay is highly sensitive and specific, can be used to detect the virus in saliva as well as NP swabs collected in different media such as VTM, saline, and commercial preservative fluid, and serves as one test for all applications. The protocol also describes an optimal laboratory setup and unidirectional workflow for detecting SARS‐CoV‐2 by RT‐qPCR. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC.

More than a year after the COVID-19 pandemic was declared, the need still exists for accurate, rapid, inexpensive and non-invasive diagnostic methods that yield high specificity and sensitivity towards the current and newly emerging SARS-CoV-2 strains. Compared to the nasopharyngeal swabs, several studies have established saliva as a more amenable specimen type for early detection of SARS-CoV-2. Considering the limitations and high demand for COVID-19 testing, we employed MALDI-ToF mass spectrometry in the analysis of 60 gargle samples from human donors and compared the resultant spectra against COVID-19 status. Several standards, including isolated human serum immunoglobulins, and controls, such as pre-COVID-19 saliva and heat inactivated SARS-CoV-2 virus, were simultaneously analyzed to provide a relative view of the saliva and viral proteome as they would appear in this workflow. Five potential biomarker peaks were established that demonstrated high concordance with COVID-19 positive individuals. Overall, the agreement of these results with RT-qPCR testing on nasopharyngeal swabs was ≥90% for the studied cohort, which consisted of young and largely asymptomatic student athletes. From a clinical standpoint, the results from this pilot study suggest that MALDI-ToF could be used to develop a relatively rapid and inexpensive COVID-19 assay.

The severe acute respiratory syndrome coronavirus type-2 (SARS-CoV-2) caused a global pandemic and immensely affected the global economy. Accurate, cost-effective, and quick tests have proven substantial in identifying infected people and mitigating the spread. Recently, multiple alternative platforms for testing coronavirus disease 2019 (COVID-19) have been published that show high agreement with current gold standard real-time polymerase chain reaction (RT-PCR) results. These new methods do away with nasopharyngeal (NP) swabs, eliminate the need for complicated reagents, and reduce the burden on RT-PCR test reagent supply. In the present work, we have designed an artificial intelligence-based (AI) testing method to provide confidence in the results. Current AI applications for COVID-19 studies often lack a biological foundation in the decision-making process, and our AI approach is one of the earliest to leverage explainable AI (X-AI) algorithms for COVID-19 diagnosis using mass spectrometry. Here, we have employed X-AI to explain the decision-making process on a local (per-sample) and global (all samples) basis underscored by biologically relevant features. We evaluated our technique with data extracted from human gargle samples and achieved a testing accuracy of 94.12 techniques would strengthen the relationship between AI and clinical diagnostics by providing biomedical researchers and healthcare workers with trustworthy and, most importantly, explainable test results

The objective of this study was to evaluate post-percutaneous coronary intervention (PCI) prescribing and clinical outcomes in patients receiving CYP2C19 genotype-guided P2Y12 therapy at an urban academic health center serving an ethnically diverse population. PCI patients who underwent CYP2C19

BACKGROUND: Current standard-of-care technologies, such as imaging and cyst fluid analysis, are unable to consistently distinguish intraductal papillary mucinous neoplasms (IPMNs) of the pancreas at high risk of pancreatic cancer from low-risk IPMNs. The objective was to create a single-platform assay to identify IPMNs that are at high risk for malignant progression. STUDY DESIGN: Building on the Verona International Consensus Conference branch duct IPMN biomarker review, additional protein, cytokine, mucin, DNA, and microRNA cyst fluid targets were identified for creation of a quantitative polymerase chain reaction-based assay. This included messenger RNA markers: ERBB2, GNAS, interleukin 1 beta, KRAS, MUCs1, 2, 4, 5AC, 7, prostaglandin E2R, PTGER2, prostaglandin E synthase 2, prostaglandin E synthase 1, TP63; microRNA targets: miRs 101, 106b, 10a, 142, 155, 17, 18a, 21, 217, 24, 30a, 342, 532, 92a, and 99b; and GNAS and KRAS mutational analysis. A multi-institutional international collaborative contributed IPMN cyst fluid samples to validate this platform. Cyst fluid gene expression levels were normalized, z-transformed, and used in classification and regression analysis by a support vector machine training algorithm. RESULTS: From cyst fluids of 59 IPMN patients, principal component analysis confirmed no institutional bias/clustering. Lasso (least absolute shrinkage and selection operator)-penalized logistic regression with binary classification and 5-fold cross-validation used area under the curve as the evaluation criterion to create the optimal signature to discriminate IPMNs as low risk (low/moderate dysplasia) or high risk (high-grade dysplasia/invasive cancer). The most predictive signature was achieved with interleukin 1 beta, MUC4, and prostaglandin E synthase 2 to accurately discriminate high-risk cysts from low-risk cysts with an area under the curve of up to 0.86 (p = 0.002). CONCLUSIONS: We have identified a single-platform polymerase chain reaction-based assay of cyst fluid to accurately predict IPMNs with high malignant potential for additional studies. (C) 2019 by the American College of Surgeons. Published by Elsevier Inc. All rights reserved.

