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Discussions surrounding the positive impacts of nature on human health and strategies to enhance our connection with the natural world have been ongoing. However, a limited number of theoretical models are available to guide research and practice in this area. Therefore, there is a pressing need for a systematic framework that outlines clear steps for conducting research implementing nature-based interventions. In this study, we investigate the relationship between health and nature through the lens of Complex Adaptive Systems. This approach involves examining the dynamic interactions between multiple interconnected elements to understand the complex emergent behaviors that arise from such relationships. Our model is designed to support nature-based interventions, considering the essential interdependence between humans and nature. This perspective aims to improve both human health and biodiversity conservation in a mutually beneficial manner. The underlying interactions that drive nature-based health interventions are thoroughly explored, leading us to propose a novel intervention model named "A time with e-Natureza" (e-Nature). This model encompasses four types of experiences, drawing from scientific literature and insights from authors engaged in an interdisciplinary research group: (1) Aesthetic and emotional experience; (2) Multisensory integration experience; (3) Knowledge experience; and (4) Engagement experience. Each experience within the model targets affective, cognitive, and behavioral aspects, with a specific focus on fostering a deeper connection with nature. Distinct activities are incorporated within each experience to promote successful outcomes. The model is grounded in existing theories that address the human-nature relationship and is informed by Nursing theories that support health promotion interventions. By presenting this new model, our aim is to contribute to the effective implementation of nature-based interventions that not only enhance human well-being but also support the conservation of nature. This integrated approach recognizes the mutual benefits of human-nature interaction and offers valuable insights for future research and practical applications in the fields of nature and health., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Leão, Hingst-Zaher, Savieto, Patricio, de Oliveira, Catissi, Lima, Borba, Bomfim and de Abreu.)

Matching of actionable tumor mutations with targeted therapy increases response rates and prolongs survival in lung cancer patients. Drug development and trials targeting genetic alterations are expanding rapidly. We describe the role of a Molecular Tumor Board (MTB) in the design of molecularly informed treatment strategies in our lung cancer patient population. Tumor DNA was sequenced using a 50-gene targeted next-generation sequencing panel. Cases were evaluated by a multidisciplinary MTB who suggested a course of treatment based on each patient's molecular findings. During a three-year period, 21 lung cancer patients were presented at the MTB. All patients lacked common activating EGFR mutations and ALK rearrangements. One patient had Stage IIIb disease; all others were Stage IV; 18 patients had received ≥1 prior line of therapy (range 0-5). Suggestions for treatment with a targeted therapy were made for 19/21 (90.5%) patients, and four patients (21%) underwent treatment with a targeted agent, two as part of a clinical trial. Identified barriers to treatment with targeted therapy included: ineligibility for clinical trials (n ​= ​2), lack of interest in study/distance to travel (n ​= ​2), lack of disease progression (n ​= ​2), poor performance status (n ​= ​5), decision to treat next with immunotherapy (n ​= ​3), and unknown (n ​= ​1). For the majority of lung cancer patients, the MTB provided recommendations based on tumor genetic profiles. Identified barriers to treatment suggest that presentation to the MTB at earlier stages of disease may increase the number of patients eligible for treatment with a genetically informed targeted agent., Competing Interests: The authors have no conflicts of interest to disclose., (© 2020 Published by Elsevier B.V.)

Background: Li-Fraumeni syndrome is a cancer predisposition syndrome caused by germline TP53 tumor suppressor gene mutations, with no previous association with pancreatic neuroendocrine tumors (PNETs). Here we present the first case of PNET associated with Li-Fraumeni syndrome., Case Presentation: This is a 43-year-old female who underwent laparoscopic distal pancreatectomy at age 39 for a well-differentiated grade 2 cystic PNET. When the patient was 41 years old, her seven-year-old daughter was found to have an astrocytoma and a germline TP53 mutation. While undergoing surveillance with 68 Gallium-DOTATATE positron emission tomography/computed tomography for her PNET, the patient was found to have a large choroid plexus papilloma in her right temporal lobe. She underwent genetic counseling and testing that identified a germline pathogenic variant in TP53, leading to the diagnosis of Li-Fraumeni syndrome. Her PNET had a hemizygous pathogenic TP53 mutation with loss of the wild-type alternate allele, consistent with loss of heterozygosity and the two-hit hypothesis. She was enrolled in a Li-Fraumeni syndrome protocol and continues surveillance screening with our service., Conclusions: This is the first PNET reported in association with Li-Fraumeni syndrome. Pancreatic cancer risk is elevated in this syndrome, and our case highlights the need for vigilance in screening for pancreatic neoplasms in these patients.

Next generation sequencing (NGS) has introduced new types of data, such as variant allele frequencies (VAFs), into the workup of acute myeloid leukemias (AML). There is interest in using NGS to prognosticate disease behavior and monitor residual disease, but the attribution of sequencing data entirely to the leukemic clone may be confounded by VAF contribution from background non-leukemic populations and undetected copy number aberrations. Sixty-eight patients with AML were evaluated by a clinically validated gene sequencing panel at our institution from 2015 to 2018. No correlation was found with a direct comparison of blast counts and VAFs in both primary- and secondary-AML ( R 2  = 0.0584 and 0.0235, respectively). Only moderate correlations were attained when evaluating against mutant NPM1 allele fraction alone ( R 2  = 0.5303) or when variants with allelic frequencies >55% of the blast burden were excluded ( R 2  = 0.4608). VAFs from regular clinical-use gene sequencing panels show poor unrestricted correlation with leukemic blast proportions in AML.

Pulmonary adenofibroma (PAF) is a rare neoplasm that may be related to solitary fibrous tumor (SFT). A subset of PAFs harbor the NAB2-STAT6 fusion that is typical of SFT, but a significant proportion do not. Their distinction is clinically important as SFTs can potentially have an aggressive clinical course, while there has been no report of a PAF behaving in a malignant fashion. We report a case of a 60-year-old male who developed a SFT and PAF in the same lung. The SFT harbored a NAB2-STAT6 fusion, while the PAF did not have any identifiable fusion. This case represents the first instance of a single patient with both of these tumors occurring simultaneously in the same lung.

