In the past decade, advances in precision oncology have resulted in an increased demand for predictive assays that enable the selection and stratification of patients for treatment. The enormous ...divergence of signalling and transcriptional networks mediating the crosstalk between cancer, stromal and immune cells complicates the development of functionally relevant biomarkers based on a single gene or protein. However, the result of these complex processes can be uniquely captured in the morphometric features of stained tissue specimens. The possibility of digitizing whole-slide images of tissue has led to the advent of artificial intelligence (AI) and machine learning tools in digital pathology, which enable mining of subvisual morphometric phenotypes and might, ultimately, improve patient management. In this Perspective, we critically evaluate various AI-based computational approaches for digital pathology, focusing on deep neural networks and 'hand-crafted' feature-based methodologies. We aim to provide a broad framework for incorporating AI and machine learning tools into clinical oncology, with an emphasis on biomarker development. We discuss some of the challenges relating to the use of AI, including the need for well-curated validation datasets, regulatory approval and fair reimbursement strategies. Finally, we present potential future opportunities for precision oncology.
Immune-checkpoint inhibitors (ICI), particularly inhibitors of the PD-1 axis, have altered the management of non-small cell lung cancer (NSCLC) over the last 10 years. First demonstrated to improve ...outcomes in second-line or later therapy of advanced disease, ICIs were shown to improve overall survival compared with chemotherapy in first-line therapy for patients whose tumors express PD-L1 on at least 50% of cells. More recently, combining ICIs with chemotherapy has been shown to improve survival in patients with both squamous and nonsquamous NSCLC, regardless of PD-L1 expression. However, PD-L1 and, more recently, tumor mutational burden have not proven to be straightforward indicative biomarkers. We describe the advances to date in utilizing these biomarkers, as well as novel markers of tumor inflammation, to ascertain which patients are most likely to benefit from ICIs. Ongoing translational work promises to improve the proportion of patients who benefit from these agents.
To update key recommendations of the American Society of Clinical Oncology/College of American Pathologists estrogen (ER) and progesterone receptor (PgR) testing in breast cancer guideline.
A ...multidisciplinary international Expert Panel was convened to update the clinical practice guideline recommendations informed by a systematic review of the medical literature.
The Expert Panel continues to recommend ER testing of invasive breast cancers by validated immunohistochemistry as the standard for predicting which patients may benefit from endocrine therapy, and no other assays are recommended for this purpose. Breast cancer samples with 1% to 100% of tumor nuclei positive should be interpreted as ER positive. However, the Expert Panel acknowledges that there are limited data on endocrine therapy benefit for cancers with 1% to 10% of cells staining ER positive. Samples with these results should be reported using a new reporting category, ER Low Positive, with a recommended comment. A sample is considered ER negative if < 1% or 0% of tumor cell nuclei are immunoreactive. Additional strategies recommended to promote optimal performance, interpretation, and reporting of cases with an initial low to no ER staining result include establishing a laboratory-specific standard operating procedure describing additional steps used by the laboratory to confirm/adjudicate results. The status of controls should be reported for cases with 0% to 10% staining. Similar principles apply to PgR testing, which is used primarily for prognostic purposes in the setting of an ER-positive cancer. Testing of ductal carcinoma in situ (DCIS) for ER is recommended to determine potential benefit of endocrine therapies to reduce risk of future breast cancer, while testing DCIS for PgR is considered optional. Additional information can be found at www.asco.org/breast-cancer-guidelines.
The presence of a high degree of tumor-infiltrating lymphocytes (TIL) has been proven to be associated with outcome in patients with non-small cell lung cancer (NSCLC). However, recent evidence ...indicates that tissue architecture is also prognostic of disease-specific survival and recurrence. We show a set of descriptors (spatial TIL, SpaTIL) that capture density, and spatial colocalization of TILs and tumor cells across digital images that can predict likelihood of recurrence in early-stage NSCLC.
The association between recurrence in early-stage NSCLC and SpaTIL features was explored on 301 patients across four different cohorts. Cohort D
(
= 70) was used to identify the most prognostic SpaTIL features and to train a classifier to predict the likelihood of recurrence. The classifier performance was evaluated in cohorts D
(
= 119), D
(
= 112), and D
(
= 112). Two pathologists graded each sample of D
and D
; intraobserver agreement and association between manual grading and likelihood of recurrence were analyzed.
