NAFLD and NASH biomarker qualification in the LITMUS consortium - Lessons learned.

Biomarkers have the potential to accelerate drug development, as early indicators of improved clinical response, to improve patient safety, and for personalised medicine. However, few have been approved through the biomarker qualification pathways of the regulatory agencies. This paper outlines how biomarkers can accelerate drug development, and reviews the lessons learned by the EU IMI2-funded LITMUS consortium, which has had several interactions with regulatory agencies in both the US and EU regarding biomarker qualification in patients with non-alcoholic fatty liver disease and non-alcoholic steatohepatitis. Sharing knowledge of such interactions with the scientific community is of paramount importance to increase the chances of qualification of relevant biomarkers that may accelerate drug development, and thereby help patients, across disease indications. A qualified biomarker enables a decision to be made that all understand and support in a common framework.

Thrombospondin 1 and cathepsin D improve prostate cancer diagnosis by avoiding potentially unnecessary prostate biopsies.

OBJECTIVES: To investigate and further validate if two novel cancer-related glycoproteins, discovered by a genetic-guided proteomics approach, can distinguish benign disease from prostate cancer (PCa) in men with enlarged prostates. PATIENTS AND METHODS: A retrospective study was performed that included men with a total prostate-specific antigen (PSA) concentration of 2.0-10 ng/mL, negative digital rectal examination and enlarged prostate (volume ≥35 mL). Serum samples were collected between 2011 and 2016 at a single centre from 474 men before they underwent prostate biopsy. Serum concentrations of thrombospondin 1 (THBS1) and cathepsin D (CTSD) glycoproteins were combined with the percentage of free PSA to total PSA ratio (%fPSA) to predict any or significant cancer at biopsy. RESULTS: The multivariable logistic regression model including THBS1, CTSD and %fPSA discriminated among biopsy-positive and biopsy-negative patients in the validation set with an area under the curve (AUC) of 0.86 (P < 0.001, 95% confidence interval (CI) 0.82-0.91), while %fPSA alone showed an AUC of 0.64 (P < 0.001, 95% CI 0.57-0.71). At 90% sensitivity for PCa, the specificity of the model was 62%, while %fPSA had a specificity of 23%. For high grade (Gleason score ≥ 7 in prostatectomy specimen) PCa, the specificity was 48% at 90% sensitivity, with an AUC of 0.83, (P < 0.001, 95% CI 0.77 to 0.88). Limitations of the study include the retrospective set-up and single-centre cohort. CONCLUSIONS: A model combining two cancer-related glycoproteins (THBS1 and CTSD) and %fPSA can improve PCa diagnosis and may reduce the number of unnecessary prostate biopsies because of its improved specificity for PCa when compared to %fPSA alone.

Development and validation of a novel multivariate risk score to guide biopsy decision for the diagnosis of clinically significant prostate cancer.

Objectives: Selecting patients suspected of having prostate cancer (PCa) for a prostate biopsy remains a challenge. Prostate-specific antigen (PSA)-based testing is hampered by its low specificity that often leads to negative biopsy results or detection of clinically insignificant cancers, especially in the 2-10 ng/mL range. The objective was to evaluate a novel diagnostic test called Proclarix incorporating thrombospondin-1 and cathepsin D alongside total and free PSA as well as age for predicting clinically significant PCa. Patients and methods: The test was developed following a retrospective study design using biobanked samples of 955 men from two reference centres. A multivariate approach was used for model development followed by validation to discriminate significant (grade group ≥2) from insignificant or no cancer at biopsy. The test specificity, positive predictive value (PPV) and negative predictive value (NPV) at a fixed sensitivity of 90% were compared to percent free PSA (%fPSA) alone. The number of avoidable prostate biopsies deemed to be representative of clinical utility was also assessed. Results: In the targeted patient population, the test displayed increased diagnostic accuracy compared to %fPSA alone. Application of the established model on 955 patients at a fixed sensitivity of 90% for significant disease resulted in a specificity of 43%, NPV of 95% and a PPV of 25%. This is in comparison to a specificity of 17%, NPV of 89% and PPV of 19% for %fPSA alone and had the potential to reduce the total number of biopsies needed to identify clinically significant cancer. Further, the test score correlated with significance of cancer assessed on prostate biopsy. Conclusions: The Proclarix test can be used as an aid in the decision-making process if to biopsy men in this challenging patient population. The use of the test could reduce the number of biopsies performed avoiding invasive procedures, anxiety, discomfort, pain and complications.

Analytical performance of thrombospondin-1 and cathepsin D immunoassays part of a novel CE-IVD marked test as an aid in the diagnosis of prostate cancer.