Malignant melanoma (MM) may display highly variable phenotypic diversity, sometimes associated with loss of immunohistochemical melanocytic markers and acquisition of nonmelanocytic lineage of differentiation. Primary cutaneous MM with rhabdomyosarcomatous differentiation is extremely rare with only 5 reported cases in the literature. To date, a chronological progression of a MM to rhabdomyosarcoma has not been conclusively documented. A 96-year-old man underwent a re-excision of an "atypical fibroxanthoma" of the forearm, which revealed a small lentigo maligna melanoma associated with a dominant dermal high-grade spindle cell nodule admixed with a population of malignant polygonal epithelioid cells. On immunohistochemical studies, the spindle cells were completely negative for all melanocytic markers, whereas a small population of polygonal neoplastic cells at the periphery was positive for Desmin and Myo-D1, supporting early rhabdomyosarcomatous transformation. Several subsequent re-excisions demonstrated merely nodules of malignant pleomorphic epithelioid cells with rhabdomyosarcomatous differentiation and devoid of melanocytic markers. In addition, both rhabdomyosarcomatous component and original MM displayed identical mutations. Therefore, the histologic, immunohistochemical, and molecular findings documented for the first time a chronological progression from an invasive MM to a pleomorphic rhabdomyosarcoma through an intermediate stage of undifferentiated sarcoma/atypical fibroxanthoma. Interestingly, subsequent recurrences of pure rhabdomyosarcomatous component displayed skip lesions/microsatellitosis, marked tumor-infiltrative lymphocytes, and rare junctional nests of rhabdomyosarcomatous cells in the epidermis, histologic features that were not described in primary cutaneous rhabdomyosarcoma and therefore could serve as morphologic clues to the diagnosis of rhabdomyosarcomatous transformation in an MM.

Purpose: Cardiorespiratory fitness is inversely associated with a high risk of cardiovascular disease, all-cause mortality, and mortality attributable to various cancers. It is often estimated indirectly using mathematical formulas for estimating oxygen uptake. Cardiopulmonary exercise testing, especially oxygen uptake, represents the "gold standard" for assessing exercise capacity. The purpose of this report was to develop reference standards for exercise capacity by establishing cardiorespiratory fitness values derived from cardiopulmonary exercise testing in a Brazilian population. We focused on oxygen uptake standards and compared the maximal oxygen uptake [mLO2·kg-1·min-1] values with those in the existing literature., Methods: A database was constructed using reports from cardiopulmonary exercise testing performed at Fleury laboratory. The final cohort included 18,189 individuals considered to be free of structural heart disease. Percentiles of maximal oxygen uptake for men and women were determined for six age groups between 7 and 84 years. We compared the values with existing reference data from patients from Norway and the United States., Results: There were significant differences in maximal oxygen uptake between sexes and across the age groups. In our cohort, the 50th percentile maximal oxygen uptake values for men and women decreased from 44.7 and 36.3 mLO2·kg-1·min-1 to 28.4 and 22.3 mLO2·kg-1·min-1 for patients aged 20-29 years to patients aged 60-69 years, respectively. For each age group, both Norwegian men and women had greater cardiorespiratory fitness than cohorts in the United States and Brazil., Conclusion: To our knowledge, our analysis represents the largest reference data for cardiorespiratory fitness based on treadmill cardiopulmonary exercise testing. Our findings provide reference values of maximal oxygen uptake measurements from treadmill tests in Brazilian populations that are more accurate than previous standard values based on workload-derived estimations. This data may also add information to the global data used for the interpretation of cardiorespiratory fitness., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: Authors are employed by and received salary from Fleury Medicina e Saúde. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Xanthomas are histiocytic lesions of the skin, soft tissue, and bone and are generally considered to be reactive in nature. When they arise in the bones of the jaw, they are referred to as central xanthomas. New evidence supports the hypothesis that central xanthomas are a separate and distinct entity from their extragnathic counterparts. Noonan syndrome (NS) is an autosomal dominant disorder that has been associated with giant cell lesions, which also commonly occur in the jaw. We present a case of a 15-year-old boy with NS who presented with a radiolucent lesion of the mandible that on excision was found to be a central xanthoma. Although giant cell lesions have been well described in NS, xanthomas of the jaw have not been reported. We will also discuss the entities that must be excluded before making a diagnosis of central xanthoma, as this can affect both treatment and follow-up., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Isocitrate dehydrogenases 1 and 2 (IDH1/2) are important metabolic enzymes that convert isocitrate to α-ketoglutarate. IDH1/2 mutations are associated with the development of multiple malignancies. In this study, we examine the prevalence and features of non-small cell lung cancers (NSCLC) with IDH1/2 mutations. From May 2013 to March 2017, 800 lung cancer samples were successfully sequenced for somatic mutations on the Ion Torrent PGM with the 50-gene AmpliSeq Cancer Hotspot Panel v2 on the Ion Torrent PGM (318 chip). Variants were identified using the Ion Torrent Variant Caller Plugin and reference genome hg19. Golden Helix's SVS software was used for variant annotation and prediction of significance. Nine samples (1.1%) from 8 patients harbored an IDH1 (3 p.R132C and 2 p.R132L) or IDH2 mutation (p.R140W, p.R172S, and p.R172T). All patients' tumors had adenocarcinoma histology, and all patients had a smoking history. Eighty-eight percent of patients' tumors had a coexisting KRAS mutation and 6 of 8 were diagnosed at greater than 70 years of age. Five patients presented with stage IV disease and 3 with stage I. After comparison to a cohort of KRAS-mutated NSCLC with a history of smoking, IDH-mutated cases were significantly older but presented with similar rates of advanced-stage disease and sex distribution. Additional studies are needed to understand the role of IDH1/2 mutations in the development of NSCLC, but such patients who have poor prognostic indicators (KRAS mutation and advanced stage at presentation) may benefit from IDH1/2-directed therapies., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The tumor suppressor gene TP53 is the most frequently mutated gene in human cancer. It encodes p53, a DNA-binding transcription factor that regulates multiple genes involved in DNA repair, metabolism, cell cycle arrest, apoptosis, and senescence. TP53 is associated with human cancer by mutations that lead to a loss of wild-type p53 function as well as mutations that confer alternate oncogenic functions that enable them to promote invasion, metastasis, proliferation, and cell survival. Identifying the discrete TP53 mutations in tumor cells may help direct therapies that are more effective. In this study, we identified the frequency of individual TP53 mutations in patients with colon adenocarcinoma (48%), non-small cell lung carcinoma (NSCLC) (36%), and glioma/glioblastoma (28%) at our institution using next-generation sequencing. We also identified the occurrence of somatic mutations in numerous actionable genes including BRAF, EGFR, KRAS, IDH1, and PIK3CA that occurred concurrently with these TP53 mutations. Of the 480 tumors examined that contained one or more mutations in the TP53 gene, 219 were colon adenocarcinomas, 215 were NSCLCs, and 46 were gliomas/glioblastomas. Among the patients positive for TP53 mutations diagnosed with colon adenocarcinoma, 50% also showed at least one mutation in pathogenic genes of which 14% were BRAF, 33% were KRAS, and 3% were NRAS. Forty-seven percent of NSCLC patients harboring TP53 mutations also had a mutation in at least one actionable pathogenic variant with the following frequencies: BRAF: 4%, EGFR: 10%, KRAS: 28%, and PIK3CA: 4%. Fifty-two percent of patients diagnosed with glioma/glioblastoma with a positive TP53 mutation had at least one concurrent mutation in a known pathogenic gene of which 9% were CDKN2A, 41% were IDH1, and 11% were PIK3CA., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: We aim to build an informatics methodology capable of identifying statistically significant associations between the clinical findings of non-small cell lung cancer (NSCLC) recorded in patient pathology reports and the various clinically actionable genetic mutations identified from next-generation sequencing (NGS) of patient tumor samples., Methods: We built an information extraction and analysis pipeline to identify the associations between clinical findings in the pathology reports of patients and corresponding genetic mutations. Our pipeline leverages natural language processing (NLP) techniques, large biomedical terminologies, semantic similarity measures, and clustering methods to extract clinical concepts in freetext from patient pathology reports and group them as salient findings., Results: In this study, we developed and applied our methodology to lobectomy surgical pathology reports of 142 NSCLC patients who underwent NGS testing and who had mutations in 4 oncogenes with clinical ramifications for NSCLC treatment ( EGFR , KRAS , BRAF , and PIK3CA ). Our approach identified 732 distinct positive clinical concepts in these reports and highlighted multiple findings with strong associations ( P -value ≤ 0.05) to mutations in specific genes. Our assessment showed that these associations are consistent with the published literature., Conclusions: This study provides an automatic pipeline to find statistically significant associations between clinical findings in unstructured text of patient pathology reports and genetic mutations. This approach is generalizable to other types of pathology and clinical reports in various disorders and can provide the first steps toward understanding the role of genetic mutations in the development and treatment of different types of cancer., Competing Interests: The authors have no competing interests to declare., (Schattauer GmbH.)

