CKS1B as a potential target for prognostic assessment and intervention in pancreatic cancer and its role in abnormal proliferation and cellular phenotype through mediation of cell cycle signaling pathways.

OBJECTIVES: To investigate the role of cell cycle protein-dependent kinase regulatory subunit 1B (CKS1B) in driving the aggressive and rapid proliferation observed in pancreatic cancer. METHODS: A comprehensive analysis was carried out using raw mRNA information and data from 2 databases: the cancer genome atlas and gene expression omnibus. The differential expression of CKS1B at the mRNA and tissue levels in cancer and adjacent paracancerous tissues were assessed. Additionally, the relationship of CKS1B expression and overall survival (OS) rate was investigated using Kaplan-Meier survival curves. Potential molecular mechanisms by which CKS1B may influence the biological characteristics of pancreatic cancer were explored using resources available within the encyclopedia of RNA interactomes database. RESULTS: The CKS1B exhibited significant differential expression at the mRNA as well as protein levels. A correlation with statistical significance between CKS1B expression and N stage, age, and alcohol consumption was observed. Notably, high CKS1B expression was determined as a predictive factor for worse OS. Furthermore, the analysis revealed a potential synergistic role between CKS1B and the molecule PKMYT1, which could impact the ATR-Chk1-Cdc25 signaling pathway and disrupt the G2/M checkpoint within the cell cycle, ultimately promoting abnormal tumor proliferation. CONCLUSION: The CKS1B may serve as a novel potential prognostic factor in pancreatic cancer and is involved in the abnormal proliferation biology phenotype by mediating cell cycle signaling pathways.

Research-based flow cytometry assays for pathogenic assessment in the human B-cell biology of gene variants revealed in the diagnosis of inborn errors of immunity: a Bruton's tyrosine kinase case-study.

Introduction: Inborn errors of immunity (IEI) are an expanding group of rare diseases whose field has been boosted by next-generation sequencing (NGS), revealing several new entities, accelerating routine diagnoses, expanding the number of atypical presentations and generating uncertainties regarding the pathogenic relevance of several novel variants. Methods: Research laboratories that diagnose and provide support for IEI require accurate, reproducible and sustainable phenotypic, cellular and molecular functional assays to explore the pathogenic consequences of human leukocyte gene variants and contribute to their assessment. We have implemented a set of advanced flow cytometry-based assays to better dissect human B-cell biology in a translational research laboratory. We illustrate the utility of these techniques for the in-depth characterization of a novel (c.1685G>A, p.R562Q) de novo gene variant predicted as probably pathogenic but with no previous insights into the protein and cellular effects, located in the tyrosine kinase domain of the Bruton's tyrosine kinase (BTK) gene, in an apparently healthy 14-year-old male patient referred to our clinic for an incidental finding of low immunoglobulin (Ig) M levels with no history of recurrent infections. Results and discussion: A phenotypic analysis of bone marrow (BM) revealed a slightly high percentage of pre-B-I subset in BM, with no blockage at this stage, as typically observed in classical X-linked agammaglobulinemia (XLA) patients. The phenotypic analysis in peripheral blood also revealed reduced absolute numbers of B cells, all pre-germinal center maturation stages, together with reduced but detectable numbers of different memory and plasma cell isotypes. The R562Q variant allows Btk expression and normal activation of anti-IgM-induced phosphorylation of Y551 but diminished autophosphorylation at Y223 after anti IgM and CXCL12 stimulation. Lastly, we explored the potential impact of the variant protein for downstream Btk signaling in B cells. Within the canonical nuclear factor kappa B (NF-κB) activation pathway, normal IκBα degradation occurs after CD40L stimulation in patient and control cells. In contrast, disturbed IκBα degradation and reduced calcium ion (Ca2+) influx occurs on anti-IgM stimulation in the patient's B cells, suggesting an enzymatic impairment of the mutated tyrosine kinase domain.

Budget impact analysis of next-generation sequencing versus sequential single-gene testing in Japanese patients with advanced non-small-cell lung cancer.

