Using a Systems Engineering Approach to Build a PCR Testing System at a Medical School During the COVID-19 Pandemic.

Background: During the COVID-19 pandemic, there was an increasing need to expand diagnostic testing in hospitals. At Keio University Hospital (KUH), clinical staff were concerned that the demand for PCR testing might exceed the capacity of the Clinical Laboratory. In response, basic researchers at Keio University School of Medicine (KUSM) set out to build a new, collaborative, PCR testing system. To be authorized to perform such diagnostic PCR testing, KUSM registered its core laboratory as an external clinical laboratory (ECL). Methods: In the pandemic, there was a pressure to build the PCR system quickly. Speed required discussions that developed a shared understanding of the unprecedented, new KUH/KUSM PCR system. To design, construct, and archive the new PCR testing system, we used a systems engineering (SE) approach. This included diagram visualization of functional flows and application of the Unified Architecture Framework (UAF), both of which are often used in system building. We considered daily demand for PCR testing at KUH and KUSM, and daily COVID-19 infections in Japan. Results: We operated the collaborative PCR testing system from August 2020 to June 2022. Given public health insurance reimbursement policies, KUH focused on individuals with suspicious symptoms, while the ECL at KUSM screened samples from asymptomatic individuals. KUSM performed about half as many tests as KUH. Interviewing KUH staff revealed that diagrams helped promote a better understanding of the KUH/KUSM PCR testing system. Conclusion: When designing temporary systems that may be repurposed in the future, we suggest using an SE approach with diagrams and UAF perspectives. This approach will enable stakeholders to understand what is being proposed to be built, and facilitate achieving an informed consensus on the proposed system. We suggest that SE approaches should be widely used in projects that involve building and operating complex, collaborative systems, and documenting the process.

Multi-modal molecular programs regulate melanoma cell state.

Melanoma cells display distinct intrinsic phenotypic states. Here, we seek to characterize the molecular regulation of these states using multi-omic analyses of whole exome, transcriptome, microRNA, long non-coding RNA and DNA methylation data together with reverse-phase protein array data on a panel of 68 highly annotated early passage melanoma cell lines. We demonstrate that clearly defined cancer cell intrinsic transcriptomic programs are maintained in melanoma cells ex vivo and remain highly conserved within melanoma tumors, are associated with distinct immune features within tumors, and differentially correlate with checkpoint inhibitor and adoptive T cell therapy efficacy. Through integrative analyses we demonstrate highly complex multi-omic regulation of melanoma cell intrinsic programs that provide key insights into the molecular maintenance of phenotypic states. These findings have implications for cancer biology and the identification of new therapeutic strategies. Further, these deeply characterized cell lines will serve as an invaluable resource for future research in the field.

SARS-CoV-2 RT-qPCR testing of pooled saliva samples: A case study of 824 asymptomatic individuals and a questionnaire survey in Japan.

From the beginning of the COVID-19 pandemic, the demand for diagnostic and screening tests has exceeded supply. Although the proportion of vaccinated people has increased in wealthier countries, breakthrough infections have occurred amid the emergence of new variants. Pooled-sample COVID-19 testing using saliva has been proposed as an efficient, inexpensive, and non-invasive method to allow larger-scale testing, especially in a screening setting. In this study, we aimed to evaluate pooled RT-qPCR saliva testing and to compare the results with individual tests. Employees of Philips Japan, Ltd. were recruited to participate in COVID-19 screening from October to December 2020. Asymptomatic individuals (n = 824) submitted self-collected saliva samples. Samples were tested for the presence of SARS-CoV-2 by RT-qPCR in both 10-sample pools and individual tests. We also surveyed participants regarding their thoughts and behaviors after the PCR screening project. Two of the 824 individuals were positive by RT-qPCR. In the pooled testing, one of these two had no measurable Ct value, but showed an amplification trend at the end of the PCR cycle. Both positive individuals developed cold-like symptoms, but neither required hospitalization. Of the 824 participants, 471 responded to our online questionnaire. Overall, while respondents agreed that PCR screening should be performed regularly, the majority were willing to undergo PCR testing only when it was provided for free or at low cost. In conclusion, pooled testing of saliva samples can support frequent large-scale screening that is rapid, efficient, and inexpensive.