Abstract DNA instability, including increased DNA double strand breaks, has recently been demonstrated in myeloproliferative neoplasms (MPN). Because PARP1 has a central role in DNA repair and maintaining genomic integrity, we tested whether DNA repair inhibition with the PARP inhibitor ABT-888 (Abbvie, Chicago, IL) could affect the growth of myeloid neoplasms in-vitro and in-vivo, and/or enhance the cytotoxic effect of a standard alkylating agent commonly used in preparative regimens for stem cell transplants. Since the JAK2V617Fmutation is common in chronic MPNs or acute myeloid leukemia (AML) secondary to MPNs, we initially utilized JAK2 mutated AML cell lines (SET2 and HEL) in-vitro and in-vivo. We then tested the effect of PARP inhibition on CD34+ cells obtained from patients with primary myelofibrosis. JAK2 mutated SET2 and HEL cells were treated with ABT-888 in liquid cultures. Both cell lines were relatively sensitive with IC50 of 11.3μM and 74.2μM respectively. When the cells were treated with a combination of increasing doses of busulfan and 8μM ABT-888, significant synergy was observed, with busulfan IC50 decreasing from 27μM to 4μM in SET2 cells and from 45.1μM to 28.1μM in HEL cells. Treatment of SET2 cells with 8μM ABT-888, or with 8μM ABT888 and 10μM busulfan, was also accompanied by marked increase in γH2AX foci compared to control at 24 hours. We subsequently showed that ABT-888, busulfan and combination treatments resulted in increasing numbers of cells in G2/M arrest (30% vs. 35% vs 53% respectively, p=0.002). Western blot analysis of cells in G/2M arrest after combination treatment demonstrated a strongly increased phosphorylation of checkpoint kinase 1 (Chk1). In an immunofluorescence assay on treated cells we then showed that activated Chk1 translocated to the nucleus, suggesting that the Chk1-ATR pathway may be responsible for cell cycle arrest. To demonstrate the in-vivo effect of PARP inhibition, we transplanted 5x106 SET2 cells into a NOD/SCID/ gamma null mice to create a xenograft model. Mice developed AML with splenomegaly and bone marrow engraftment resulting in a median survival of 36 days. In mice that were treated with daily intraperitoneal injections of low dose ABT-888 (1.5mg/kg), starting 14 days after leukemic cell injection, survival increased to a median of 40 days (p=0.002). We subsequently tested the effect of PARP inhibition on primary myelofibrosis CD34+ cells obtained from the bone marrow or peripheral blood of 3 patients with JAK2V617Fmutation and 2 with CALR mutation. Control experiments were performed with normal CD34+ progenitors. In a standard clonogenic assay in methylcellulose, cells were plated with or without 4μM ABT-888, 5μM busulfan or a combination of both. Compared to untreated cells, ABT-888, busulfan and the combination of the two drugs reduced colony formation by 39%, 70% and 89%, respectively (p=0.01). Both CALR and JAK2 mutated cells were sensitive to ABT-888. On the contrary, treatment of normal CD34+ cells with ABT-888 did not affect colony growth. Here we describe the potential for PARP inhibition to exploit the genomic instability of MPNs. Potential clinical applications include the addition of PARP inhibition to busulfan in pretransplant conditioning or novel drug combinations including PARP inhibitors. Disclosures No relevant conflicts of interest to declare.

The mitogen-activated protein kinase (MAPK) signaling pathway is a cascade of protein kinases that act in a sequential and predominantly linear fashion, albeit displaying some cross talk with other signaling cascades. Mutations in proteins integral to the MAPK signaling pathway are present in more than 50% of cutaneous melanomas. The most frequently mutated protein is v-raf murine sarcoma viral oncogene homolog B (BRAF), followed by neuroblastoma Ras viral oncogene homolog (NRAS). Recently, the development of targeted drugs for the treatment of BRAF-mutant melanoma has led to the widespread implementation of molecular assays for the detection of specific BRAF mutations. There have been some attempts to standardize testing of BRAF mutations, but this has not been achieved so far. Here we provide an updated review on the role of the MAPK signaling pathway in the pathogenesis of cutaneous melanoma, focusing on several different BRAF mutations and their diagnostic and therapeutic implications.