SpaTIL was associated with likelihood of recurrence in all test sets (log-rank
< 0.02). A multivariate Cox proportional hazards analysis revealed an HR of 3.08 (95% confidence interval, 2.1-4.5,
= 7.3 × 10
). In contrast, agreement among expert pathologists using tumor grade was moderate (Kappa = 0.5), and the manual TIL grading was only prognostic for one reader in D
(
= 8.0 × 10
).
A set of features related to density and spatial architecture of TILs was found to be associated with a likelihood of recurrence of early-stage NSCLC. This information could potentially be used for helping in treatment planning and management of early-stage NSCLC.
.
Many tumor cells are fueled by altered metabolism and increased glutamine (Gln) dependence. We identify regulation of the L-glutamine carrier proteins SLC1A5 and SLC38A2 (SLC1A5/38A2) by the ...ubiquitin ligase RNF5. Paclitaxel-induced ER stress to breast cancer (BCa) cells promotes RNF5 association, ubiquitination, and degradation of SLC1A5/38A2. This decreases Gln uptake, levels of TCA cycle components, mTOR signaling, and proliferation while increasing autophagy and cell death. Rnf5-deficient MMTV-PyMT mammary tumors were less differentiated and showed elevated SLC1A5 expression. Whereas RNF5 depletion in MDA-MB-231 cells promoted tumorigenesis and abolished paclitaxel responsiveness, SLC1A5/38A2 knockdown elicited opposing effects. Inverse RNF5hi/SLC1A5/38A2lo expression was associated with positive prognosis in BCa. Thus, RNF5 control of Gln uptake underlies BCa response to chemotherapies.
Display omitted
•Gln carrier proteins SLC1A5 and SLC38A2 are regulated by ubiquitin ligase RNF5•Glutamine uptake and mTOR activity following ERS is RNF5-SLC1A5 dependent•RNF5 inhibition of SLC1A5 is required for paclitaxel-induced apoptosis of BCa cells•Low SLC1A5 expression in BCa TMA and RPPA associates with good prognosis
Jeon et al. show that paclitaxel-induced ER stress in BCa cells promotes the ubiquitin ligase RNF5 to associate with, ubiquitinate, and degrade the Gln carriers SLC1A5 and SLC38A2, thereby leading to decreased Gln uptake and increased autophagy and cell death.
A proposal for validation of antibodies Uhlen, Mathias; Bandrowski, Anita; Carr, Steven ...
Nature methods,
10/2016, Letnik:
13, Številka:
10
Journal Article
Recenzirano
We convened an ad hoc International Working Group for Antibody Validation in order to formulate the best approaches for validating antibodies used in common research applications and to provide ...guidelines that ensure antibody reproducibility. We recommend five conceptual pillars for antibody validation to be used in an application-specific manner. Antibodies are among the most frequently used tools in basic science research and in clinical assays. Despite their widespread use, as well as extensive and valuable discourse in the literature16, a comprehensive scientific framework for antibody validation across research applications is lacking. As a result, the quality and consistency of data generated through the use of antibodies vary greatly. This poses an impediment to the rigor and reproducibility that are the cornerstones of the advancement of science.
Tumor-infiltrating lymphocytes (TILs) are usually measured using subjective methods. Studies suggest that TIL subtypes have independent roles in cancer and that they could support the use of novel ...immunostimulatory therapies. We simultaneously measured TIL subtypes in non-small cell lung cancer (NSCLC) samples using objective methods and determined their relationship with clinico-pathologic characteristics and survival.
Using multiplexed quantitative fluorescence (QIF), we measured the levels of CD3, CD8, and CD20 in 552 NSCLC from two independent collections represented in tissue microarrays (YTMA79, n = 202 and YTMA140, n = 350). The level of TILs was obtained in different tumor compartments using cytokeratin stain to define tumor cells and 4',6-Diamidino-2-Phenylindole. Association of TILs with clinical parameters was determined using univariate and multivariable analyses. All statistical tests were two-sided.
In both NSCLC collections there was a low correlation between the three TIL markers (linear regression coefficients (R(2)) = 0.19-0.22, P < .001 for YTMA79 and R(2) = 0.23-0.32, P < .001 for YTMA140). No consistent association between the level of TIL subtypes and age, sex, smoking history, tumor size, stage, and histology type was found. In univariate analysis, an elevated CD3 or CD8 signal was statistically significantly associated with longer survival in both collections. However, only CD8 was independent from age, tumor size, histology, and stage in multivariable analysis. High CD20 was associated with longer survival in the YTMA79 cohort.