Reprogramming of bivalent chromatin states in NRAS mutant melanoma suggests PRC2 inhibition as a therapeutic strategy.

The dynamic evolution of chromatin state patterns during metastasis, their relationship with bona fide genetic drivers, and their therapeutic vulnerabilities are not completely understood. Combinatorial chromatin state profiling of 46 melanoma samples reveals an association of NRAS mutants with bivalent histone H3 lysine 27 trimethylation (H3K27me3) and Polycomb repressive complex 2. Reprogramming of bivalent domains during metastasis occurs on master transcription factors of a mesenchymal phenotype, including ZEB1, TWIST1, and CDH1. Resolution of bivalency using pharmacological inhibition of EZH2 decreases invasive capacity of melanoma cells and markedly reduces tumor burden in vivo, specifically in NRAS mutants. Coincident with bivalent reprogramming, the increased expression of pro-metastatic and melanocyte-specific cell-identity genes is associated with exceptionally wide H3K4me3 domains, suggesting a role for this epigenetic element. Overall, we demonstrate that reprogramming of bivalent and broad domains represents key epigenetic alterations in metastatic melanoma and that EZH2 plus MEK inhibition may provide a promising therapeutic strategy for NRAS mutant melanoma patients.

The genetic and epigenetic basis of distinct melanoma types.

Melanoma represents the deadliest skin cancer. Recent therapeutic developments, including targeted and immune therapies have revolutionized clinical management and improved patient outcome. This progress was achieved by rigorous molecular and functional studies followed by robust clinical trials. The identification of key genomic alterations and gene expression profiles have propelled the understanding of distinct characteristics within melanoma subtypes. The aim of this review is to summarize and highlight the main genetic and epigenetic findings of melanomas and highlight their pathological and therapeutic importance.

Estimating copy number using next-generation sequencing to determine ERBB2 amplification status.

Assessing Erb-b2 receptor tyrosine kinase 2 (ERBB2) amplification status in breast and gastric cancer is necessary for deciding the best therapeutic strategy. Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are currently used for assessing protein levels and gene copy number (CN), respectively. The use of next-generation sequencing (NGS) to measure ERBB2 CN in breast cancer is approved by the United States Federal Drug Administration as a companion diagnostic. However, a CN of less than 8 is evaluated as "equivocal", which means that some ERBB2 amplification cases diagnosed as "HER2 negative" might be false-negative cases. We reviewed the results of gene profiling targeting 160 cancer-related genes in breast (N = 90) and non-breast (N = 19) cancer tissue, and compared the ERBB2 CN results with the IHC/FISH scores. We obtained an estimated CN from the measured CN by factoring in the histological proportion of tumor cells and found that an ERBB2-estimated CN of 3.2 or higher was concordant with the combined IHC/FISH outcome in 98.4% (88/90) of breast cancer cases, while this was not always evident among non-breast cancer cases. Therefore, NGS-estimated ERBB2 CN could be considered a diagnostic test for anti-HER2 therapy after the completion of adequate prospective clinical trials.

RT-PCR Screening Tests for SARS-CoV-2 with Saliva Samples in Asymptomatic People: Strategy to Maintain Social and Economic Activities while Reducing the Risk of Spreading the Virus.

The year 2020 will be remembered for the coronavirus disease 2019 (COVID-19) pandemic, which continues to affect the whole world. Early and accurate identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is fundamental to combat the disease. Among the current diagnostic tests, real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) is the most reliable and frequently used method. Herein, we discuss the interpretation of RT-qPCR results relative to viral infectivity. Although nasopharyngeal swab samples are often used for RT-qPCR testing, they require collection by trained medical staff. Saliva samples are emerging as an inexpensive and efficient alternative for large-scale screening. Pooled-sample testing of saliva has been applied for mass screening of SARS-CoV-2 infection. Current policies recommend isolating people with borderline cycle threshold (Ct) values (35

Pulmonary metastasis secondary to abiraterone-resistant prostate cancer with homozygous deletions of BRCA2: First Japanese case.