BACKGROUND: Identification of genomic alterations (e.g., EGFR, ALK, ROS1, BRAF, NTRK, and MET) is essential for initiating targeted therapy in patients with advanced non-small-cell lung cancer (aNSCLC). This study estimated the budget impact of using the sequential single-gene (SSG) test, which tests for each mutation one at a time, versus next-generation sequencing (NGS), which tests for all mutations at the same time, among newly diagnosed patients with aNSCLC from a Japanese healthcare payer's perspective. METHODS: A budget impact model (BIM) was used to determine the expected budget impact associated with NGS for newly diagnosed aNSCLC in Japan over a 3-year period. The BIM compared the total costs (biopsy, testing, and treatment) and average turnaround time of "future NGS" and "current NGS" versus SSG testing. RESULTS: The adoption of current NGS over SSG testing had a budget impact of -0.24%, but adoption of future NGS over SSG testing had a budget impact of +4.33% across a 3-year time horizon on the Japanese budget for aNSCLC treatment. The adoption of current or future NGS over SSG testing would shorten the average turnaround time for testing. CONCLUSIONS: The adoption of current NGS over SSG testing would slightly decrease the yearly costs. However, the adoption of future or current NGS over SSG testing would shorten the average turnaround time, enabling faster identification of genomic alterations and earlier initiation of treatment for aNSCLC patients in Japan.

Comprehensive assessment of anaplastic lymphoma kinase in localized and metastatic prostate cancer reveals targetable alterations.

Anaplastic lymphoma kinase (ALK) is a tyrosine kinase with genomic and expression changes in many solid tumors. ALK inhibition is first line therapy for lung cancers with ALK alterations, and an effective therapy in other tumor types, but has not been well-studied in prostate cancer. Here, we aim to delineate the role of ALK genomic and expression changes in primary and metastatic prostate cancer. We determined ALK expression by immunohistochemistry and RNA-Seq, and genomic alterations by NGS. We assessed functional consequences of ALK overexpression and pharmacological ALK inhibition by cell proliferation and cell viability assays. Among 372 primary prostate cancer cases we identified one case with uniformly high ALK protein expression. Genomic analysis revealed a SLC45A3-ALK fusion which promoted oncogenesis in in vitro assays. We observed ALK protein expression in 5/52 (9%) of metastatic prostate cancer cases, of which 4 of 5 had neuroendocrine features. ALK-expressing neuroendocrine prostate cancer had a distinct transcriptional program, and earlier disease progression. An ALK-expressing neuroendocrine prostate cancer model was sensitive to pharmacological ALK inhibition. In summary, we found that ALK overexpression is rare in primary prostate cancer, but more frequent in metastatic prostate cancers with neuroendocrine differentiation. Further, ALK fusions similar to lung cancer are an occasional driver in prostate cancer. Our data suggest that ALK-directed therapies could be an option in selected patients with advanced prostate cancer.

Pre-and Postoperative Circulating Tumour DNA in Patients With Gastrointestinal Stromal Tumour - A Methodological Assessment Study.

BACKGROUND/AIM: Gastrointestinal stromal tumours (GISTs) harbour genetic aberrations in receptor tyrosine kinase KIT (KIT) or platelet-derived growth factor receptor A (PDGFRA) in 85-90% of the patients. Circulating tumour DNA (ctDNA) is a potential biomarker in patients with GIST. Previous studies investigating ctDNA around surgery in patients with GIST presented divergent results regarding the proportion of patients with detectable ctDNA. This study aimed to 1) investigate the feasibility of detecting and monitoring ctDNA pre-and postoperative, 2) compare two different circulating free DNA (cfDNA) extraction methods, and validate results obtained by next-generation sequencing (NGS) using Real-Time PCR technology. PATIENTS AND METHODS: Eight patients planned for immediate surgery or surgery after neoadjuvant oncological treatment were included in the study, from whom blood collection was performed pre- and postoperatively for ctDNA analysis. Furthermore, blood samples from six patients with GIST harbouring a point mutation in KIT or PDGFRA in tissues from primary tumours were used for comparison and validation sub-study. RESULTS: In this explorative study, none of the patients with very low to intermediate risk GIST harboured KIT, or PDGFRA mutated ctDNA in pre-or postoperative blood samples. The methods used for cfDNA extraction gave similar output, and the two methods for ctDNA analysis gave identical results. CONCLUSION: There is no benefit in analysing ctDNA around surgery in very low to intermediate-risk GIST patients. Larger studies investigating ctDNA in patients with high-risk GIST around surgery are warranted.

