A key constraint in genomic testing in oncology is that matched normal specimens are not commonly obtained in clinical practice. Thus, while well-characterized genomic alterations do not require ...normal tissue for interpretation, a significant number of alterations will be unknown in whether they are germline or somatic, in the absence of a matched normal control. We introduce SGZ (somatic-germline-zygosity), a computational method for predicting somatic vs. germline origin and homozygous vs. heterozygous or sub-clonal state of variants identified from deep massively parallel sequencing (MPS) of cancer specimens. The method does not require a patient matched normal control, enabling broad application in clinical research. SGZ predicts the somatic vs. germline status of each alteration identified by modeling the alteration's allele frequency (AF), taking into account the tumor content, tumor ploidy, and the local copy number. Accuracy of the prediction depends on the depth of sequencing and copy number model fit, which are achieved in our clinical assay by sequencing to high depth (>500x) using MPS, covering 394 cancer-related genes and over 3,500 genome-wide single nucleotide polymorphisms (SNPs). Calls are made using a statistic based on read depth and local variability of SNP AF. To validate the method, we first evaluated performance on samples from 30 lung and colon cancer patients, where we sequenced tumors and matched normal tissue. We examined predictions for 17 somatic hotspot mutations and 20 common germline SNPs in 20,182 clinical cancer specimens. To assess the impact of stromal admixture, we examined three cell lines, which were titrated with their matched normal to six levels (10-75%). Overall, predictions were made in 85% of cases, with 95-99% of variants predicted correctly, a significantly superior performance compared to a basic approach based on AF alone. We then applied the SGZ method to the COSMIC database of known somatic variants in cancer and found >50 that are in fact more likely to be germline.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Microsatellite instability (MSI) is an important biomarker for predicting response to immune checkpoint inhibitor therapy, as emphasized by the recent checkpoint inhibitor approval for MSI-high ...(MSI-H) solid tumors. Herein, we describe and validate a novel method for determining MSI status from a next-generation sequencing comprehensive genomic profiling assay using formalin-fixed, paraffin-embedded samples. This method is 97% (65/67) concordant with current standards, PCR and immunohistochemistry. We further apply this method to >67,000 patient tumor samples to identify genes and pathways that are enriched in MSI-stable or MSI-H tumor groups. Data show that although rare in tumors other than colorectal and endometrial carcinomas, MSI-H samples are present in many tumor types. Furthermore, the large sample set revealed that MSI-H tumors selectively share alterations in genes across multiple common pathways, including WNT, phosphatidylinositol 3-kinase, and NOTCH. Last, MSI is sufficient, but not necessary, for a tumor to have elevated tumor mutation burden. Therefore, MSI can be determined from comprehensive genomic profiling with high accuracy, allowing for efficient MSI-H detection across all tumor types, especially those in which routine use of immunohistochemistry or PCR-based assays would be impractical because of a rare incidence of MSI. MSI-H tumors are enriched in alterations in specific signaling pathways, providing a rationale for investigating directed immune checkpoint inhibitor therapies in combination with pathway-targeted therapies.
Tumor-infiltrating lymphocytes (TIL) in the residual disease (RD) of triple-negative breast cancers (TNBC) after neoadjuvant chemotherapy (NAC) are associated with improved survival, but insight into ...tumor cell-autonomous molecular pathways affecting these features are lacking.
We analyzed TILs in the RD of clinically and molecularly characterized TNBCs after NAC and explored therapeutic strategies targeting combinations of MEK inhibitors with PD-1/PD-L1-targeted immunotherapy in mouse models of breast cancer.
Presence of TILs in the RD was significantly associated with improved prognosis. Genetic or transcriptomic alterations in Ras-MAPK signaling were significantly correlated with lower TILs. MEK inhibition upregulated cell surface MHC expression and PD-L1 in TNBC cells both in vivo and in vitro. Moreover, combined MEK and PD-L1/PD-1 inhibition enhanced antitumor immune responses in mouse models of breast cancer.