INTRODUCTION: Most metastatic prostate cancers acquire the capacity for androgen-independent growth and become resistant to androgen deprivation therapy. A patient-focused treatment strategy is needed for aggressive castration-resistant prostate cancer. CASE PRESENTATION: We report the case of a 62-year-old man who presented with prostatic adenocarcinoma who was treated by radiation and combined androgen blockade. After completion of first-line therapy, he was diagnosed with multiple metastatic castration-resistant prostate cancer in the lung. Second-line therapy with abiraterone acetate resulted in partial remission of the lung metastases. Thoracic surgery was performed to remove the single lung metastasis remaining. Next-generation sequencing of the specimens demonstrated homozygous loss of BRCA2. We note in this case a heterogeneous response to abiraterone acetate may be related to the somatic BRCA2 deletions. CONCLUSIONS: We present the first Japanese case of a metastatic abiraterone acetate-resistant castration-resistant prostate cancer accompanied by BRCA2 mutation.

The Expression of CD74-Regulated Inflammatory Markers in Stage IV Melanoma: Risk of CNS Metastasis and Patient Survival.

Innate inflammatory features have been found in melanoma tumors from patients at all stages, and molecular analysis has identified definitive inflammatory proteins expressed by tumors cells in patients who presents the worst prognosis. We have previously observed weakened outcomes in patients with constitutive expression of inducible nitric oxide synthase (iNOS), macrophage migration inhibitory factor (MIF) and improved outcomes with CD74 expression in stage III melanoma. In our current study, we tested our hypothesis on CD74-regulated inflammatory markers' expression in stage IV melanoma tumors whether the signature is associated with survival outcome and/or risk of developing CNS metastasis. We retrospectively identified 315 patients with stage IV melanoma. In a tissue microarray (TMA), we examined the expression of cells with CD74, its receptor MIF, and downstream inflammatory markers iNOS, nitrotyrosine (NT), cyclooxygenase (COX)-2 and microsomal prostaglandin E synthase-1 (mPGES1). We analyzed the association of those inflammatory markers with overall survival time (OS) and time to CNS metastasis using Kaplan-Meier survival analyses. Our data validates CD74 as a useful prognostic tumor cell protein marker associated with favorable OS as in stage III melanomas, while the tumor NT expression strongly predicts an increased risk of developing CNS metastasis (p = 0.0008) in those patients.

Elevated Endogenous SDHA Drives Pathological Metabolism in Highly Metastatic Uveal Melanoma.

Purpose: Metastatic uveal melanoma (UM) has a very poor prognosis and no effective therapy. Despite remarkable advances in treatment of cutaneous melanoma, UM remains recalcitrant to chemotherapy, small-molecule kinase inhibitors, and immune-based therapy. Methods: We assessed two sets of oxidative phosphorylation (OxPhos) genes within 9858 tumors across 31 cancer types. An OxPhos inhibitor was used to characterize differential metabolic programming of highly metastatic monosomy 3 (M3) UM. Seahorse analysis and global metabolomics profiling were done to identify metabolic vulnerabilities. Analyses of UM TCGA data set were performed to determine expressions of key OxPhos effectors in M3 and non-M3 UM. We used targeted knockdown of succinate dehydrogenase A (SDHA) to determine the role of SDHA in M3 UM in conferring resistance to OxPhos inhibition. Results: We identified UM to have among the highest median OxPhos levels and showed that M3 UM exhibits a distinct metabolic profile. M3 UM shows markedly low succinate levels and has highly increased levels of SDHA, the enzyme that couples the tricarboxylic acid cycle with OxPhos by oxidizing (lowering) succinate. We showed that SDHA-high M3 UM have elevated expression of key OxPhos molecules, exhibit abundant mitochondrial reserve respiratory capacity, and are resistant to OxPhos antagonism, which can be reversed by SDHA knockdown. Conclusions: Our study has identified a critical metabolic program within poor prognostic M3 UM. In addition to the heightened mitochondrial functional capacity due to elevated SDHA, M3 UM SDHA-high mediate resistance to therapy that is reversible with targeted treatment.

CD38-Mediated Immunosuppression as a Mechanism of Tumor Cell Escape from PD-1/PD-L1 Blockade.