Presently, the ability to study disease at the most fundamental molecular level has led to a reclassification of human cancers into numerous subtypes that vary in disease progression and response to therapy. Similar to most solid tumors, breast cancer is a heterogeneous disease with considerable variation in histologic and biological features. Triple-negative breast cancer (TNBC) is a subtype of breast cancer in which the estrogen receptor and progesterone receptor are not expressed, and human epidermal growth factor-receptor 2 is not amplified or overexpressed. Data derived from highly complex molecular technologies, such as microarrays and next-generation sequencing, have identified gene expression and somatic mutation profile subsets of TNBC that reflect biological behavior more accurately and may lead to further effective therapeutic targets, better prognosis, and improved outcomes. Herein, we review the genomic findings of TNBC and discuss current efforts in precision medicine as they relate to TNBC., (Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Advanced stage malignant melanoma often responds poorly to therapy with low survival rates. New therapeutic approaches are based upon a growing understanding of the underlying molecular abnormalities. We demonstrate the feasibility of a next generation sequencing (NGS) assay, which targets hotspots in 50 cancer genes, to assess genotypes that may influence therapeutic selection and response. DNA was extracted from formalin fixed paraffin embedded (FFPE) melanoma specimens to create multiplexed libraries which were sequenced. Of the 121 cases, BRAF mutations were present in 48 cases (40%) and NRAS mutations in 24 cases (20%). We identified other gene variants in 20 BRAF-mutated cases. Additional gene variants were also identified in the 57 BRAF wild-type cases. Four patients harbored different gene mutations at metastatic sites as compared to their primary lesions or metastasis from different sites. Concurrent gene variants may provide additional targets for future therapies and may suggest alternative mechanisms of secondary resistance., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Next-generation sequencing (NGS) assays are highly complex tests that can vary substantially in both their design and intended application. Despite their innumerous advantages, NGS assays present some unique challenges associated with the preanalytical process, library preparation, data analysis, and reporting. According to a number of professional laboratory organization, control materials should be included both during the analytical validation phase and in routine clinical use to guarantee highly accurate results. The SeraseqTM Solid Tumor Mutation Mix AF10 and AF20 control materials consist of 26 biosynthetic DNA constructs in a genomic DNA background, each containing a specific variant or mutation of interest and an internal quality marker at 2 distinct allelic frequencies of 10% and 20%, respectively. The goal of this interlaboratory study was to evaluate the Seraseq AF10 and AF20 control materials by verifying their performance as control materials and by evaluating their ability to measure quality metrics essential to a clinical test., Methods: Performance characteristics were assessed within and between 6 CLIA-accredited laboratories and 1 research laboratory., Results: Most laboratories detected all 26 mutations of interest; however, some discrepancies involving the internal quality markers were observed., Conclusion: This interlaboratory study showed that the Seraseq AF10 and AF20 control materials have high quality, stability, and genomic complexity in variant types that are well suited for assisting in NGS assay analytical validation and monitoring routine clinical applications., (© 2017 American Association for Clinical Chemistry.)