Economic Analysis of Exclusionary EGFR Test Versus Up-Front NGS for Lung Adenocarcinoma in High EGFR Mutation Prevalence Areas.

BACKGROUND: This study sought to determine whether exclusionary EGFR mutation testing followed by next-generation sequencing (NGS) is a cost-efficient and timely strategy in areas with high prevalence rates of EGFR mutation. METHODS: We developed a decision tree model to compare exclusionary EGFR testing followed by NGS and up-front NGS. Patients entered the model upon diagnosis of metastatic lung adenocarcinoma. Gene alterations with FDA-approved targeted therapies included EGFR, ALK, ROS1, BRAF, RET, MET, NTRK, and KRAS. Model outcomes were testing-related costs; time-to-test results; monetary loss, taking both costs and time into consideration; and percentage of patients who could be treated by FDA-approved therapies. Stacked 1-way and 3-way sensitivity analyses were performed. RESULTS: Exclusionary EGFR testing incurred testing-related costs of US $1,387 per patient, a savings of US $1,091 compared with the costs of up-front NGS. The time-to-test results for exclusionary EGFR testing and up-front NGS were 13.0 and 13.6 days, respectively. Exclusionary EGFR testing resulted in a savings of US $1,116 in terms of net monetary loss, without a reduction of patients identified with FDA-approved therapies. The EGFR mutation rate and NGS cost had the greatest impact on minimizing monetary loss. Given that the tissue-based NGS turnaround time was shortened to 7 days, up-front NGS testing would become the best strategy if its price could be reduced to US $568 in Taiwan. CONCLUSIONS: In areas with high prevalence rates of EGFR mutation, exclusionary EGFR testing followed by NGS, rather than up-front NGS, is currently a cost-efficient strategy for metastatic lung adenocarcinoma.

Total cost of testing for genomic alterations associated with next-generation sequencing versus polymerase chain reaction testing strategies among patients with metastatic non-small cell lung cancer.

BACKGROUND: To assess the total cost of testing associated with next-generation sequencing (NGS) versus polymerase chain reaction (PCR) testing strategies among patients with metastatic non-small cell lung cancer (mNSCLC) from a Medicare and US commercial payer's perspective. MATERIALS AND METHODS: A decision tree model considered testing for genomic alterations in EGFR, ALK, ROS1, BRAF, KRAS, MET, HER2, RET, NTRK1 among patients with newly diagnosed mNSCLC using (1) liquid or tissue biopsy NGS tests, (2) exclusionary mutation (KRAS) test followed by sequential PCR tests, (3) sequential PCR tests, or (4) hotspot panel PCR tests. The alteration test sequence followed clinical guideline recommendations. Inputs based on literature, expert opinion, or assumptions included prevalence of mNSCLC, proportion of patients using each testing strategy (50% NGS [90% tissue, 10% liquid], 10% exclusionary, 10% sequential, 30% hotspot), proportion testing positive for each genomic mutation, rebiopsy rates, and costs for testing and associated medical care. Time to appropriate targeted therapy initiation and total costs were calculated for NGS, each PCR testing strategy, and all PCR strategies combined. RESULTS: Among a hypothetical plan of 1,000,000 members (75% commercial, 25% Medicare), 1,119 patients were estimated to have mNSCLC and be eligible for testing. Estimated mean time to appropriate targeted therapy was 2 weeks for NGS and 6 weeks for PCR (sequential: 9 weeks, exclusionary: 8 weeks, hotspot: 3 weeks). Mean per patient costs were $4,932 for NGS and $6,605 for PCR (exclusionary: $5,563, sequential: $6,263, hotspot: $7,066). Per patient costs were higher from a commercial perspective (NGS: $6,225; PCR: $8,430) relative to Medicare (NGS: $2,099; PCR: $2,646); nevertheless, NGS was the least costly testing strategy across plan types. CONCLUSION: NGS was associated with the fastest time to appropriate targeted therapy initiation and lowest total cost of testing compared to PCR testing strategies for newly diagnosed patients with mNSCLC.

Assessment of ALK Fusions in Uncommon Inflammatory Myofibroblastic Tumors With ALK IHC Positivity but FISH-Equivocal Findings by Targeted RNA Sequencing.

