Enhancing saliva diagnostics: The impact of amylase depletion on MALDI-ToF MS profiles as applied to COVID-19.

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.

MALDI-ToF protein profiling as a potential rapid diagnostic platform for COVID-19.

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.

A Multiplex One-Step RT-qPCR Protocol to Detect SARS-CoV-2 in NP/OP Swabs and Saliva.

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. Basic Protocol 1: Manual viral nucleic acid extraction from NP/OP swabs collected in different media, and from saliva Alternate Protocol 1: Low-throughput automated extraction on the Qiagen EZ1 Advanced XL machine (1-14 samples) Alternate Protocol 2: High-throughput automated extraction on the Kingfisher Flex machine (1-96 samples) Basic Protocol 2: Multiplex RT-qPCR protocol to detect SARS-CoV-2 Alternate Protocol 3: Multiplex one-step RT-qPCR protocol to detect SARS-CoV-2 with S and E gene probes labeled with the same fluorochrome.

Development and validation of viral load assays to quantitate SARS-CoV-2.

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.

NGS Analysis of Clonality and Minimal Residual Disease in a Patient with Concurrent Richter's Transformation and CLL/SLL.

B-cell lymphomas are neoplastic proliferations of clonal B lymphocytes. Clonality is generally determined by PCR amplification of VDJ rearrangements in the IgH heavy chain or VJ rearrangements in Igκ/Igλ light chain genes followed by capillary electrophoresis. More recently, next-generation sequencing (NGS) has been used to detect clonality in B-cell lymphomas because of the exponential amount of information that is obtained beyond just detecting a clonal population. The additional information obtained is useful for diagnostic confirmation, prognosis assessment, and response to therapy. In this study, we utilized NGS analysis to characterize two histologically distinct lymphomas (DLBCL and CLL/SLL) that were detected contemporaneously in an asymptomatic patient. NGS analysis showed that the same VDJ rearrangement was present in nodal (DLBCL) and marrow (CLL/SLL) biopsies confirming that the DLBCL resulted from Richter's transformation of a subclinical CLL/SLL. The V region of the rearrangement remained unmutated without somatic hypermutation. In silico analysis showed that the HCDR3 sequence was heterogeneous and not stereotypic. Minimal residual disease analysis by NGS showed that the tumor clone decreased by 2.84 logs in the bone marrow after R-CHOP therapy. However, a small number of tumor cells were still detected in the peripheral blood after R-CHOP therapy. Subsequent allogeneic transplantation was successful in eradicating the tumor clone and achieving deep molecular remission. We show that NGS analysis facilitated clinical management in our patient by helping to characterize the VDJ rearrangement in detail and by tracking minimal residual disease with high sensitivity and specificity.

Cyst Fluid Biosignature to Predict Intraductal Papillary Mucinous Neoplasms of the Pancreas with High Malignant Potential.

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 1ß, 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 1ß, 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.

Mitogen-Activated Protein Kinase Signaling Pathway in Cutaneous Melanoma: An Updated Review.

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.

Tumor stiffness is unrelated to myosin light chain phosphorylation in cancer cells.

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.

Feasibility of implementing a comprehensive warfarin pharmacogenetics service.

STUDY OBJECTIVE: To determine the procedural feasibility of a pharmacist-led interdisciplinary service for providing genotype-guided warfarin dosing for hospitalized patients newly starting warfarin. DESIGN: Prospective observational study. SETTING: A 438-bed tertiary care hospital affiliated with a large academic institution. PATIENTS: Eighty patients who started warfarin therapy and were managed by a newly implemented pharmacogenetics service. INTERVENTION: All patients received routine warfarin genotyping and clinical pharmacogenetics consultation. MEASUREMENTS AND MAIN RESULTS: The primary outcomes were percentage of genotype-guided dose recommendations available prior to the second warfarin dose and adherence of the medical staff to doses recommended by the pharmacogenetics service. Of 436 genotype orders placed during the first 6 months of the service, 190 (44%) were deemed appropriate. For the 80 patients on the service who consented to data collection, 76% of the genotypes were available prior to the second warfarin dose. The median (range) time from genotype order to genotype result was 26 hours (7-80 hrs), and the time to genotype-guided dose recommendation was 30 hours (7-80 hrs). A total of 73% of warfarin doses ordered by the medical staff were within 0.5 mg of the daily dose recommended by the pharmacogenetics consult service. CONCLUSION: Providing routine genotype-guided warfarin dosing supported by a pharmacogenetics consult service is feasible from a procedural standpoint, with most genotypes available prior to the second warfarin dose and good adherence to genotype-guided dose recommendations by the medical staff.

