Signal transduction pathways are important in physiology and pathophysiology. Targeted drugs aim at modifying pathogenic pathway activity, e.g., in cancer. Optimal treatment choice requires assays to ...measure pathway activity in individual patient tissue or cell samples. We developed a method enabling quantitative measurement of functional pathway activity based on Bayesian computational model inference of pathway activity from measurements of mRNA levels of target genes of the pathway-associated transcription factor. Oestrogen receptor, Wnt, and PI3K-FOXO pathway assays have been described previously. Here, we report model development for androgen receptor, Hedgehog, TGFβ, and NFκB pathway assays, biological validation on multiple cell types, and analysis of data from published clinical studies (multiple sclerosis, amyotrophic lateral sclerosis, contact dermatitis, Ewing sarcoma, lymphoma, medulloblastoma, ependymoma, skin and prostate cancer). Multiple pathway analysis of clinical prostate cancer (PCa) studies showed increased AR activity in hyperplasia and primary PCa but variable AR activity in castrate resistant (CR) PCa, loss of TGFβ activity in PCa, increased Wnt activity in TMPRSS2:ERG fusion protein-positive PCa, active PI3K pathway in advanced PCa, and active PI3K and NFκB as potential hormonal resistance pathways. Potential value for future clinical practice includes disease subtyping and prediction and targeted therapy response prediction and monitoring.
Androgen deprivation therapy (ADT) is first‐line palliative treatment in androgen receptor‐positive (AR+) salivary duct carcinoma (SDC), and response rates are 17.6–50.0%. We investigated potential ...primary ADT resistance mechanisms for their predictive value of clinical benefit from ADT in a cohort of recurrent/metastatic SDC patients receiving palliative ADT (n = 30). We examined mRNA expression of androgen receptor (AR), AR splice variant‐7, intratumoral androgen synthesis enzyme‐encoding genes AKR1C3, CYP17A1, SRD5A1 and SRD5A2, AR protein expression, ERBB2 (HER2) gene amplification and DNA mutations in driver genes. Furthermore, functional AR pathway activity was determined using a previously reported Bayesian model which infers pathway activity from AR target gene expression levels. SRD5A1 expression levels and AR pathway activity scores were significantly higher in patients with clinical benefit from ADT compared to those without benefit. Survival analysis showed a trend toward a longer median progression‐free survival for patients with high SRD5A1 expression levels and high AR pathway activity scores. The AR pathway activity analysis, and not SRD5A1 expression, also showed a trend toward better disease‐free survival in an independent cohort of locally advanced SDC patients receiving adjuvant ADT (n = 14) after surgical tumor resection, and in most cases a neck dissection (13/14 patients) and postoperative radiotherapy (13/14 patients). In conclusion, we are the first to describe that AR pathway activity may predict clinical benefit from ADT in SDC patients, but validation in a prospective study is needed.
What's new?
Androgen deprivation therapy (ADT) is a leading treatment strategy in the palliative care of patients with androgen receptor (AR)‐positive salivary duct carcinoma (SDC). However, while as many as half of patients may respond to ADT, resistance frequently emerges, undermining its use. In this investigation of primary ADT resistance mechanisms, expression of the androgen synthesis enzyme‐encoding gene SRD5A1 and functional activity of the AR pathway were found to predict clinical benefit from ADT in SDC patients. High AR pathway activity scores were further linked to improved disease‐free survival in SDC patients with locally advanced disease who received adjuvant ADT.
•Novel technology to quantitatively measure activity of signal transduction pathways (STP) in individual stem cell samples.•Pluripotency STP activity profile characterized by active PI3K, MAPK, ...Hedgehog, Notch, TGFβ, and NFκB pathway and inactive Wnt pathway.•Measuring STP activity improves standardization of pluripotency.•Measuring STP activity improves experimental reproducibility.•Measuring STP activity enables controlled manipulation of pluripotency/differentiation.
Important challenges in stem cell research and regenerative medicine are reliable assessment of pluripotency state and purity of differentiated cell populations. Pluripotency and differentiation are regulated and determined by activity of developmental signal transduction pathways (STPs). To date activity of these STPs could not be directly measured on a cell sample.