CONTEXT.­: Fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) are common methods to detect ALK status in inflammatory myofibroblastic tumors (IMTs). However, equivocal ALK FISH signals and inconsistency between FISH and IHC are occasionally observed. OBJECTIVE.­: To study the inconsistency between FISH and IHC, and clarify ALK status in IMT by targeted RNA sequencing (RNAseq). DESIGN.­: In this study, 12 consultation cases preliminarily diagnosed as uncommon IMTs with ALK IHC positivity but FISH negativity, plus 3 ALK-positive and 3 ALK-negative IMTs, were re-analyzed by IHC, FISH, and RNAseq. RESULTS.­: As a result, 1 case with FUS-TFCP2 fusion was detected by RNAseq, which was previously misdiagnosed as IMT. In the other 11 uncommon IMTs, 90.9% (10 of 11) showed equivocal ALK FISH signals, and all were confirmed to harbor ALK fusion by RNAseq, except for 1 failure, suggesting that a low threshold for ALK FISH might be proposed in IMT. Furthermore, RNAseq also identified IGFBP5-ALK in 1 case with ALK IHC positivity but typical FISH negativity, suggesting the possibility of false negatives for ALK FISH. For the typical IMTs, ALK fusion was identified by RNAseq in all 3 ALK-positive IMTs as expected, and additionally FN1-ROS1 fusions were identified in 2 of 3 ALK-negative IMTs. CONCLUSIONS.­: These findings indicated that RNAseq can simultaneously detect multiple gene fusions and provide fusion forms and breakpoints, which is of great value for differential diagnosis, especially for those uncommon IMTs with equivocal FISH findings or inconsistency between IHC and FISH.

Cost-effectiveness analysis of first-line treatment with crizotinib in ROS1-rearranged advanced non-small cell lung cancer (NSCLC) in Canada.

INTRODUCTION: While no direct comparative data exist for crizotinib in ROS1+ non-small cell lung cancer (NSCLC), studies have suggested clinical benefit with this targeted agent. The objective of this study was to assess the cost-effectiveness of crizotinib compared to standard platinum-doublet chemotherapy for first-line treatment of ROS1+ advanced NSCLC. METHODS: A Markov model was developed with a 10-year time horizon from the perspective of the Canadian publicly-funded health care system. Health states included progression-free survival (PFS), up to two further lines of therapy post-progression, palliation and death. Given a lack of comparative data and small study samples, crizotinib or chemotherapy studies with advanced ROS1+ NSCLC patients were identified and time-to-event data from digitized Kaplan-Meier curves were collected to pool PFS data. Costs of drugs, treatment administration, monitoring, adverse events and palliative care were included in 2018 Canadian dollars, with 1.5% discounting. An incremental cost-effectiveness ratio (ICER) was estimated probabilistically using 5000 simulations. RESULTS: In the base-case probabilistic analysis, crizotinib produced additional 0.885 life-years and 0.772 quality-adjusted life-years (QALYs) at an incremental cost of $238,077, producing an ICER of $273,286/QALY gained. No simulations were found to be cost-effective at a willingness-to-pay threshold of $100,000/QALY gained. A scenario analysis assuming efficacy equivalent to the ALK+ NSCLC population showed a slightly more favorable cost-effectiveness profile for crizotinib. CONCLUSIONS: Available data appear to support superior activity of crizotinib compared to chemotherapy in ROS1+ advanced NSCLC. At the list price, crizotinib was not cost-effective at commonly accepted willingness-to-pay thresholds across a wide range of sensitivity analyses.

Cost-effectiveness analysis comparing companion diagnostic tests for EGFR, ALK, and ROS1 versus next-generation sequencing (NGS) in advanced adenocarcinoma lung cancer patients.