Increased neurotensin receptor-1 expression during progression of colonic adenocarcinoma.

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.

Reduction of pp32 expression in poorly differentiated pancreatic ductal adenocarcinomas and intraductal papillary mucinous neoplasms with moderate dysplasia.

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.

MLL-SEPT6 fusion transcript with a novel sequence in an infant with acute myeloid leukemia.

The MLL gene at 11q23 is a site of frequent rearrangement in acute leukemia with multiple fusion partners. A relatively uncommon rearrangement, associated with infant AML-M4, fuses the MLL and SEPT6 genes. SEPT6, located at Xq24, is a member of a family of mammalian septins involved in diverse functions such as cytokinesis, cell polarity, and oncogenesis. We describe the case of an infant with acute myelogenous leukemia who showed cytogenetic evidence of rearrangement between 11q23 and Xq24 regions. Fluorescence in situ hybridization analysis suggested a possible break in the MLL gene, and molecular analysis using reverse transcriptase-polymerase chain reaction followed by sequencing confirmed the expression of an MLL-SEPT6 fusion transcript with a novel sequence. The findings emphasize the importance of combined cytogenetic and molecular analyses in the workup of acute leukemia, especially in those leukemias that occur infrequently.

Osteopontin expression and serum levels in metastatic uveal melanoma: a pilot study.

PURPOSE: This was a pilot study conducted to examine the expression of osteopontin in uveal melanoma and to determine whether serum osteopontin can be used in detecting metastatic uveal melanoma. METHODS: Osteopontin mRNA was measured in three uveal melanoma cell lines of various invasive potential by real-time PCR. Tissue sections of primary and metastatic uveal melanomas were stained for osteopontin. Serum osteopontin levels were measured by ELISA assays in 15 patients with metastatic uveal melanoma and in 37 patients who were disease-free for at least 10 years after treatment of the primary tumor. Paired serum samples drawn from eight patients before and after development of metastasis were analyzed. RESULTS: By real-time PCR, highly invasive primary and metastatic uveal melanoma cells expressed 6- and 250-fold excess osteopontin mRNA, respectively, compared with poorly invasive primary uveal melanoma cells. Tissue sections of primary uveal melanomas lacking looping vasculogenic mimicry patterns either did not stain for osteopontin or exhibited weak, diffuse staining. In primary melanomas containing looping vasculogenic mimicry patterns, strong osteopontin staining was detected in the tumor periphery where patterns were located. Diffuse strong expression of osteopontin was detected in eight samples of uveal melanomas metastatic to the liver. Serum osteopontin levels were significantly higher in patients with metastatic uveal melanoma than in patients who had been disease free for at least 10 years after treatment (P = 0.0001) or in age-matched control subjects. Serum osteopontin levels were significantly higher (P = 0.008) after metastasis than before the detection of metastasis in eight patients. When a cutoff of 10 ng/mL was used, the sensitivity and specificity of serum osteopontin in detecting metastatic melanoma was 87.5%, and the area under the receiver operator characteristic curve was 96%. CONCLUSIONS: Osteopontin is expressed diffusely in tissue sections of hepatic metastases from uveal melanoma, and increased serum osteopontin levels correlate with melanoma metastasis to the liver with high specificity and sensitivity.

Identification of a functional mutation in pp32r1 (ANP32C).

No mutations or polymorphisms have previously been reported in pp32r1 (ANP32C; GenBank: AF008216.1). pp32r1 is part of the highly conserved ANP32 family, some of whose members are associated with control of histone acetylation, mRNA stability, and specialized forms of apoptosis. Although 87.6% identical at the protein level, pp32r1 is functionally distinct from pp32 (ANP32A) in its failure to suppress oncogenesis in in vitro transformation systems and its tumorigenicity in in vivo assays. The present study found that pp32r1 expression levels vary among human tumor cell lines, with the highest levels found in prostatic adenocarcinoma cell lines. pp32r1 also appears to be polymorphic at nucleotide g.4520 and nucleotide g.4664 in human tobacco-associated oral mucosal lesions, human fibroblast cell lines, and several carcinoma cell lines. PC-3 human prostatic adenocarcinoma cells likewise appear to be polymorphic at these loci, but additionally contain a g.4870T>C transversion mutation. The mutation results in a p.Tyr140His substitution, which lies in a functionally important region of the molecule. In the PC-3 prostate cancer line, the mutation is either homozygous, or hemizygous accompanied by loss of heterozygosity. ACHN cells stably transfected with pp32r1 containing this mutation showed a markedly increased rate of growth. The pp32r1 mutation could thus be causally associated with the neoplastic growth properties of PC-3, and be of potential clinical significance.