Although treatment with immune checkpoint inhibitors provides promising benefit for patients with cancer, optimal use is encumbered by high resistance rates and requires a thorough understanding of resistance mechanisms. We observed that tumors treated with PD-1/PD-L1 blocking antibodies develop resistance through the upregulation of CD38, which is induced by all-trans retinoic acid and IFNß in the tumor microenvironment. In vitro and in vivo studies demonstrate that CD38 inhibits CD8+ T-cell function via adenosine receptor signaling and that CD38 or adenosine receptor blockade are effective strategies to overcome the resistance. Large data sets of human tumors reveal expression of CD38 in a subset of tumors with high levels of basal or treatment-induced T-cell infiltration, where immune checkpoint therapies are thought to be most effective. These findings provide a novel mechanism of acquired resistance to immune checkpoint therapy and an opportunity to expand their efficacy in cancer treatment.Significance: CD38 is a major mechanism of acquired resistance to PD-1/PD-L1 blockade, causing CD8+ T-cell suppression. Coinhibition of CD38 and PD-L1 improves antitumor immune response. Biomarker assessment in patient cohorts suggests that a combination strategy is applicable to a large percentage of patients in whom PD-1/PD-L1 blockade is currently indicated. Cancer Discov; 8(9); 1156-75. ©2018 AACR.See related commentary by Mittal et al., p. 1066This article is highlighted in the In This Issue feature, p. 1047.

Non-corticosteroid adherence and itch severity influence perception of itch in atopic dermatitis.

Topical corticosteroid phobia is an important problem in the treatment of atopic dermatitis as it can affect the ability to control disease severity and itch by reducing treatment adherence. Topical corticosteroid phobia often ends up even non-corticosteroid adherence. As such, non-corticosteroid adherence, disease severity and itch are likely to be associated with each other, but their relationship has yet to be thoroughly investigated. Thus, the purpose of this study is to investigate it in atopic dermatitis. Using data from 1190 participants in an Internet survey, we identified 255 non-corticosteroid users and 225 with moderate to severe itch who were defined as non-corticosteroid adherents. Corticosteroid users with the same itch categories (n = 878) served as controls. We also examined how itch severity affects the perception of itch in atopic dermatitis. Unexpectedly, non-corticosteroid adherents were less sensitive to the conditions to elicit itch such as perspiring, commuting homeward, drinking alcohol and wearing woolen clothes compared with the control. We also found that patients with severer itch were more sensitive to itch during/after bathing, when lying in bed, commuting homeward, studying/working, drinking alcohol, undressing, getting up in the morning, after a meal, ingesting piquant foods and when they were unoccupied, angry, busy, nervous, sad or enjoying themselves. In conclusion, we found that non-corticosteroid adherence and itch severity influence perception of itch in atopic dermatitis and discuss possible mechanisms underlying these results. The information obtained in this study may be useful for communication with and education of atopic dermatitis patients and their treatment in outpatient clinics.

Integrative Analysis Identifies Four Molecular and Clinical Subsets in Uveal Melanoma.

Comprehensive multiplatform analysis of 80 uveal melanomas (UM) identifies four molecularly distinct, clinically relevant subtypes: two associated with poor-prognosis monosomy 3 (M3) and two with better-prognosis disomy 3 (D3). We show that BAP1 loss follows M3 occurrence and correlates with a global DNA methylation state that is distinct from D3-UM. Poor-prognosis M3-UM divide into subsets with divergent genomic aberrations, transcriptional features, and clinical outcomes. We report change-of-function SRSF2 mutations. Within D3-UM, EIF1AX- and SRSF2/SF3B1-mutant tumors have distinct somatic copy number alterations and DNA methylation profiles, providing insight into the biology of these low- versus intermediate-risk clinical mutation subtypes.

Trends in hepatocyte growth factor, insulin-like growth factor 1, thyroid-stimulating hormone, and leptin expression levels in uveal melanoma patient serum and tumor tissues: correlation to disease progression.

This exploratory study was carried out to determine the expression levels of hepatocyte growth factor (HGF), insulin-like growth factor 1, thyroid-stimulating hormone (TSH), and leptin in serum and tumor samples from patients with uveal melanoma and to investigate the potential association of these expression levels with disease progression and patient survival. Seventeen patients, including nine nonmetastatic and eight metastatic, were included in the study. Eighteen healthy individuals served as controls. The levels of these four proteins in serum and tissue samples were determined by enzyme-linked immunosorbent assays and immunohistochemical staining, respectively. Associations between protein levels and survival, disease progression, and other clinicopathological factors were analyzed statistically. Serum levels of HGF were significantly higher and TSH levels were lower in uveal melanoma patients than in healthy individuals, but the level of neither protein differed significantly between metastatic and nonmetastatic groups. Of the four proteins tested, only serum TSH was significantly associated with patient survival. No correlation was observed between the tissue and serum levels of each protein. The levels of HGF in serum may be markers of uveal melanoma development. The prognostic and predictive values of these potential markers need to be determined in a larger cohort.