Here we validate a novel assay platform for measurement of activity of developmental STPs (STP) for use in stem cells and stem cell derivatives. In addition to previously developed STP assays, we report development of an additional STP assay for the MAPK-AP1 pathway. Subsequently, activity of Notch, Hedgehog, TGFβ, Wnt, PI3K, MAPK-AP1, and NFκB signaling pathways was calculated from Affymetrix transcriptome data of human pluripotent embryonic (hES) and iPS cell lines under different culture conditions, organ-derived multipotent stem cells, and differentiated cell types, to generate quantitative STP activity profiles.
Results show that the STP assay technology enables reliable and quantitative measurement of multiple STP activities simultaneously on any individual cell sample. Using the technology, we found that culture conditions dominantly influence the pluripotent stem cell STP activity profile, while the origin of the stem cell line was a minor variable. A pluripotency STP activity profile (Pluripotency qPAP) was defined (active PI3K, MAPK, Hedgehog, Notch, TGFβ, and NFκB pathway, inactive Wnt pathway). Differentiation of hES cells to intestinal progenitor cells resulted in an STP activity profile characterized by active PI3K, Wnt and Notch pathways, comparable to the STP activity profile measured on primary intestinal crypt stem cells. Quantitative STP activity measurement is expected to improve experimental reproducibility and standardization of pluripotent and multipotent stem cell culture/differentiation, and enable controlled manipulation of pluripotency/differentiation state using pathway targeting compounds.
Sepsis is a life-threatening complication of a bacterial infection. It is hard to predict which patients with a bacterial infection will develop sepsis, and accurate and timely diagnosis as well as ...assessment of prognosis is difficult. Aside from antibiotics-based treatment of the causative infection and supportive measures, treatment options have remained limited. Better understanding of the immuno-pathophysiology of sepsis is expected to lead to improved diagnostic and therapeutic solutions. Functional activity of the innate (inflammatory) and adaptive immune response is controlled by a dedicated set of cellular signal transduction pathways, that are active in the various immune cell types. To develop an immune response-based diagnostic assay for sepsis and provide novel therapeutic targets, signal transduction pathway activities have been analyzed in whole blood samples from patients with sepsis.
A validated and previously published set of signal transduction pathway (STP) assays, enabling determination of immune cell function, was used to analyze public Affymetrix expression microarray data from clinical studies containing data from pediatric and adult patients with sepsis. STP assays enable quantitative measurement of STP activity on individual patient sample data, and were used to calculate activity of androgen receptor (AR), estrogen receptor (ER), JAK-STAT1/2, JAK-STAT3, Notch, Hedgehog, TGFβ, FOXO-PI3K, MAPK-AP1, and NFκB signal transduction pathways.
Activity of AR and TGFβ pathways was increased in children and adults with sepsis. Using the mean plus two standard deviations of normal pathway activity (in healthy individuals) as threshold for abnormal STP activity, diagnostic assay parameters were determined. For diagnosis of pediatric sepsis, the AR pathway assay showed high sensitivity (77%) and specificity (97%), with a positive prediction value (PPV) of 99% and negative prediction value (NPV) of 50%. For prediction of favorable prognosis (survival), PPV was 95%, NPV was 21%. The TGFβ pathway activity assay performed slightly less for diagnosing sepsis, with a sensitivity of 64% and specificity of 98% (PPV 99%, NPV 39%).
The AR and TGFβ pathways have an immunosuppressive role, suggesting a causal relation between increased pathway activity and sepsis immunopathology. STP assays have been converted to qPCR assays for further evaluation of clinical utility for sepsis diagnosis and prediction of prognosis, as well as for prediction of risk at developing sepsis in patients with a bacterial infection. STPs may present novel therapeutic targets in sepsis.
Cytostretch, an Organ-on-Chip Platform Gaio, Nikolas; van Meer, Berend; Quirós Solano, William ...