Expression of GCMB by intrathymic parathyroid hormone-secreting adenomas indicates their parathyroid cell origin.

GCMA and GCMB are related transcription factors that are critically important for embryological development of the placenta and parathyroid glands, respectively. Mice in which parathyroid glands have been surgically removed or fail to develop due to genetic loss of GCMB show continued production of PTH from a subset of thymic cells that express GCMA. In this study we examined whether human thymus produces PTH and/or GCMA and whether intrathymic PTH-secreting adenomas express GCMA or GCMB to determine the embryological origin of the secretory cells. By contrast to mouse thymus, analysis of 22 samples of human thymus tissue by RT-PCR and/or immunohistochemistry failed to demonstrate the expression of either PTH or GCMA. RT-PCR analysis of 16 intrathymic adenomas from patients with surgically cured primary hyperparathyroidism showed that these tumors expressed PTH and GCMB and not GCMA. We conclude that the normal human thymus does not express GCMA or PTH, and therefore, in contrast to the mouse, the human thymus is not a source of PTH production. Finally, intrathymic PTH-secreting adenomas express the parathyroid-specific GCMB gene, which suggests that these tumors were derived from parathyroid cells that migrated errantly during embryogenesis.

Comprehensive analysis of CBFbeta-MYH11 fusion transcripts in acute myeloid leukemia by RT-PCR analysis.

CBFbeta-MYH11 fusion transcripts are expressed in acute myeloid leukemias of the M4Eo subtype. Patients who express CBFbeta-MYH11 fusion transcripts respond favorably to high-dose chemotherapy and are generally spared allogeneic bone marrow transplantation. Hence it is important to identify this fusion in all patients with acute myeloid leukemia M4Eo leukemia. The fusion can be detected by cytogenetics, fluorescence in-situ hybridization (FISH), or by molecular analysis with RT-PCR. Multiple fusion transcripts arising as a result of various breakpoints in the CBFbeta and MYH11 have been identified. In this report we describe a comprehensive RT-PCR assay to identify all known fusion transcripts and provide an algorithm for molecular analysis of CBFbeta-MYH11 fusions from patient specimens. Further, identification of the fusion transcript by such an assay would help in the diagnosis and follow up of patients with cryptic inversion 16 translocations (such as patient 2 in this report) not detected by standard cytogenetics or FISH and for rational design of probes for quantitative analysis by real-time PCR.

pp32 reduction induces differentiation of TSU-Pr1 cells.

pp32 (ANP32A) is a nuclear phosphoprotein expressed as a nonmutated form in self-renewing cell populations and neoplastic cells. Mechanistically, pp32 may regulate pathways important in the process of differentiation as part of separate complexes inhibiting histone acetylation and regulating immediate-early and cytokine mRNA stability. Prostatic adenocarcinomas express pp32 in a differentiation related manner-well-differentiated tumors express lower levels of pp32 than poorly differentiated tumors. In benign prostate, pp32 is expressed in basal cells but not in terminally differentiated glandular cells. Based on these observations, we hypothesized that reduction of pp32 expression might be an important differentiation signal. We used anti-sense pp32 and RNAi transfection to study the effects of reduced pp32 expression in the TSU-Pr1 carcinoma cell line. pp32 reduction induced TSU-Pr1 cells to differentiate into neuronal-like cells with associated inhibition of growth. Reduction of pp32 and consequent differentiation were accompanied by a marked reduction in expression of SET, which complexes with pp32, by a marked change in acetylation status of histone H4, and by further differential expression of genes in differentiation pathways. Thus, reduction of pp32 in the undifferentiated TSU-Pr1 neoplastic cell line induces differentiation and thus may be an element of a differentiation control pathway in both normal and neoplastic cells.