Purpose: To assess the clinical and pharmacodynamic activity of dovitinib in a treatment-resistant, molecularly enriched non-muscle-invasive urothelial carcinoma of the bladder (NMIUC) population. Experimental Design: A multi-site pilot phase II trial was conducted. Key eligibility criteria included the following: Bacillus Calmette-Guerin (BCG)-unresponsive NMIUC ( > 2 prior intravesical regimens) with increased phosphorylated FGFR3 (pFGFR3) expression by centrally analyzed immunohistochemistry (IHC+) or FGFR3 mutations (Mut+) assessed in a CLIA-licensed laboratory. Patients received oral dovitinib 500 mg daily (5 days on/2 days off). The primary endpoint was 6-month TURBT-confirmed complete response (CR) rate. Results: Between 11/2013 and 10/2014, 13 patients enrolled (10 IHC+ Mut-, 3 IHC+ Mut+). Accrual ended prematurely due to cessation of dovitinib clinical development. Demographics included the following: median age 70 years; 85% male; carcinoma in situ (CIS; 3 patients), Ta/T1 (8 patients), and Ta/T1 + CIS (2 patients); median prior regimens 3. Toxicity was frequent with all patients experiencing at least one grade 3-4 event. Six-month CR rate was 8% (0% in IHC+ Mut-; 33% in IHC+ Mut+). The primary endpoint was not met. Pharmacodynamically active (94-5,812 nmol/L) dovitinib concentrations in urothelial tissue were observed in all evaluable patients. Reductions in pFGFR3 IHC staining were observed post-dovitinib treatment. Conclusions: Dovitinib consistently achieved biologically active concentrations within the urothelium and demonstrated pharmacodynamic pFGFR3 inhibition. These results support systemic administration as a viable approach to clinical trials in patients with NMIUC. Long-term dovitinib administration was not feasible due to frequent toxicity. Absent clinical activity suggests that patient selection by pFGFR3 IHC alone does not enrich for response to FGFR3 kinase inhibitors in urothelial carcinoma. Clin Cancer Res; 23(12); 3003-11. ©2016 AACR ., (©2016 American Association for Cancer Research.)

Targeted genomic profiling (TGP) using massively parallel DNA sequencing is becoming the standard methodology in clinical laboratories for detecting somatic variants in solid tumors. The variety of methodologies and sequencing platforms in the marketplace for TGP has resulted in a variety of clinical TGP laboratory developed tests (LDT). The variability of LDTs is a challenge for test-to-test and laboratory-to-laboratory reliability. At the University of Vermont Medical Center (UVMMC), we validated a TGP assay for solid tumors which utilizes DNA hybridization capture and complete exon and selected intron sequencing of 29 clinically actionable genes. The validation samples were run on the Illumina MiSeq platform. Clinical specificity and sensitivity were evaluated by testing samples harboring genomic variants previously identified in CLIA-approved, CAP accredited laboratories with clinically validated molecular assays. The Molecular Laboratory at Dartmouth Hitchcock Medical Center (DHMC) provided 11 FFPE specimens that had been analyzed on AmpliSeq Cancer Hotspot Panel version 2 (CHPv2) and run on the Ion Torrent PGM. A Venn diagram of the gene lists from the two institutions is shown. This provided an excellent opportunity to compare the inter-laboratory reliability using two different target sequencing methods and sequencing platforms. Our data demonstrated an exceptionally high level of concordance with respect to the sensitivity and specificity of the analyses. All clinically-actionable SNV and InDel variant calls in genes covered by both panels (n=17) were identified by both laboratories. This data supports the proposal that distinct gene panel designs and sequencing workflows are capable of making consistent variant calls in solid tumor FFPE-derived samples., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Context: - At our medical center, cytopathologists perform rapid on-site evaluation for specimen adequacy of fine-needle aspiration and touch imprint of needle core biopsy lung cancer samples. Two years ago the molecular diagnostics laboratory at our institution changed to next-generation sequencing using the Ion Torrent PGM and the 50-gene AmpliSeq Cancer Hotspot Panel v2 for analyzing mutations in a 50-gene cancer hot spot panel. This was associated with a dramatic fall in adequacy rate (68%)., Objective: - To improve the adequacy rate to at least 90% for molecular testing using next-generation sequencing for all specimens collected by rapid on-site evaluation by the cytology laboratory., Design: - After baseline data on adequacy rate of cytology specimens with rapid on-site evaluation for molecular testing had been collected, 2 changes were implemented. Change 1 concentrated all the material in one block but did not produce desired results; change 2, in addition, faced the block only once with unstained slides cut up front for molecular testing. Data were collected in an Excel spreadsheet and adequacy rate was assessed., Results: - Following process changes 1 and 2 we reached our goal of at least 90% adequacy rate for molecular testing by next-generation sequencing on samples collected by rapid on-site evaluation including computed tomography-guided needle core biopsies (94%; 17 of 18) and fine-needle aspiration samples (94%; 30 of 32)., Conclusion: - This study focused on factors that are controllable in a pathology department and on maximizing use of scant tissue. Optimizing the adequacy of the specimen available for molecular tests avoids the need for a second procedure to obtain additional tissue.