BACKGROUND: The treatment of choice for advanced non-small cell lung cancer is selected according to the presence of specific alterations. Patients should undergo molecular testing for relevant modifications and the mutational status of EGFR and translocation of ALK and ROS1 are commonly tested to offer the best intervention. In addition, the tests costs should also be taken in consideration. Therefore, this work was performed in order to evaluate the cost-effectiveness of a unique exam using NGS (next generation sequencing) versus other routinely used tests which involve RT-PCR and FISH. METHODS: The target population was NSCLC, adenocarcinoma, and candidates to first-line therapy. Two strategies were undertaken, strategy 1 corresponded to sequential tests with EGFR RT-PCR, then FISH for ALK and ROS1. Strategy 2 differed from 1 in that ALK and ROS1 translocation testing were performed simultaneously by FISH. Strategy 3 considered single test next-generation sequencing, a platform that includes EGFR, ALK and ROS1 genes. A decision tree analysis was used to model genetic testing options. From the test results, a microsimulation model was nested to estimate survival outcomes and costs of therapeutic options. RESULTS: The use of NGS added 24% extra true cases as well as extra costs attributed to the molecular testing. The ICER comparing NGS with sequential tests was US$ 3479.11/correct case detected. The NGS improved a slight gain in life years and QALYs. CONCLUSION: Our results indicated that, although precise, the molecular diagnosis by NGS of patients with advanced stage NSCLC adenocarcinoma histology was not cost-effective in terms of quality-adjusted life years from the perspective of the Brazilian supplementary health system.

RET/PTC Gene Rearrangements in Thyroid Carcinogenesis: Assessment and Clinico-Pathological Correlations.

Rearranged during transfection (RET) is a proto oncogene implicated in thyroid carcinogenesis of papillary type (PTC). The RET proto-oncogene in PTC is constitutively activated by fusion of its tyrosine kinase domain with the 5 ´region of another gene thereby generating chimeric products collectively named RET/PTCs. RET/PTC1 and RET/PTC3 are best characterized among all RET/PTC rearrangements. Kashmir valley has witnessed an alarming increase in thyroid cancer incidence in young women. Therefore, we investigated the occurrence of RET/PTC 1 & 3 rearrangements by semi quantitative and qPCR in thyroid cancer patients (n = 48) of Kashmiri population and interrelated results with various clinicopathological characteristics. We observed that all the RET/PTC rearrangements were confined to PTC cases (10/40). Presence of RET/PTC rearrangement significantly correlated with gender, elevated TSH levels and lymph node metastasis. Overall, our study advocates that RET/PTC3 rearrangement is a frequent event in the carcinogenesis of thyroid gland in Kashmiri population although a study with a larger sample size is needed to get a clear scenario.

Assessment of a New ROS1 Immunohistochemistry Clone (SP384) for the Identification of ROS1 Rearrangements in Patients with Non-Small Cell Lung Carcinoma: the ROSING Study.

INTRODUCTION: The ROS1 gene rearrangement has become an important biomarker in NSCLC. The College of American Pathologists/International Association for the Study of Lung Cancer/Association for Molecular Pathology testing guidelines support the use of ROS1 immunohistochemistry (IHC) as a screening test, followed by confirmation with fluorescence in situ hybridization (FISH) or a molecular test in all positive results. We have evaluated a novel anti-ROS1 IHC antibody (SP384) in a large multicenter series to obtain real-world data. METHODS: A total of 43 ROS1 FISH-positive and 193 ROS1 FISH-negative NSCLC samples were studied. All specimens were screened by using two antibodies (clone D4D6 from Cell Signaling Technology and clone SP384 from Ventana Medical Systems), and the different interpretation criteria were compared with break-apart FISH (Vysis). FISH-positive samples were also analyzed with next-generation sequencing (Oncomine Dx Target Test Panel, Thermo Fisher Scientific). RESULTS: An H-score of 150 or higher or the presence of at least 70% of tumor cells with an intensity of staining of 2+ or higher by the SP384 clone was the optimal cutoff value (both with 93% sensitivity and 100% specificity). The D4D6 clone showed similar results, with an H-score of at least 100 (91% sensitivity and 100% specificity). ROS1 expression in normal lung was more frequent with use of the SP384 clone (p < 0.0001). The ezrin gene (EZR)-ROS1 variant was associated with membranous staining and an isolated green signal FISH pattern (p = 0.001 and p = 0.017, respectively). CONCLUSIONS: The new SP384 ROS1 IHC clone showed excellent sensitivity without compromising specificity, so it is another excellent analytical option for the proposed testing algorithm.