Increased levels of CD3 and CD8 + TILs are associated with better outcome in NSCLC, but only CD8 is independent from other prognostic variables. Objective measurement of TIL subpopulations could be useful to predict response or evaluate the local immune effect of anticancer immune checkpoint inhibitors.
Antibodies are among the most frequently used tools in basic science research and in clinical assays, but there are no universally accepted guidelines or standardized methods for determining the ...validity of these reagents. Furthermore, for commercially available antibodies, it is clear that what is on the label does not necessarily correspond to what is in the tube. To validate an antibody, it must be shown to be specific, selective, and reproducible in the context for which it is to be used. In this review, we highlight the common pitfalls when working with antibodies, common practices for validating antibodies, and levels of commercial antibody validation for seven vendors. Finally, we share our algorithm for antibody validation for immunohistochemistry and quantitative immunofluorescence.
Because durable response to programmed cell death 1 (PD-1) inhibition is limited to a subset of melanoma patients, new predictive biomarkers could have clinical utility. We hypothesize that ...pretreatment tumor-infiltrating lymphocyte (TIL) profiles could be associated with response.
Pretreatment whole tissue sections from 94 melanoma patients treated with anti-PD-1 therapy were profiled by multiplex immunofluorescence to perform TIL quantification (CD4, CD8, CD20) and assess TIL activation (CD3, GZMB, Ki67). Two independent image analysis technologies were used: inForm (PerkinElmer) to determine cell counts, and AQUA to measure protein by quantitative immunofluorescence (QIF). TIL parameters by both methodologies were correlated with objective response or disease control rate (ORR/DCR) by RECIST 1.1 and survival outcome.
Pretreatment lymphocytic infiltration, by cell counts or QIF, was significantly higher in complete or partial response than in stable or progressive disease, particularly for CD8 (
< 0.0001). Neither TIL activation nor dormancy was associated with outcome. CD8 associations with progression-free survival (HR > 3) were independently significant in multivariable analyses and accounted for similar CD3 associations in anti-PD-1-treated patients. CD8 was not associated with melanoma prognosis in the absence of immunotherapy. Predictive performance of CD8 cell count (and QIF) had an area under the ROC curve above 0.75 (ORR/DCR), which reached 0.83 for ipilimumab plus nivolumab.
Pretreatment lymphocytic infiltration is associated with anti-PD-1 response in metastatic melanoma. Quantitative TIL analysis has potential for application in digital precision immuno-oncology as an "indicative" companion diagnostic.
Anaplastic thyroid carcinoma (ATC) is a frequently lethal malignancy that is often unresponsive to available therapeutic strategies. The tumorigenesis of ATC and its relationship to the widely ...prevalent well-differentiated thyroid carcinomas are unclear. We have analyzed 22 cases of ATC as well as 4 established ATC cell lines using whole-exome sequencing. A total of 2674 somatic mutations (121/sample) were detected. Ontology analysis revealed that the majority of variants aggregated in the MAPK, ErbB and RAS signaling pathways. Mutations in genes related to malignancy not previously associated with thyroid tumorigenesis were observed, including mTOR, NF1, NF2, MLH1, MLH3, MSH5, MSH6, ERBB2, EIF1AX and USH2A; some of which were recurrent and were investigated in 24 additional ATC cases and 8 ATC cell lines. Somatic mutations in established thyroid cancer genes were detected in 14 of 22 (64%) tumors and included recurrent mutations in BRAF, TP53 and RAS-family genes (6 cases each), as well as PIK3CA (2 cases) and single cases of CDKN1B, CDKN2C, CTNNB1 and RET mutations. BRAF V600E and RAS mutations were mutually exclusive; all ATC cell lines exhibited a combination of mutations in either BRAF and TP53 or NRAS and TP53. A hypermutator phenotype in two cases with >8 times higher mutational burden than the remaining mean was identified; both cases harbored unique somatic mutations in MLH mismatch-repair genes. This first comprehensive exome-wide analysis of the mutational landscape of ATC identifies novel genes potentially associated with ATC tumorigenesis, some of which may be targets for future therapeutic intervention.
PDF