Objectives: To describe three methods used to screen for frameshift mutations in exon 9 of the CALR gene., Methods: Genomic DNA from 47 patients was extracted from peripheral blood and bone marrow using the EZ1 DNA Blood Kit (Qiagen, Valencia, CA) and quantified by the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, San Diego, CA). After clinical history, cytogenetics, and molecular tests, patients were diagnosed with either clonal or nonclonal hematologic diseases. CALR screening was primarily performed using fragment analysis polymerase chain reaction, then next-generation sequencing and Sanger sequencing., Results: Among the 18 patients diagnosed with clonal diseases, one had acute myeloid leukemia (positive for trisomy 8), and 17 had myeloproliferative neoplasms (MPNs), including chronic myeloid leukemia (CML), essential thrombocythemia (ET), primary myelofibrosis (PMF), and polycythemia vera (PV). Patients with CML were positive for the BCR-ABL1 fusion. Ten patients were positive for JAK2 (PMF, n = 1; ET, n = 2; PV, n = 7), and three were CALR positive (ET, n = 1; PMF, n = 2). Patients diagnosed with a nonclonal disease were negative for JAK2, BCR-ABL, and CALR mutations., Conclusions: Screening for CALR mutations is essential in BCR-ABL-negative MPNs since it not only provides valuable diagnostic and prognostic information but also identifies potential treatment targets. Since this study describes the importance of screening for known and novel biomarkers, we described in detail three methods that could be easily integrated into a clinical laboratory., (© American Society for Clinical Pathology, 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Metastatic breast cancer is a genetically heterogeneous disease and effective therapies for advanced stage disease are limited., Methods: In this study, distant metastases of 22 formalin-fixed, paraffin-embedded (FFPE) breast cancer samples were sequenced using the Ion Torrent PGM and the 50 gene AmpliSeq Cancer Hotspot Panel v2 from 10ng of extracted DNA using 318 chips. Data analysis was performed with the Ion Torrent Variant Caller Plugin (hg19) and Golden Helix's SVS software for annotation and prediction of the significance of the variants., Results: All patients were female with a median age of 61years (range 37-85years). Metastatic sites included liver (n=6, 27%), skin (n=5, 23%), brain (n=4, 18%), lymph node (n=3, 14%), lung (n=2, 9%), retroperitoneum (n=1, 4.5%), and colon (n=1, 4.5%). Overall, 28 variants in 11 genes were observed. Five (23%) samples showed no alterations and 17 (77%) showed at least one potentially biologically significant variant (BSV) defined as having FDA-approved drugs or clinical trials evaluating their significance. BSVs included mutations in the following genes: TP53 (n=8), APC (n=4), PIK3CA (n=5), MET (n=2), ERBB2 (n=2), AKT1 (n=1), CDKN2A (n=1), KRAS (n=1), and FGFR3 (n=1)., Conclusions: Potentially actionable mutations were identified in the majority of breast cancer metastases. Evaluating metastatic breast tumors using a NGS approach provides a better understanding of the mechanisms behind tumor progression and evolution and also identifies additional potentially beneficial therapeutic targets for patient management or eligibility for clinical trials., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Molecular technologies have allowed laboratories to detect and establish the profiles of human cancers by identifying a variety of somatic variants. In order to improve personalized patient care, we have established a next-generation sequencing (NGS) test to screen for somatic variants in primary or advanced cancers. In this study, we describe the laboratory quality management program for NGS testing, and also provide an overview of the somatic variants identified in over 1000 patient samples as well as their implications in clinical practice., Methods: Over the past one-and-a-half years, our laboratory received a total of 1028 formalin-fixed, paraffin-embedded (FFPE) tumor tissues, which consisted of non-small-cell lung carcinomas (NSCLCs), colon adenocarcinomas, glioma/glioblastomas, melanomas, breast carcinomas, and other tumor types. During this time period, we implemented a series of quality control (QC) checks that included (1) pre-DNA extraction, (2) DNA quantification, (3) DNA quality, (4) library quantification, (5) post-emulsification PCR, and (6) post-sequencing metrics. At least 10 ng of genomic DNA (gDNA) were used to prepare barcoded libraries using the AmpliSeq CHPv2. Samples were multiplexed and sequenced on Ion Torrent 318 chips using the Ion PGM System. Variants were identified using the Variant Caller Plugin, and annotation and functional predictions were performed using the Golden Helix SVS., Results: A total of 1005 samples passed QC1-3, and following additional library preparation QC checkpoints, 877 samples were sequenced. Samples were classified into two categories: wild-type (127) and positive for somatic variants (750). Somatic variants were classified into clinically actionable (60%) and non-actionable (40%)., Conclusions: The use of NGS in routine clinical laboratory practice allowed for the detection of tumor profiles that are essential for the selection of targeted therapies and identification of applicable clinical trials, contributing to the improvement of personalized patient care in oncology.

Accurate identification of somatic mutations in formalin-fixed, paraffin-embedded tumor tissue is required for enrollment into clinical trials for many novel targeted therapeutics, including trials requiring EGFR mutation status in non-small-cell lung carcinomas. Central clinical trial laboratories contracted to perform this analysis typically rely on US Food and Drug Administration-approved targeted assays to identify these mutations. We present two cases in which central laboratories inaccurately reported EGFR mutation status because of improper identification and isolation of tumor material and failure to accurately report assay limitations, resulting in enrollment denial. Such cases highlight the need for increased awareness by clinical trials of the limitation of these US Food and Drug Administration-approved assays and the necessity for a mechanism to reevaluate discordant results by alternative laboratory-developed procedures, including clinical next-generation sequencing., (Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

KRAS mutant non-small cell lung cancers (NSCLCs) vary in clinical outcome depending on which specific KRAS mutation is present. Shorter progression free survival has been associated with KRAS variants G12C and G12V. Cell lines with these variants depend to a greater extent on the RAS/RAF/MEK/ERK signaling pathway and become more susceptible to MEK inhibition. Because different KRAS mutations may lead to altered drug sensitivity, we aimed to determine specific KRAS mutation status in a NSCLC patient cohort at our institution. A total of 502 NSCLC samples were screened for somatic mutations using the 50 gene AmpliSeq™ Cancer Hotspot Panel v2 (CHPv2). However only samples positive for variants in the KRAS gene were included in this study. Variants identified in the KRAS genes were curated using publicly available databases. The overall mutation rate in the KRAS gene was 32.7% (164/502). The most common KRAS mutations were G12C (41%), G12V (19%), and G12D (14%) along with less frequent variants. After re-mining our sequencing data, we found that more than a half of our KRAS mutant NSCLC patients could potentially benefit from the addition of a MEK inhibitor such as selumetinib to standard chemotherapeutic agents. Due to mutated KRAS, these patients will likely fail traditional anti-EGFR therapies but be eligible for newer combination therapies., (Copyright © 2016 Elsevier Inc. All rights reserved.)