These data suggest the possibility that Ras-MAPK pathway activation promotes immune-evasion in TNBC, and support clinical trials combining MEK- and PD-L1-targeted therapies. Furthermore, Ras/MAPK activation and MHC expression may be predictive biomarkers of response to immune checkpoint inhibitors.
In this work, we report a method to acquire and analyze hyperspectral coherent anti-Stokes Raman scattering (CARS) microscopy images of organic materials and biological samples resulting in an ...unbiased quantitative chemical analysis. The method employs singular value decomposition on the square root of the CARS intensity, providing an automatic determination of the components above noise, which are retained. Complex CARS susceptibility spectra, which are linear in the chemical composition, are retrieved from the CARS intensity spectra using the causality of the susceptibility by two methods, and their performance is evaluated by comparison with Raman spectra. We use non-negative matrix factorization applied to the imaginary part and the nonresonant real part of the susceptibility with an additional concentration constraint to obtain absolute susceptibility spectra of independently varying chemical components and their absolute concentration. We demonstrate the ability of the method to provide quantitative chemical analysis on known lipid mixtures. We then show the relevance of the method by imaging lipid-rich stem-cell-derived mouse adipocytes as well as differentiated embryonic stem cells with a low density of lipids. We retrieve and visualize the most significant chemical components with spectra given by water, lipid, and proteins segmenting the image into the cell surrounding, lipid droplets, cytosol, and the nucleus, and we reveal the chemical structure of the cells, with details visualized by the projection of the chemical contrast into a few relevant channels.
The uptake of microfluidics by the wider scientific community has been limited by the fabrication barrier created by the skills and equipment required for the production of traditional microfluidic ...devices. Here we present simple 3D printed microfluidic devices using an inexpensive and readily accessible printer with commercially available printer materials. We demonstrate that previously reported limitations of transparency and fidelity have been overcome, whilst devices capable of operating at pressures in excess of 2000 kPa illustrate that leakage issues have also been resolved. The utility of the 3D printed microfluidic devices is illustrated by encapsulating dental pulp stem cells within alginate droplets; cell viability assays show the vast majority of cells remain live, and device transparency is sufficient for single cell imaging. The accessibility of these devices is further enhanced through fabrication of integrated ports and by the introduction of a Lego®-like modular system facilitating rapid prototyping whilst offering the potential for novices to build microfluidic systems from a database of microfluidic components.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Targetable oncogenic alterations are detected more commonly in patients with non-small cell lung cancer (NSCLC) who never smoked cigarettes. For such patients, specific kinase inhibitors have emerged ...as effective clinical treatments. However, the currently known oncogenic alterations do not account for all never smokers who develop NSCLC. We sought to identify additional oncogenic alterations from patients with NSCLC to define additional treatment options.
We analyzed 576 lung adenocarcinomas from patients of Asian and Caucasian ethnicity. We identified a subset of cancers that did not harbor any known oncogenic alteration. We performed targeted next-generation sequencing (NGS) assay on 24 patients from this set with >75% tumor cell content.
EGFR mutations were the most common oncogenic alteration from both Asian (53%) and Caucasian (41.6%) patients. No known oncogenic alterations were present in 25.7% of Asian and 31% of Caucasian tumor specimens. We identified a FGFR3-TACC3 fusion event in one of 24 patients from this subset using targeted NGS. Two additional patients harboring FGFR3-TACC3 were identified by screening our entire cohort (overall prevalence, 0.5%). Expression of FGFR3-TACC3 led to IL3 independent growth in Ba/F3 cells. These cells were sensitive to pan-fibroblast growth factor receptor (pan-FGFR) inhibitors but not the epidermal growth factor (EGFR) inhibitor gefitinib.
FGFR3-TACC3 rearrangements occur in a subset of patients with lung adenocarcinoma. Such patients should be considered for clinical trials featuring FGFR inhibitors.