A 53-year-old female renal transplant patient presented with a 3-year history of painful verrucous lesions on both palms (Fig. 1). The patient’s post-transplant immunosuppression regimen had included tacrolimus, prednisone, and mycophenolic acid. A skin biopsy from the right palm revealed verruca vulgaris (Fig. 2a). Despite treatment with topical urea (30%), topical salicylic acid (12%), and oral acitretin (25 mg/day), the patient’s verrucae continued to progress. A second biopsy demonstrated full-thickness atypia of keratinocytes consistent with squamous cell carcinoma (SCC) in situ (Fig. 2b). In situ hybridization revealed the presence of HPV subtype 1 (Fig. 2c). Mycophenolic acid and tacrolimus were substituted with sirolimus to decrease immunosuppression, and intralesional bleomycin, intralesional 5-flourouracil (5-FU), and oral epidermal growth factor receptor (EGFR) inhibitors were considered. Meanwhile, the patient was started on R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) chemotherapy for a diffuse large B cell lymphoma. At follow-up one month after the first round of R-CHOP therapy, a dramatic regression in the skin lesions was noted. Complete resolution of the lesions on the hands was noted after three rounds of R-CHOP (Fig. 3), and the patient’s hands remained clinically clear at the 10month follow-up visit.

Many tumors are stiffer than their surrounding tissue. This increase in stiffness has been attributed, in part, to a Rho-dependent elevation of myosin II light chain phosphorylation. To characterize this mechanism further, we studied myosin light chain kinase (MLCK), the main enzyme that phosphorylates myosin II light chains. We anticipated that increases in MLCK expression and activity would contribute to the increased stiffness of cancer cells. However, we find that MLCK mRNA and protein levels are substantially less in cancer cells and tissues than in normal cells. Consistent with this observation, cancer cells contract 3D collagen matrices much more slowly than normal cells. Interestingly, inhibiting MLCK or Rho kinase did not affect the 3D gel contractions while blebbistatin partially and cytochalasin D maximally inhibited contractions. Live cell imaging of cells in collagen gels showed that cytochalasin D inhibited filopodia-like projections that formed between cells while a MLCK inhibitor had no effect on these projections. These data suggest that myosin II phosphorylation is dispensable in regulating the mechanical properties of tumors.

Introduction: African American (AA) men have an increased death rate compared to Caucasian men (15.4 per 100,000 persons versus 12.1 per 100,000 persons respectively) after gemcitabine-based standard of care therapy for pancreatic ductal adenocarcinoma (PDA). HuR, an mRNA binding protein, creates a favorable ‘therapeutic window’ for gemcitabine efficacy against cancer cells (while sparing normal cells) by regulating its metabolic enzyme, deoxycytidine kinase (dCK) mRNA (Cancer Research 2009;69:4567-72). Computational analysis identified a single nucleotide polymorphism (SNP) residing in the regulatory binding site of the dCK gene. This germline SNP has a prevalence of ∼65% in the AA population, in contrast to only 5% in Caucasians. We hypothesized that the presence of this SNP may significantly affect gemcitabine metabolism and contribute to the survival disparity that is seen in the AA patient population. Methods: We designed a 33 base pair probe that encompassed the dCK-SNP to test whether this region of dCK mRNA could bind to HuR. We cloned the wild type (T) and the SNP (C) sequences into luciferase reporter constructs in order to quantitate any effect the single nucleotide change had on protein expression in an unbiased manner. We also developed a facile genotyping assay to determine dCK-SNP genotypes in a group of 47 patients with resected PDA and correlated the genotypes with clinical outcome. Results: We identified the short sequence motif in which HuR regulates dCK. HuR preferentially and robustly binds to the dCK mRNA sequence (33bps) that harbors the SNP. A single nucleotide change (T to C) in this binding region induced a 2.7-fold increase in protein expression. One hundred percent of our AA patient population (n=13) had the presence of the SNP in at least one allele (39% homozygous for the SNP) while only 11% of Caucasian patients (n=37) had one SNP allele (none of these patients were homozygous for the SNP) (p=0.0001). Of the patients treated with gemcitabine, patients with a wild type dCK-genotype (n=27, all Caucasian) had a median survival of 20.7 months, compared to 10.4 months in patients (n=11, all AA patients) that harbored at least one SNP allele (hazard ratio 2.5, p=0.058 by Gehan-Breslow-Wilcoxon test). Conclusion: Taken together, our data show that: 1) the dCK-SNP is frequently detected in the AA population; 2) the dCK-SNP functionally affects protein translation; and 3) the presence of the SNP is associated with poor clinical outcome after gemcitabine therapy. We hypothesize that patients who harbor the germline dCK-SNP improperly metabolize gemcitabine in normal cells, and thus negate the favorable ‘gemcitabine-therapeutic window’ that preferentially destroys tumor cells. Detection of the dCK-SNP genotype in addition to other validated biomarkers such as HuR status will help guide adjuvant treatment options and ultimately enhance outcomes in the AA, PDA patient population.