N-of-1 trials target actionable mutations, yet such approaches do not test genomically-informed therapies in patient tumor models prior to patient treatment. To address this, we developed patient-derived xenograft (PDX) models from fine needle aspiration (FNA) biopsies (FNA-PDX) obtained from primary pancreatic ductal adenocarcinoma (PDAC) at the time of diagnosis. Here, we characterize PDX models established from one primary and two metastatic sites of one patient. We identified an activating KRAS G12R mutation among other mutations in these models. In explant cells derived from these PDX tumor models with a KRAS G12R mutation, treatment with inhibitors of CDKs (including CDK9) reduced phosphorylation of a marker of CDK9 activity (phospho-RNAPII CTD Ser2/5) and reduced viability/growth of explant cells derived from PDAC PDX models. Similarly, a CDK inhibitor reduced phospho-RNAPII CTD Ser2/5, increased apoptosis, and inhibited tumor growth in FNA-PDX and patient-matched metastatic-PDX models. In summary, PDX models can be constructed from FNA biopsies of PDAC which in turn can enable genomic characterization and identification of potential therapies.

Cholangiocarcinoma is a heterogeneous malignant process, which is further classified into intrahepatic cholangiocarcinoma (ICC) and extrahepatic cholangiocarcinoma (ECC). The poor prognosis of the disease is partly due to the lack of understanding of the disease mechanism. Multiple gene alterations identified by various molecular techniques have been described recently. As a result, multiple targeted therapies for ICC and ECC are being developed. In this study, we identified and compared somatic mutations in ICC and ECC patients using next generation sequencing (NGS) (Ampliseq Cancer Hotspot Panel v2 and Ion Torrent 318v2 chips). Eleven of 16 samples passed internal quality control established for NGS testing. ICC cases (n=3) showed IDH1 (33.3%) and NRAS (33.3%) mutations. Meanwhile, TP53 (75%), KRAS (50%), and BRAF (12.5%) mutations were identified in ECC cases (n=8). Our study confirmed the molecular heterogeneity of ICC and ECC using NGS. This information will be important for individual patients as targeted therapies for ICC and ECC become available in the future., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Although genetic profiling of tumors is a potentially powerful tool to predict drug sensitivity and resistance, its routine use has been limited because clinicians are often unfamiliar with interpretation and incorporation of the information into practice. We established a Molecular Tumor Board (MTB) to interpret individual patients' tumor genetic profiles and provide treatment recommendations., Patients and Methods: DNA from tumor specimens was sequenced in a Clinical Laboratory Improvement Amendments-certified laboratory to identify coding mutations in a 50-gene panel (n = 34) or a 255-gene panel (n = 1). Cases were evaluated by a multidisciplinary MTB that included pathologists, oncologists, hematologists, basic scientists, and genetic counselors., Results: During the first year, 35 cases were evaluated by the MTB, with 32 presented for recommendations on targeted therapies, and 3 referred for potential germline mutations. In 56.3% of cases, MTB recommended treatment with a targeted agent based on evaluation of tumor genetic profile and treatment history. Four patients (12.5%) were subsequently treated with a MTB-recommended targeted therapy; 3 of the 4 patients remain on therapy, 2 of whom experienced clinical benefit lasting >10 months., Conclusion: For the majority of cases evaluated, the MTB was able to provide treatment recommendations based on targetable genetic alterations. The most common reasons that MTB-recommended therapy was not administered stemmed from patient preferences and genetic profiling at either very early or very late stages of disease; lack of drug access was rarely encountered. Increasing awareness of molecular profiling and targeted therapies by both clinicians and patients will improve acceptance and adherence to treatments that could significantly improve outcomes., Implications for Practice: Case evaluation by a multidisciplinary Molecular Tumor Board (MTB) is critical to benefit from individualized genetic data and maximize clinical impact. MTB recommendations shaped treatment options for the majority of cases evaluated. In the few patients treated with MTB-recommended therapy, disease outcomes were positive and support genetically informed treatment., (©AlphaMed Press.)

Background: Regulatory T cells (Tregs) are potentially prognostic indicators in patients with glioblastoma. If differences in frequency of Tregs in tumor or blood account for substantial variation in patient survival, then reliably measuring Tregs may enhance treatment selection and improve outcomes., Methods: We measured Tregs and CD3+ T cells in tumors and blood from 25 patients with newly diagnosed glioblastoma. Tumor-infiltrating Tregs and CD3+ T cells, measured by quantitative DNA demethylation analysis (epigenetic qPCR) and by immunohistochemistry, and peripheral blood Treg proportions measured by flow cytometry were correlated with patient survival. Additionally, we analyzed data from The Cancer Genome Atlas (TCGA) to correlate the expression of Treg markers with patient survival and glioblastoma subtypes., Results: Tregs, as measured in tumor tissue and peripheral blood, did not correlate with patient survival. Although there was a correlation between tumor-infiltrating Tregs expression by epigenetic qPCR and immunohistochemistry, epigenetic qPCR was more sensitive and specific. Using data from TCGA, mRNA expression of Forkhead box protein 3 (FoxP3) and Helios and FoxP3 methylation level did not predict survival. While the classical glioblastoma subtype corresponded to lower expression of Treg markers, these markers did not predict survival in any of the glioblastoma subtypes., Conclusions: Although immunosuppression is a hallmark of glioblastoma, Tregs as measured in tissue by gene expression, immunohistochemistry, or demethylation and Tregs in peripheral blood measured by flow cytometry do not predict survival of patients. Quantitative DNA demethylation analysis provides an objective, sensitive, and specific way of identifying Tregs and CD3+ T cells in glioblastoma., (© The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