Background
Parathyroid carcinoma (PC) is a rare endocrine malignancy that can cause life‐threatening hypercalcemia. We queried whether comprehensive genomic profiling (CGP) of PC might identify ...genomic alterations (GAs), which would suggest benefit from rationally matched therapeutics.
Methods
We performed hybrid‐capture‐based CGP to identify GAs and tumor mutational burden (TMB) in tumors from patients with this malignancy.
Results
There were 85 total GAs in 16 cases (5.3 GAs per case), and the median TMB was 1.7 mutations per megabase (m/Mb), with three cases having >20 m/Mb (18.7%). The genes most frequently harboring GA were CDC73 (38%), TP53 (38%), and MEN1 (31%). All MEN1‐mutated cases also had loss of heterozygosity at that locus, but in contrast all CDC73‐mutated cases retained heterozygosity. GAs suggesting potential benefit from matched targeted therapy were identified in 11 patients (69%) and most frequently found in PTEN (25%), NF1 (12.5%), KDR (12.5%), PIK3CA (12.5%), and TSC2 (12.5%). A patient whose tumor harbored KDR T668 K and who was treated with cabozantinib experienced a > 50% drop in parathyroid hormone level and radiographic partial response of 5.4 months with duration limited by toxicity.
Conclusion
CGP identified GAs in PC that suggest benefit from targeted therapy, as supported by an index case of response to a matched tyrosine kinase inhibitor. Moreover, the unexpectedly high frequency of high TMB (>20 m/Mb) suggests a subset of PC may benefit from immune checkpoint inhibitors.
Implications for Practice
Parathyroid carcinoma (PC) is a rare endocrine malignancy that can cause life‐threatening hypercalcemia. However, its molecular characteristics remain unclear, with few systemic therapeutic options available for this tumor. Hybrid‐capture‐based comprehensive genomic profiling of 16 primary cancers demonstrated presence of potentially actionable genomic alterations, including PTEN, NF1, KDR, PIK3CA, and TSC2, and a subset of hypermutated cancers with more than 20 mutations per megabase, the latter of which could benefit from immune checkpoint inhibitor therapy. A case benefiting from rationally matched targeted therapy for activating KDR mutation is also presented. These findings should be further investigated for their therapeutic potential.
Results from comprehensive genomic profiling in a cohort of patients with advanced parathyroid carcinoma are reported.
Introduction.
For patients with non‐small cell lung cancer (NSCLC) to benefit from ALK inhibitors, sensitive and specific detection of ALK genomic rearrangements is needed. ALK break‐apart ...fluorescence in situ hybridization (FISH) is the U.S. Food and Drug Administration approved and standard‐of‐care diagnostic assay, but identification of ALK rearrangements by other methods reported in NSCLC cases that tested negative for ALK rearrangements by FISH suggests a significant false‐negative rate. We report here a large series of NSCLC cases assayed by hybrid‐capture‐based comprehensive genomic profiling (CGP) in the course of clinical care.
Materials and Methods.
Hybrid‐capture‐based CGP using next‐generation sequencing was performed in the course of clinical care of 1,070 patients with advanced lung cancer. Each tumor sample was evaluated for all classes of genomic alterations, including base‐pair substitutions, insertions/deletions, copy number alterations and rearrangements, as well as fusions/rearrangements.
Results.
A total of 47 patients (4.4%) were found to harbor ALK rearrangements, of whom 41 had an EML4‐ALK fusion, and 6 had other fusion partners, including 3 previously unreported rearrangement events: EIF2AK‐ALK, PPM1B‐ALK, and PRKAR1A‐ALK. Of 41 patients harboring ALK rearrangements, 31 had prior FISH testing results available. Of these, 20 were ALK FISH positive, and 11 (35%) were ALK FISH negative. Of the latter 11 patients, 9 received crizotinib based on the CGP results, and 7 achieved a response with median duration of 17 months.
Conclusion.