Micromachines (Basel),
07/2016, Letnik:
7, Številka:
7
Journal Article
Recenzirano
Odprti dostop
Organ-on-Chips (OOCs) are micro-fabricated devices which are used to culture cells in order to mimic functional units of human organs. The devices are designed to simulate the physiological ...environment of tissues in vivo. Cells in some types of OOCs can be stimulated in situ by electrical and/or mechanical actuators. These actuations can mimic physiological conditions in real tissue and may include fluid or air flow, or cyclic stretch and strain as they occur in the lung and heart. These conditions similarly affect cultured cells and may influence their ability to respond appropriately to physiological or pathological stimuli. To date, most focus has been on devices specifically designed to culture just one functional unit of a specific organ: lung alveoli, kidney nephrons or blood vessels, for example. In contrast, the modular Cytostretch membrane platform described here allows OOCs to be customized to different OOC applications. The platform utilizes silicon-based micro-fabrication techniques that allow low-cost, high-volume manufacturing. We describe the platform concept and its modules developed to date. Membrane variants include membranes with (i) through-membrane pores that allow biological signaling molecules to pass between two different tissue compartments; (ii) a stretchable micro-electrode array for electrical monitoring and stimulation; (iii) micro-patterning to promote cell alignment; and (iv) strain gauges to measure changes in substrate stress. This paper presents the fabrication and the proof of functionality for each module of the Cytostretch membrane. The assessment of each additional module demonstrate that a wide range of OOCs can be achieved.
Oestrogen receptor (ER) expression is a prognostic biomarker in endometrial cancer (EC). However, expression does not provide information about the functional activity of the ER pathway. We evaluated ...a model to quantify ER pathway activity in EC, and determined the prognostic relevance of ER pathway activity.
ER pathway activity was measured in two publicly available datasets with endometrial and EC tissue, and one clinical cohort with 107 samples from proliferative and hyperplastic endometrium and endometrioid-type EC (EEC) and uterine serous cancer (USC). ER pathway activity scores were inferred from ER target gene mRNA levels from Affymetrix microarray data (public datasets), or measured by qPCR on formalin-fixed paraffin-embedded samples (clinical cohort) and related to ER expression and outcome.
ER pathway activity scores differed significantly throughout the menstrual cycle supporting the validity of the pathway test. The highest ER pathway scores were found in proliferative and hyperplastic endometrium and stage I EEC, whereas stage II-IV EEC and USCs had significantly lower levels. Low ER pathway activity was associated with recurrent disease, and added prognostic value in patients with low ER expression.
The ER pathway test reflects activity of the ER pathway, and may improve prediction of outcome in EC patients.
Approximately 20% of women with endometrial cancer have advanced-stage disease or suffer from a recurrence. For these women, prognosis is poor, and palliative treatment options include hormonal ...therapy and chemotherapy. Lack of predictive biomarkers and suboptimal use of existing markers for response to hormonal therapy have resulted in overall limited efficacy.
This study aimed to improve the efficacy of hormonal therapy by relating immunohistochemical expression of estrogen and progesterone receptors and estrogen receptor pathway activity scores to response to hormonal therapy.
Patients with advanced or recurrent endometrial cancer and available biopsies taken before the start of hormonal therapy were identified in 16 centers within the European Network for Individualized Treatment in Endometrial Cancer and the Dutch Gynecologic Oncology Group. Tumor tissue was analyzed for estrogen and progesterone receptor expressions and estrogen receptor pathway activity using a quantitative polymerase chain reaction–based messenger RNA model to measure the activity of estrogen receptor–related target genes in tumor RNA. The primary endpoint was response rate defined as complete and partial response using the Response Evaluation Criteria in Solid Tumors. The secondary endpoints were clinical benefit rate and progression-free survival.