We report a unique case of a 59-year-old woman diagnosed with a benign phyllodes tumor (PT), which recurred twice in the same location over a 7-year period: first as a malignant PT and then as a malignant PT with coexisting spindle cell metaplastic breast carcinoma (MBC). The MBC was differentiated from the malignant PT by expression of cytokeratins (CKs) AE1/AE3, CK MNF-116, CK 5/6, and p63. Somatic mutation analysis using a next-generation sequencing platform revealed a shared mutation in F-box and WD repeat domain containing 7, a tumor suppressor gene that encodes a ubiquitin ligase-associated protein, in the original benign PT and the first recurrent malignant PT. Chromosomal microarray analysis showed shared genetic gains and losses between the malignant PT and MBC. This case highlights the utility of immunohistochemistry to differentiate malignant PT from spindle cell MBC, describes a novel mutation in PT, and demonstrates a biologic relationship between these 2 entities., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Breast cancer is a complex disease characterized by many morphological, clinical and molecular features. For many years, this disease has been classified according to histopathologic criteria, known as the tumor, node and metastasis (TNM) staging system. Clinical criteria that include immunohistochemical markers, such as the estrogen receptor (ER), the progesterone receptor (PR), and the human epidermal growth factor receptor 2 (HER2), provide a classification of breast cancer and dictates the optimal therapeutic approach for treatment. With genomic techniques, such as real-time reverse transcriptase PCR (RT-PCR), microarrays, next-generation sequencing, and whole-exome sequencing, breast cancer diagnostics is going through a significant evolution. Genomic and transcriptomic technologies make the analysis of gene expression signatures and mutation status possible so that tumors may now be classified more accurately with respect to diagnosis and prognosis. The -omic era has also made the possible identification of new biomarkers involved in breast cancer development, survival and invasion that can be gradually incorporated either into clinical testing or clinical trials. Together, clinical and molecular criteria can contribute to a more personalized management of the breast cancer patient. This article will present the progress made in the diagnosis and management of breast cancer using molecular information provided by genomic and transcriptomic technologies., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Some epithelial neoplasms of the appendix, including low-grade appendiceal mucinous neoplasm and adenocarcinoma, can result in pseudomyxoma peritonei (PMP). Little is known about the mutational spectra of these tumor types and whether mutations may be of clinical significance with respect to therapeutic selection. In this study, we identified somatic mutations using the Ion Torrent AmpliSeq Cancer Hotspot Panel v2., Methods: Specimens consisted of 3 nonneoplastic retention cysts/mucocele, 15 low-grade mucinous neoplasms (LAMNs), 8 low-grade/well-differentiated mucinous adenocarcinomas with pseudomyxoma peritonei, and 12 adenocarcinomas with/without goblet cell/signet ring cell features. Barcoded libraries were prepared from up to 10 ng of extracted DNA and multiplexed on single 318 chips for sequencing. Data analysis was performed using Golden Helix SVS. Variants that remained after the analysis pipeline were individually interrogated using the Integrative Genomics Viewer., Results: A single Janus kinase 3 (JAK3) mutation was detected in the mucocele group. Eight mutations were identified in the V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and GNAS complex locus (GNAS) genes among LAMN samples. Additional gene mutations were identified in the AKT1 (v-akt murine thymoma viral oncogene homolog 1), APC (adenomatous polyposis coli), JAK3, MET (met proto-oncogene), phosphatidylinositol-4,5-bisphosphate 3-kinase (PIK3CA), RB1 (retinoblastoma 1), STK11 (serine/threonine kinase 11), and tumor protein p53 (TP53) genes. Among the PMPs, 6 mutations were detected in the KRAS gene and also in the GNAS, TP53, and RB1 genes. Appendiceal cancers showed mutations in the APC, ATM (ataxia telangiectasia mutated), KRAS, IDH1 [isocitrate dehydrogenase 1 (NADP+)], NRAS [neuroblastoma RAS viral (v-ras) oncogene homolog], PIK3CA, SMAD4 (SMAD family member 4), and TP53 genes., Conclusions: Our results suggest molecular heterogeneity among epithelial tumors of the appendix. Next generation sequencing efforts have identified mutational spectra in several subtypes of these tumors that may suggest a phenotypic heterogeneity showing mutations that are relevant for targeted therapies., (© 2014 The American Association for Clinical Chemistry.)

O-6-methylguanine-DNA methyltransferase (MGMT) has been associated with resistance to alkylating agent cancer therapy in Glioblastoma (GBM), the most common and aggressive primary brain tumor in adults. Lower expression or silencing of the MGMT protein by promoter methylation has been reported to improve survival in patients with GBM [1]. This protocol describes bisulfite conversion, methylation sensitive PCR amplification and data analysis/interpretation. This protocol differs from published protocols in that it:•Describes a detailed method to measure MGMT using DNA extracted from solid tumor tissue. We have optimized the DNA extraction by using FFPE tissue blocks that contain greater than 50% tumor tissue, when non-tumor tissue was also present. Performance of this assay is compromised when lower quantities of tumor cells are used as the methylation status of tumor cells is diluted out by methylation status of normal cells.•The measurement of MGMT could be further (enhanced) optimized using a percentage of methylation ration cutoff of 2 as methylated.•The machine specifications detailed here are specific to measuring MGMT from PPFE tumor tissue.

Background: Somatic mutation analysis is standard of practice for solid tumors in order to identify therapeutic sensitizing and resistance mutations. Our laboratory routinely performed standalone PCR-based methods for mutations in several genes. Rapid discovery and introduction of new therapeutics has demanded additional genomic information for adequate management of the cancer patient. We evaluated a next generation sequencing assay, the Ion Torrent AmpliSeq Cancer Hotspot Panelv2 (CHPv2), capable of identifying multiple somatic mutations in 50 genes in a single assay., Methods: Accuracy, precision, limit of detection, and specificity were evaluated using DNA from well-characterized cell lines, genetically engineered cell lines fixed and embedded in paraffin, and previously tested mutation positive or negative, formalin-fixed, paraffin-embedded (FFPE) tissues. Normal kidney, tonsil and colon FFPE tissues were used as controls., Results: Accuracy studies showed 100% concordance in each patient sample between previous PCR results and the corresponding variants identified using the Ion Torrent panel. Precision studies gave consistent results when libraries were prepared from the same original DNA and were run on multiple 316 chips. The limit of detection was determined to be 5% for single nucleotide variants (SNVs) and 20% for insertions and deletions (indels). Specificity studies using normal FFPE tissue previously tested by PCR methods were also 100%., Conclusions: We have evaluated the performance of the AmpliSeq Cancer Panel Hotspotv2 and show that it is suitable for clinical testing. This next generation sequencing panel has allowed the laboratory to consolidate a broader range of molecular oncology testing to a single platform and single assay.