Novel algorithmic approach predicts tumor mutation load and correlates with immunotherapy clinical outcomes using a defined gene mutation set.

BACKGROUND: While clinical outcomes following immunotherapy have shown an association with tumor mutation load using whole exome sequencing (WES), its clinical applicability is currently limited by cost and bioinformatics requirements. METHODS: We developed a method to accurately derive the predicted total mutation load (PTML) within individual tumors from a small set of genes that can be used in clinical next generation sequencing (NGS) panels. PTML was derived from the actual total mutation load (ATML) of 575 distinct melanoma and lung cancer samples and validated using independent melanoma (n = 312) and lung cancer (n = 217) cohorts. The correlation of PTML status with clinical outcome, following distinct immunotherapies, was assessed using the Kaplan-Meier method. RESULTS: PTML (derived from 170 genes) was highly correlated with ATML in cutaneous melanoma and lung adenocarcinoma validation cohorts (R2 = 0.73 and R2 = 0.82, respectively). PTML was strongly associated with clinical outcome to ipilimumab (anti-CTLA-4, three cohorts) and adoptive T-cell therapy (1 cohort) clinical outcome in melanoma. Clinical benefit from pembrolizumab (anti-PD-1) in lung cancer was also shown to significantly correlate with PTML status (log rank P value < 0.05 in all cohorts). CONCLUSIONS: The approach of using small NGS gene panels, already applied to guide employment of targeted therapies, may have utility in the personalized use of immunotherapy in cancer.

Elevated Serum Leptin Levels are Associated With an Increased Risk of Sentinel Lymph Node Metastasis in Cutaneous Melanoma.

The metabolic hormone leptin has been implicated in the pathogenesis of various malignancies and may contribute to the high rate of cancer in obese individuals. We reported that leptin and its receptor are expressed by melanoma tumors and cell lines, and that leptin stimulates proliferation of cultured melanoma cells. Here, we tested the hypothesis that leptin contributes to early melanoma progression by assessing its association with sentinel node positivity in cutaneous melanoma patients. The study enrolled 72 patients who were scheduled to undergo lymphatic mapping and sentinel node biopsy. Fasting blood was obtained before surgery, and serum leptin levels were measured by enzyme-linked immunosorbent assay (ELISA) with a "raw" (assay value) and an "adjusted" value (raw value divided by body mass index). Leptin levels and other clinicopathologic parameters were compared between sentinel node positive and negative groups. Logistic regression models were used to predict sentinel node status using leptin and other relevant clinical parameters. The raw and adjusted leptin levels were significantly higher in the 15 patients with positive sentinel nodes. These findings could not be attributed to differences in body mass indices. Univariate models revealed raw leptin, adjusted leptin, Breslow thickness, and mitotic rate as significant predictors of sentinel node status. Leptin levels and Breslow thickness remained significant in multivariate models. Survival and follow-up analysis revealed more aggressive disease in diabetic patients. Elevated serum leptin levels predict sentinel node metastasis in melanoma. Validation of this finding in larger cohorts should enable better stratification of early stage melanoma patients.

Uveal melanoma: From diagnosis to treatment and the science in between.

Melanomas of the choroid, ciliary body, and iris of the eye are collectively known as uveal melanomas. These cancers represent 5% of all melanoma diagnoses in the United States, and their age-adjusted risk is 5 per 1 million population. These less frequent melanomas are dissimilar to their more common cutaneous melanoma relative, with differing risk factors, primary treatment, anatomic spread, molecular changes, and responses to systemic therapy. Once uveal melanoma becomes metastatic, therapy options are limited and are often extrapolated from cutaneous melanoma therapies despite the routine exclusion of patients with uveal melanoma from clinical trials. Clinical trials directed at uveal melanoma have been completed or are in progress, and data from these well designed investigations will help guide future directions in this orphan disease. Cancer 2016;122:2299-2312. © 2016 American Cancer Society.