Chaihu-Shugan-San and absorbed meranzin hydrate induce anti-atherosclerosis and behavioral improvements in high-fat diet ApoE-/- mice via anti-inflammatory and BDNF-TrkB pathway.

The comorbidity of coronary heart disease (CHD) and depression in patients is extremely prevalent, with a rate from 20 to 50%, while depression-like behaviors exist in a larger percentage of patients. Therefore, the study of comorbidities is particularly urgent. Chaihu-Shugan-San (CSS), a classical traditional Chinese medicine formula, has been used to treat CHD with depression for hundreds of years. However, the mechanism of its action on comorbidities remains unclear. Here, we focused on the behavioral changes in ApoE-/- mice fed a high-fat diet (HFD) and elucidated the mechanism of CSS and its main absorbed component, meranzin hydrate (MH), as an anti-atherosclerosis and anti-depression agent. In the present study, mice were fed an HFD for 16 weeks and were intragastrically administered high and low doses of CSS and MH. Depressive-like behaviors were evaluated by the sucrose preference test, the open-field test, the light-dark test and the tail-suspension test, after which atherosclerotic plaques, plasma lipids, inflammatory cytokine levels and the expression of BDNF/TrkB were measured. We demonstrated that the atherosclerosis model group exhibited significant depressant behaviors. Moreover, CSS inhibited depressive-like behavioral changes, reduced atherosclerotic plaque areas, plasma total cholesterol, triglycerides, LDL-cholesterol levels and inflammatory cytokines including TNF-α, IL-1ß, and IL-6 in plasma and hippocampi, increased the protein and mRNA expression of BDNF and TrkB in the aorta and the hippocampus. Interestingly, MH, the main component in CSS that is absorbed in the plasma and hippocampus, exerted effects similar to those of CSS. In addition, MH increased the protein and mRNA expression of BDNF and TrkB in human umbilical vein endothelial cells (HUVECs) induced by LPS. Collectively, these results suggest that MH represents the CSS decoction, induces anti-atherosclerosis effects and improves depression-like behaviors in HFD-fed ApoE-/- mice. Moreover, the regulation of proinflammatory factors and BDNF-TrkB signaling are possibly involved in this process. Our findings indicate that MH is a potential phytochemical compound for the prevention of the comorbidity of atherosclerosis and depression.

[Precision medicine: A major step forward in specific situations, a myth in refractory cancers?] / Médecine de précision : une avancée majeure dans des situations spécifiques, un mythe dans les cancers réfractaires ?

In recent years, high-throughput sequencing techniques have been developed for cancerology and many clinical trials are currently structured around biomarkers that can guide specific treatment choices. This approach is characteristic of precision medicine, which is actually a concept initiated several decades ago with, for example, retinoic acid in promyelocytic leukemia. This paper will review the different types of molecular alterations and « -omics ¼ biological analyses, bioinformatics tools, coupled drug/biomarkers already validated, the ethical issues of whole genomic sequencing of an individual as part of an inclusion in a clinical trial and finally the first results of precision medicine trials. The AcSé crizotinib program, supported by the Inca (french Cancer National Institute), is emblematic of a success of this personalized medicine illustrated by 4 points: the discovery of a cohort of patients with lung cancer with a ROS1 rearrangement characteristic of a sensitivity to crizotinib, a rapid availability of this innovation through the implementation of a temporary recommendation for use (ANSM), the obtention of a conditional marketing authorization by the pharmaceutical industry and finally, financial assumption of responsibility by French social security (HAS), despite preliminary and non-comparative data. In the case of cancers refractory to standard chemotherapy, and regarding our system of access to drugs illustrated by the PROFILER clinical trial, this approach allows the access to a therapeutic drug targeting specific biomarkers only in 7% of patients included. This does not bode well for efficient treatment and even less for survival. Allowing patients to be included in trials that identify molecular targets by molecular screening, and not being able to propose the drug of interest is a traumatic event for those patients who live in the hope of an immediate future. In refractory disease we must rethink precision medicine in a more humanistic vision for our patients and not only in a dimension of medico-industrial promotion. The implementation of a new multi-drug/multi-molecular target program could address this issue.

Economic analysis of BRAF gene mutation testing in real world practice using claims data: costs of single gene versus panel tests in patients with lung cancer.