Comprehensive genomic profiling detected canonical ALK rearrangements and ALK rearrangements with noncanonical fusion partners in a subset of patients with NSCLC with previously negative ALK FISH results. In this series, such patients had durable responses to ALK inhibitors, comparable to historical response rates for ALK FISH‐positive cases.
Implications for Practice:
Comprehensive genomic profiling (CGP) that includes hybrid capture and specific baiting of intron 19 of ALK is a highly sensitive, alternative method for identification of drug‐sensitive ALK fusions in patients with non‐small cell lung cancer (NSCLC) who had previously tested negative using standard ALK fluorescence in situ hybridization (FISH) diagnostic assays. Given the proven benefit of treatment with crizotinib and second‐generation ALK inhibitors in patients with ALK fusions, CGP should be considered in patients with NSCLC, including those who have tested negative for other alterations, including negative results using ALK FISH testing.
摘要
引言. 敏感且特异性地检测到ALK基因组重排为能够从ALK抑制剂治疗获益的非小细胞肺癌 (NSCLC) 患者所必需。ALK分离荧光原位杂交 (FISH) 是获得美国食品和药物管理局批准的标准诊断方法, 但使用其他方法在ALK重排FISH阴性的NSCLC病例中发现了ALK重排, 提示这种方法的假阴性率相当高。本文报告了在临床治疗过程中采用基于杂交捕获法的综合基因组分析 (CGP) 在一个NSCLC病例大型队列中进行检测的结果。
材料与方法. 临床诊疗过程中, 在1 070例晚期肺癌患者中使用应用下一代测序技术的基于杂交捕获法的CGP。对每个肿瘤标本都进行所有种类基因改变的评价, 包括碱基替换、插入/缺失、拷贝数改变和重排, 以及融合/重排。
结果. 共发现47例患者 (4.4%) 携带ALK重排, 其中41例有EML4‐ALK融合, 其余6例为其他融合配偶体, 包括3例过去未报告的重排事件: EIF2AK‐ALK、PPM1B‐ALK和PRKAR1A‐ALK。在41例携带ALK重排的患者中, 31例有既往FISH检验结果。其中20例为ALK FISH阳性, 11例 (35%) 为ALK FISH阴性。在后面这11例患者中, 9例依据CGP结果接受了克唑替尼治疗, 其中7例中位缓解时间达到17个月。
结论. 综合基因组分析在既往ALK FISH检验结果为阴性的NSCLC亚组患者中发现了经典ALK重排和非经典融合配偶体。在这一病例系列中, 此类患者对ALK抑制剂治疗产生了持久应答, 缓解率与ALK FISH阳性病例的历史缓解率相当。The Oncologist 2016;21:762–770
对临床实践的提示: 综合基因组分析 (CGP) 包括杂交捕获法以及 ALK 内含子 19 的特异性诱饵, 是在使用标准 ALK 荧光原位杂交法 (FISH) 诊断为阴性的非小细胞肺癌 (NSCLC) 患者中, 鉴别药物敏感性 ALK 融合的可选方法。鉴于已证实克唑替尼和第二代 ALK 抑制剂治疗在 ALK 融合患者中的获益, 应考虑对 NSCLC 患者使用 CGP 检测, 包括那些其他可选方法检验结果阴性的患者 (也包括使用 ALK FISH 检验结果阴性的患者)。
Of a subset of 41 patients with advanced non‐small cell lung cancer with ALK rearrangements detected by hybrid capture‐based comprehensive genomic profiling, 11 previously had tested negative by ALK break‐apart fluorescence in situ hybridization. Of these 11 patients, 9 were treated with crizotinib based on comprehensive genomic profiling results; 7 of these achieved a median response duration of 17 months.
Purpose
Amplifications of receptor tyrosine kinases (RTKS) are therapeutic targets in multiple tumor types (e.g. HER2 in breast cancer), and amplification of the chromosome 4 segment harboring the ...three RTKs KIT, PDGFRA, and KDR (4q12amp) may be similarly targetable. The presence of 4q12amp has been sporadically reported in small tumor specific series but a large‐scale analysis is lacking. We assess the pan‐cancer landscape of 4q12amp and provide early clinical support for the feasibility of targeting this amplicon.