The high affinity neurotensin receptor (NTSR1) mediates most of the biologic effects of neurotensin (NT), a 13-amino acid peptide that stimulates growth in certain cell types. NT is expressed in fetal but not differentiated colonic epithelium and is re-expressed in colonic adenocarcinoma. The cognate receptor, NTSR1, is also not expressed or is present at a low level in adult colonic epithelial cells but is expressed in most colon cancer cell lines. These observations suggest that altered NT-NTSR1 signaling may be associated with malignant transformation in the colon. To further understand the possible role of NTSR1 expression in colonic tumorigenesis and progression, we examined NTSR1 mRNA by in situ hybridization in normal colonic mucosa, adenomas, and colonic adenocarcinomas. NTSR1 mRNA expression was undetectable or weak in superficial differentiated epithelial cells of normal colonic epithelium, but adenomas and adenocarcinomas showed moderate to strong expression (p < 0.05). Adenocarcinomas showed a higher level of expression compared to adenomas (p < 0.05). Furthermore, adenocarcinomas that infiltrated into and beyond the muscularis propria showed a higher intensity of NTSR1 expression compared with tumors that were localized to the mucosa or submucosa. In some cases, infiltrating margins and foci of lymphovascular invasion showed a higher intensity of expression than the main mass of the tumor. These results suggest that increased NTSR1 expression may be an early event during colonic tumorigenesis and also contribute to tumor progression and aggressive behavior in colonic adenocarcinomas. NTSR1 may thus be a potential target for preventive or therapeutic strategies in colon cancer.

Authentication of cell lines in biomedical research has been elevated to a very high priority. From a review of the literature, Lacroix1 reviewed the issue of cross-contamination of cell lines including the well known contamination of cell lines with HeLa cells,2 and the mis-identification of the ECV304 cell line as “immortalized endothelial cells” when these cells in fact originated from T24 bladder carcinoma cells.3 Lacroix 1 estimated that between 18 and 36% of cell lines have been misclassified. One survey at a large research institution suggested that fewer than 50% of researchers authenticate their cell lines.4 Nardone5 proposed recently that identification of cell lines be required of investigators before grants are awarded, and the National Institutes of Health subsequently called for researchers to authenticate cell lines as a prerequisite for grant funding.6

IPMNs and show overlapping and distinct characteristics compared to PDAC.Conclusion: This study describes a novel xenograft model and cell line deriving from adenocarcinoma arising in IPMN.Characterization of the model shows similarities to the parent tumor in accord with previously published data on IPMNs.Compared to PDAC, this model shows shared aberrations as well as evidence for distinct genomic changes.More importantly, the xenograft model may be useful for future preclinical chemotherapy studies In Vivo.

Nuclear phosphoprotein 32 (pp32) inhibits K-ras induced transformation in experimental models. pp32 mRNA expression correlates with differentiation status in breast and prostate cancers. In this study, we evaluated pp32 protein expression in relation to the differentiation status of pancreatic ductal adenocarcinomas and precursor lesions of the pancreatic cancers. pp32 expression showed strong nuclear staining in normal pancreatic acini and ducts. The intensity of this staining was maintained in pancreatic intraepithelial neoplasia, intraductal papillary mucinous neoplasms with mild dysplasia, well-differentiated adenocarcinomas, and in a subset of moderately differentiated adenocarcinomas. pp32 staining was absent or reduced in poorly differentiated tumors and in intraductal papillary mucinous neoplasms with moderate dysplasia. We validated pp32 expression by a second technique, immunoblot analysis of lysates from resected pancreatic ductal adenocarcinomas and pancreatic cancer cell lines. The well-differentiated pancreatic cancer cell line HPAC expressed high amounts of pp32, as compared to the poorly differentiated pancreatic cancer cell lines MiaPaCa2, Pl19, and Pl21 cells. Artificial introduction of pp32 expression into a poorly differentiated cell line, MiaPaCa2, caused an increase in G1 arrest compared to control cells. On the basis of this study and previous functional work that shows pp32 can inhibit K-ras transformation, we propose that reduction in pp32 expression levels may be a critical event in the progression of pancreatic tumorigenesis in an aggressive subset of pancreatic ductal adenocarcinomas.