AIMS: To assess the time to BRAF testing, compare the characteristics of tested vs not-tested patients, and describe the costs for sequential vs next-generation sequencing (NGS) BRAF testing. METHODS: Patients diagnosed with lung cancer after December 1, 2013 were identified from two US claims databases; their characteristics were assessed during the 12 months before diagnosis (index date). Testing modalities were analyzed from the index date to end of continuous health plan enrollment or data availability (December 2015), based on combinations of Current Procedural Terminology (CPT) procedure codes. Time to BRAF testing was assessed using Kaplan-Meier analysis. Costs were analyzed from a payer's perspective. RESULTS: A total of 28,011 patients newly-diagnosed with lung cancer were identified. Of them, 1,260 (4.5%) were tested for BRAF: 3.2% and 4.2% were tested at 6 and 12 months, respectively, after the index date. Compared to non-tested patients, tested patients were younger (58.3 vs 65.3 years; p < .001), had a lower Charlson Comorbidity Index (2.8 vs 2.9; p = .005), and a higher proportion had metastases (70.9% vs 43.4%; p < .001). In 76.0% of cases, BRAF was tested along with KRAS. BRAF was tested using NGS in 6.6% of cases. The average reimbursed amounts for the 10 most common CPT code combinations were $207-$2,074. Using the average costs of individual mutation tests, the total cost of sequential testing comprising KRAS, EGFR, ALK, ROS1, and BRAF tests was $3,763 ($464, $696, $1,070, $1,127, and $406, respectively), that of NGS was $2,860. LIMITATIONS: Claims data did not include BRAF test results. CONCLUSIONS: Among patients newly-diagnosed with lung cancer, 4.5% were tested for BRAF. Tested patients were younger and had a lower comorbidity burden, but more advanced disease. While reimbursed amounts varied greatly based on combinations of testing procedures, NGS testing was associated with cost savings compared to sequential testing of individual mutations.

Ligation-dependent RT-PCR: a new specific and low-cost technique to detect ALK, ROS, and RET rearrangements in lung adenocarcinoma.

Detection of anaplastic lymphoma kinase (ALK), ROS proto-oncogene 1 (ROS1), and rearranged during transfection (RET) gene rearrangements in lung adenocarcinoma is usually performed by immunohistochemistry (IHC) screening followed by fluorescence in situ hybridization (FISH), which is an expensive and difficult technique. Ligation-dependent reverse transcription polymerase chain reaction (RT-PCR) multiplex technique can detect gene rearrangements using probes specifically hybridized to either side of the break point. PCR products are then sequenced by pyrosequencing or high throughput sequencing in order to identify the two genes involved. The reagent cost is <15 dollars per patient and results are available in 2 days. We have developed a 47-probe LD-RT-PCR kit especially for lung adenocarcinomas. Thirty-nine lung adenocarcinomas were studied: 24 ALK+, 14 ROS1+, and 1 RET+. ALK+ and ROS1+ were IHC+ (D5F3 Ventana for ALK and D4D6 Cell Signaling Technology for ROS1) and all cases were FISH+ (Vysis ALK Breakapart Probe Abbott for ALK, Zytolight SPEC ROS1 Dualcolor Breakapart Probe for ROS1 and Zytolight SPEC RET Dual Color Breakapart for RET); 14 wild type samples were included as negative controls. Using LD-RT-PCR, 15 rearrangements (63%) were detected in the ALK cases (gene partner: EML4 in all cases), 9 rearrangements (64%) in the ROS1 cases (gene partners: CD74 in 8 cases and SLC34A2 in 1 case) and 1 (100%) in the single RET case (gene partner: KIF5B). No rearrangement was found in the 14 negative control cases. Negative cases using LD-RT-PCR could be explained by the fact that some partner genes were not included in our assay and therefore could not be detected. Because it is an affordable, fast, and very simple technique, we propose using LD-RT-PCR when ALK immunostaining is positive. For LD-RT-PCR-negative cases, samples should then be analyzed by FISH.

Kinase impact assessment in the landscape of fusion genes that retain kinase domains: a pan-cancer study.