Experimental Design
Tumor specimens from 132,872 patients with advanced cancer were assayed with hybrid capture based comprehensive genomic profiling which assays 186–315 genes for all classes of genomic alterations, including amplifications. Baseline demographic data were ed, and presence of 4q12amp was defined as 6 or more copies of KIT/KDR/PDGFRA. Concurrent alterations and treatment outcomes with matched therapies were explored in a subset of cases.
Results
Overall 0.65% of cases harbored 4q12amp at a median copy number of 10 (range 6–344). Among cancers with >100 cases in this series, glioblastomas, angiosarcomas, and osteosarcomas were enriched for 4q12amp at 4.7%, 4.8%, and 6.4%, respectively (all p < 0.001), giving an overall sarcoma (n = 6,885) incidence of 1.9%. Among 99 pulmonary adenocarcinoma cases harboring 4q12amp, 50 (50%) lacked any other known driver of NSLCC. Four index cases plus a previously reported case on treatment with empirical TKIs monotherapy had stable disease on average exceeding 20 months.
Conclusion
We define 4q12amp as a significant event across the pan‐cancer landscape, comparable to known pan‐cancer targets such as NTRK and microsatellite instability, with notable enrichment in several cancers such as osteosarcoma where standard treatment is limited. The responses to available TKIs observed in index cases strongly suggest 4q12amp is a druggable oncogenic target across cancers that warrants a focused drug development strategy.
Implications for Practice
Coamplification of the receptor tyrosine kinases (rtks) KIT/KDR/PDGFRA (4q12amp) is present broadly across cancers (0.65%), with enrichment in osteosarcoma and gliomas. Evidence for this amplicon having an oncogenic role is the mutual exclusivity of 4q12amp to other known drivers in 50% of pulmonary adenocarcinoma cases. Furthermore, preliminary clinical evidence for driver status comes from four index cases of patients empirically treated with commercially available tyrosine kinase inhibitors with activity against KIT/KDR/PDGFRA who had stable disease for 20 months on average. The sum of these lines of evidence suggests further clinical and preclinical investigation of 4q12amp is warranted as the possible basis for a pan‐cancer drug development strategy.
Matching treatment strategy to tumor biology is central to precision medicine. The co‐amplification of three distinct RTK encoding genes (KIT, KDR, and PDGFRA) on chromosome 4q12 (4q12amp) may be an oncogenic driver and thus a target for therapy. This article assesses the pan‐cancer landscape of 4q12amp and provides clinical support for the feasibility of targeting this amplicon.
Chronic skin-healing defects are one of the leading challenges to lifelong well-being, affecting 2–5% of populations. Chronic wound formation is linked to age and diabetes and frequently leads to ...major limb amputation. Here we identify a strategy to reverse fibroblast senescence and improve healing rates. In healthy skin, fibronectin activates Rac1 in fibroblasts, causing migration into the wound bed, and driving wound contraction. We discover that mechanical stimulation of the skin with ultrasound can overturn healing defects by activating a calcium/CamKinaseII/Tiam1/Rac1 pathway that substitutes for fibronectin-dependent signaling and promotes fibroblast migration. Treatment of diabetic and aged mice recruits fibroblasts to the wound bed and reduces healing times by 30%, restoring healing rates to those observed in young, healthy animals. Ultrasound treatment is equally effective in rescuing the healing defects of animals lacking fibronectin receptors, and can be blocked by pharmacological inhibition of the CamKinaseII pathway. Finally, we discover that the migration defects of fibroblasts from human venous leg ulcer patients can be reversed by ultrasound, demonstrating that the approach is applicable to human chronic samples. By demonstrating that this alternative Rac1 pathway can substitute for that normally operating in the skin, we identify future opportunities for management of chronic wounds.
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