Breast cancer is a complex disease characterized by many morphological, clinical, and molecular features. For many years, breast cancer has been classified according to traditional parameters, such as histological type, grade, tumor size, lymph node involvement and vascular invasion, and biomarkers (eg, estrogen receptor, progesterone receptor, and epidermal growth factor receptor 2), which are used in patient management. With emerging imaging techniques (ie, digital mammography, tomosynthesis, ultrasonography, magnetic resonance imaging, nuclear medicine, and genomic techniques, such as real-time RT-PCR and microarrays), breast cancer diagnostics is going through a significant evolution. Imaging technologies have improved breast cancer diagnosis, survival, and treatment by early detection of primary or metastatic lesions, differentiating benign from malignant lesions and promoting intraoperative surgical guidance and postoperative specimen evaluation. Genomic and transcriptomic technologies make the analysis of gene expression signatures and mutation status possible so that tumors may be classified more accurately with respect to diagnosis and prognosis. The -omic era has also made possible the identification of new biomarkers involved in breast cancer development, survival, and invasion that can be gradually incorporated into clinical testing. These advances in both imaging and genomics contribute to more personalized and predictive patient management. We review the progress made in breast cancer diagnosis and management using these new tools., (Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: A genetic TA repeat length polymorphism in the UGT1A1 promoter has been shown to affect UDP-glucuronosyltyransferase (UGT1A1) expression levels with significant clinical implications. The presence of 7 TA repeats has been associated with lowered UGT1A1 expression and the mild hyperbilinrubinemia manifested in Gilbert's syndrome. Furthermore, cancer patients carrying this variant exhibit irinotecan-related toxicity and require lower doses of this chemotherapeutic agent compared to patients carrying the 6 TA repeat allele. This polymorphism is very common and, therefore, necessitates the development of reliable means of detecting it in the clinical laboratory to deliver better personalized therapy regimens., Methods: We used 45 whole blood samples from patients previously tested with the FDA-approved Invader UGT1A1 assay (Hologic, Madison, WI) to assess extraction method, analytical sensitivity, accuracy and precision of this assay. In addition, cell line controls were used to test for the common and rare alleles of this polymorphism. The assay was based on PCR amplification and capillary electrophoresis for accurate sizing of the TA repeat copy number., Results: All samples tested and controls gave the expected results., Conclusions: We have developed and validated a simple and sensitive PCR fragment analysis assay for accurately determining TA repeat length in the UGT1A1 promoter. At our medical center, this testing is used primarily for guiding irinotecan dosing decisions for our cancer patients., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Aims: This study aimed to evaluate the copy number alteration on 2q24, its association with ACVR1 transcript expression and the prognostic value of these data in head and neck squamous cell carcinomas., Methods and Results: Twenty-eight samples of squamous cell carcinoma were evaluated by fluorescence in situ hybridization (FISH) using the probes RP11-546J1 (2q24) and RP11-21P18 (internal control). Significant gains at 2q24 were detected in most cases at frequencies varying from 3 to 35%. ACVR1 gains and amplifications were associated with longer overall survival (P = 0.022). ACVR1 mRNA expression analysis in 78 cases revealed overexpression in 44% (34 of 78) of these tumours, suggesting that gene copy number alterations could be involved in gene overexpression. In laryngeal carcinomas, overexpression of ACVR1 mRNA levels was associated with longer overall survival (P = 0.013). Multivariate analysis revealed that ACVR1 is an independent prognostic marker in laryngeal carcinomas (P = 0.012, hazard ratio = 0.165, 95% confidence interval =0.041-0.668)., Conclusions: These findings suggest that copy number alterations at 2q24 can be involved in ACVR1 overexpression, which is associated with longer overall survival in laryngeal carcinomas. To our knowledge, this is the first report indicating the relevance of ACVR1 expression in head and neck cancers., (© 2011 Blackwell Publishing Limited.)

Background: Previous reports into the role of [CAG]n repeat lengths in the androgen receptor (AR) gene indicate that these may play an important part in the development and progression of breast cancer, however, knowledge regarding benign breast lesions is limited., Patients and Methods: PCR-based GeneScan analysis was used to investigate the [CAG]n repeat length at exon 1 of the AR gene in 59 benign breast lesions (27 fibroadenomas, 18 atypical hyperplasias, and 14 hyperplasias without atypia) and 54 ductal breast carcinomas. Seventy-two cancer-free women were used as a control group. In addition, [CAG]n repeats were evaluated for the presence of loss of heterozygosity (LOH) and microsatellite instability (MSI) in a subset of these samples (27 fibroadenomas, 14 hyperplasias without atypia and 22 breast carcinomas)., Results: Shorter [CAG]n repeat lengths were strongly correlated with atypical hyperplasias (p = 0.0209) and carcinomas (p < 0.0001). LOH was found in 1/12 and 4/20 informative cases of hyperplasias without atypia and breast carcinomas, respectively. Three patients with breast carcinoma who had previously presented atypical hyperplasia showed a reduction in the [CAG]n repeat length in their carcinomas., Conclusion: Short [CAG]n repeat length (< or = 20) polymorphisms are strongly associated with breast carcinomas and atypical hyperplasias. Although non-significant, a subgroup of patients with breast carcinoma and genotype SS showed an association with parameters of worse outcome.