Pretreatment biopsies with sufficient endometrial cancer tissue and complete response evaluation were available in 81 of 105 eligible cases. Here, 22 of 81 patients (27.2%) with a response had estrogen and progesterone receptor expressions of >50%, resulting in a response rate of 32.3% (95% confidence interval, 20.9–43.7) for an estrogen receptor expression of >50% and 50.0% (95% confidence interval, 35.2–64.8) for a progesterone receptor expression of >50%. Clinical benefit rate was 56.9% for an estrogen receptor expression of >50% (95% confidence interval, 44.9–68.9) and 75.0% (95% confidence interval, 62.2–87.8) for a progesterone receptor expression of >50%. The application of the estrogen receptor pathway test to cases with a progesterone receptor expression of >50% resulted in a response rate of 57.6% (95% confidence interval, 42.1–73.1). After 2 years of follow-up, 34.3% of cases (95% confidence interval, 20–48) with a progesterone receptor expression of >50% and 35.8% of cases (95% confidence interval, 20–52) with an estrogen receptor pathway activity score of >15 had not progressed.
The prediction of response to hormonal treatment in endometrial cancer improves substantially with a 50% cutoff level for progesterone receptor immunohistochemical expression and by applying a sequential test algorithm using progesterone receptor immunohistochemical expression and estrogen receptor pathway activity scores. However, results need to be validated in the prospective Prediction of Response to Hormonal Therapy in Advanced and Recurrent Endometrial Cancer (PROMOTE) study.
Background
Advanced low‐grade ovarian carcinoma (LGOC) is difficult to treat. In several studies, high estrogen receptor (ER) protein expression was observed in patients with LGOC, which suggests ...that antihormonal therapy (AHT) is a treatment option. However, only a subgroup of patients respond to AHT, and this response cannot be adequately predicted by currently used immunohistochemistry (IHC). A possible explanation is that IHC only takes the ligand, but not the activity, of the whole signal transduction pathway (STP) into account. Therefore, in this study, the authors assessed whether functional STP activity can be an alternative tool to predict response to AHT in LGOC.
Methods
Tumor tissue samples were obtained from patients with primary or recurrent LGOC who subsequently received AHT. Histoscores of ER and progesterone receptor (PR) were determined. In addition, STP activity of the ER STP and of six other STPs known to play a role in ovarian cancer was assessed and compared with the STP activity of healthy postmenopausal fallopian tube epithelium.
Results
Patients who had normal ER STP activity had a progression‐free survival (PFS) of 16.1 months. This was significantly shorter in patients who had low and very high ER STP activity, with a median PFS of 6.0 and 2.1 months, respectively (p < .001). Unlike ER histoscores, PR histoscores were strongly correlated to the ER STP activity and thus to PFS.
Conclusions
Aberrant low and very high functional ER STP activity and low PR histoscores in patients with LGOC indicate decreased response to AHT. ER IHC is not representative of functional ER STP activity and is not related to PFS.
Patients who have low‐grade ovarian carcinoma with a functionally low or high active estrogen receptor signaling pathway have a decreased response to antihormonal therapy. Estrogen receptor immunohistochemistry does not have predictive value for antihormonal therapy response and is not representative of functional estrogen receptor signaling.
The phosphoinositide 3-kinase (PI3K) growth factor signaling pathway plays an important role in embryonic development and in many physiological processes, for example the generation of an immune ...response. The pathway is frequently activated in cancer, driving cell division and influencing the activity of other signaling pathways, such as the MAPK, JAK-STAT and TGFβ pathways, to enhance tumor growth, metastasis, and therapy resistance. Drugs that inhibit the pathway at various locations, e.g., receptor tyrosine kinase (RTK), PI3K, AKT and mTOR inhibitors, are clinically available. To predict drug response versus resistance, tests that measure PI3K pathway activity in a patient sample, preferably in combination with measuring the activity of other signaling pathways to identify potential resistance pathways, are needed. However, tests for signaling pathway activity are lacking, hampering optimal clinical application of these drugs. We recently reported the development and biological validation of a test that provides a quantitative PI3K pathway activity score for individual cell and tissue samples across cancer types, based on measuring Forkhead Box O (FOXO) transcription factor target gene mRNA levels in combination with a Bayesian computational interpretation model. A similar approach has been used to develop tests for other signaling pathways (e.g., estrogen and androgen receptor, Hedgehog, TGFβ, Wnt and NFκB pathways). The potential utility of the test is discussed, e.g., to predict response and resistance to targeted drugs, immunotherapy, radiation and chemotherapy, as well as (pre-) clinical research and drug development.
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