Assessing the impact of kinase in gene fusion is essential for both identifying driver fusion genes (FGs) and developing molecular targeted therapies. Kinase domain retention is a crucial factor in kinase fusion genes (KFGs), but such a systematic investigation has not been done yet. To this end, we analyzed kinase domain retention (KDR) status in chimeric protein sequences of 914 KFGs covering 312 kinases across 13 major cancer types. Based on 171 kinase domain-retained KFGs including 101 kinases, we studied their recurrence, kinase groups, fusion partners, exon-based expression depth, short DNA motifs around the break points and networks. Our results, such as more KDR than 5'-kinase fusion genes, combinatorial effects between 3'-KDR kinases and their 5'-partners and a signal transduction-specific DNA sequence motif in the break point intronic sequences, supported positive selection on 3'-kinase fusion genes in cancer. We introduced a degree-of-frequency (DoF) score to measure the possible number of KFGs of a kinase. Interestingly, kinases with high DoF scores tended to undergo strong gene expression alteration at the break points. Furthermore, our KDR gene fusion network analysis revealed six of the seven kinases with the highest DoF scores (ALK, BRAF, MET, NTRK1, NTRK3 and RET) were all observed in thyroid carcinoma. Finally, we summarized common features of 'effective' (highly recurrent) kinases in gene fusions such as expression alteration at break point, redundant usage in multiple cancer types and 3'-location tendency. Collectively, our findings are useful for prioritizing driver kinases and FGs and provided insights into KFGs' clinical implications.

Variations in COMT and NTRK2 Influence Symptom Burden in Women Undergoing Breast Cancer Treatment.

Women with breast cancer frequently report distressing symptoms during and after treatment that can significantly erode quality of life (QOL). Symptom burden among women with breast cancer is of complex etiology and is likely influenced by disease, treatment, and environmental factors as well as individual genetic differences. The purpose of the present study was to examine the relationships between genetic polymorphisms within Neurotrophic tyrosine kinase receptor 1 (NTRK1), Neurotrophic tyrosine kinase receptor 2 (NTRK2), and catechol-O-methyltransferase ( COMT) and patient symptom burden of QOL, pain, fatigue, anxiety, depression, and sleep disturbance before, during, and after treatment for breast cancer in a subset of participants ( N = 51) in a randomized clinical trial of a novel symptom-management modality for women with breast cancer undergoing chemotherapy. Patients were recruited at the time of initial breast cancer diagnosis and completed all survey measures at the time of recruitment, after the initiation of treatment (surgery and/or chemotherapy), and then following treatment conclusion. Multiple linear regression analyses revealed significant associations between NTRK2 and COMT single nucleotide polymorphism (SNP) genotype and symptom burden. Two COMT variants were associated with the specific symptoms of anxiety and QOL measures prior to the initiation of chemotherapy as well as pain interference and severity during and after treatment. Genotype at the NTRK2 SNP rs1212171 was associated with both sleep disturbance and fatigue. These findings, while exploratory, indicate that the genotypes of NTRK2 and COMT may contribute to relative risk for symptom burden during and shortly after the period of chemotherapy in women with early stage breast cancer.

Classification and Lineage Tracing of SH2 Domains Throughout Eukaryotes.

Today there exists a rapidly expanding number of sequenced genomes. Cataloging protein interaction domains such as the Src Homology 2 (SH2) domain across these various genomes can be accomplished with ease due to existing algorithms and predictions models. An evolutionary analysis of SH2 domains provides a step towards understanding how SH2 proteins integrated with existing signaling networks to position phosphotyrosine signaling as a crucial driver of robust cellular communication networks in metazoans. However organizing and tracing SH2 domain across organisms and understanding their evolutionary trajectory remains a challenge. This chapter describes several methodologies towards analyzing the evolutionary trajectory of SH2 domains including a global SH2 domain classification system, which facilitates annotation of new SH2 sequences essential for tracing the lineage of SH2 domains throughout eukaryote evolution. This classification utilizes a combination of sequence homology, protein domain architecture and the boundary positions between introns and exons within the SH2 domain or genes encoding these domains. Discrete SH2 families can then be traced across various genomes to provide insight into its origins. Furthermore, additional methods for examining potential mechanisms for divergence of SH2 domains from structural changes to alterations in the protein domain content and genome duplication will be discussed. Therefore a better understanding of SH2 domain evolution may enhance our insight into the emergence of phosphotyrosine signaling and the expansion of protein interaction domains.