The Prostate Specific Antigen (PSA) test suffers from low specificity for the diagnosis of Prostate Cancer (PCa). We originally discovered two cancer-related proteins thrombospondin-1 (THBS1) and cathepsin D (CTSD) using a mass-spectrometry-based proteomics approach. The two serum proteins were shown to improve the diagnosis of high-grade PCa. Thus, we developed quantitative ELISAs for the determination of their concentration in human serum. Here we report their analytical performance in terms of limit of detection, specificity, precision, linearity and interferences, which were determined based on CLSI guidelines. Further, we investigated the influence of pre-analytical factors on concentration measurements. For this, blood from 4-6 donors was collected in different tubes and stored at room temperature for different times prior to centrifugation at different centrifugal forces and temperatures. Stability of THBS1 and CTSD under different storage temperatures was also evaluated. Our results show that the assays are specific, linear and sensitive enough to allow measurement of clinical samples. Precision in terms of repeatability and total within-laboratory coefficient of variation (CV) are 5.5% and 8.1% for THBS1 and 4.3% and 7.2% for CTSD, respectively. Relative laboratory-to-laboratory differences were -6.3% for THBS1 and -3% for CTSD. Both THBS1 and CTSD were stable in serum samples, with 80-120% recoveries of concentrations across donors, sample preparation and storage. In conclusion, the ELISAs as part of the novel commercial in vitro diagnostic test Proclarix are suitable for the use in clinical practice. THBS1 and CTSD can be accurately measured for their intended use independent of the lot and laboratory when conditions consistent with routine practice for PSA sampling and storage are used.

Retinal input directs the recruitment of inhibitory interneurons into thalamic visual circuits.

Inhibitory interneurons (INs) critically control the excitability and plasticity of neuronal networks, but whether activity can direct INs into specific circuits during development is unknown. Here, we report that in the dorsal lateral geniculate nucleus (dLGN), which relays retinal input to the cortex, circuit activity is required for the migration, molecular differentiation, and functional integration of INs. We first characterize the prenatal origin and molecular identity of dLGN INs, revealing their recruitment from an Otx2(+) neuronal pool located in the adjacent ventral LGN. Using time-lapse and electrophysiological recordings, together with genetic and pharmacological perturbation of retinal waves, we show that retinal activity directs the navigation and circuit incorporation of dLGN INs during the first postnatal week, thereby regulating the inhibition of thalamocortical circuits. These findings identify an input-dependent mechanism regulating IN migration and circuit inhibition, which may account for the progressive recruitment of INs into expanding excitatory circuits during evolution.

In vivo reprogramming of circuit connectivity in postmitotic neocortical neurons.

The molecular mechanisms that control how progenitors generate distinct subtypes of neurons, and how undifferentiated neurons acquire their specific identity during corticogenesis, are increasingly understood. However, whether postmitotic neurons can change their identity at late stages of differentiation remains unknown. To study this question, we developed an electrochemical in vivo gene delivery method to rapidly manipulate gene expression specifically in postmitotic neurons. Using this approach, we found that the molecular identity, morphology, physiology and functional input-output connectivity of layer 4 mouse spiny neurons could be specifically reprogrammed during the first postnatal week by ectopic expression of the layer 5B output neuron-specific transcription factor Fezf2. These findings reveal a high degree of plasticity in the identity of postmitotic neocortical neurons and provide a proof of principle for postnatal re-engineering of specific neural microcircuits in vivo.

Pre-treatment waking cortisol response and vulnerability to interferon α induced depression.

Depressive disorder is a common consequence of interferon α treatment. An understanding of the aetiological processes involved is evolving. HPA axis abnormalities are clearly described in community depressive disorder and represent vulnerability to depression development. We explored whether pre-treatment HPA axis abnormalities influence depression emergence during interferon α treatment. We examined waking HPA axis response via salivary cortisol sampling in 44 non-depressed, chronic hepatitis C infected patients due to commence standard interferon α treatment. Hamilton depression scales and the structured clinical interview for DSM-IV major depressive disorder status were administered monthly during treatment. Major depressive disorder developed in 26 of 44 subjects during interferon-α treatment. The pre-treatment waking cortisol response over 1h was significantly greater in the subsequent switch to depression group (F=4.23, p=0.046). The waking cortisol response pre-treatment with interferon α appears greater in those subsequently switching to depressive disorder during treatment. This waking response may join other vulnerability factors for depression emergence in this group. This model could prove a valuable tool in understanding non-iatrogenic depressive disorder in the general population and notably the role of cytokines.

Strain differences and the role of AT(1) receptor expression in anxiety.

This study investigated strain specific differences to the anxiolytic response to losartan focusing on genetic variation that may influence such responses. This included: AT(1) receptor sequence variation, angiotensin II receptor associated protein (ATRAP) and receptor expression between strains. Sequencing of exon 3 of AT(1a)R revealed no differences between BKW mice (n=6) and C57 and DBA(2) strains (n=3). Comparisons of AT(1) expression do show significant differences, whereby BKW mice showed the highest levels of expression and DBA(2) mice intermediate levels when compared to the C57 strain. Sequencing of sections of the Angiotensin receptor associated protein (ATRAP) identified a non-synonymous point mutation- (T/C) transversion (position 109-161) (SNP id = rs13467517) resulting in a Valine → Alanine (V157A) amino acid change in the BKW and DBA(2) strains. Our results indicate that the previously reported strain dependent effects are not due to variation in AT(1a) receptor sequence. Differences in AT(1)gene expression levels between strains, which mirror their anxiety phenotype, are observed. This is coupled with a non-synonymous single nucleotide polymorphism in ATRAP, a negative regulator of AT(1) signalling.

Assessment of biological activity of novel peptide analogues of angiotensin IV.

OBJECTIVES: Angiotensin IV (Ang IV) is a metabolite of angiotensin II which acts on specific AT(4) receptors identified as the enzyme insulin regulated aminopeptidase (IRAP). The transduction process of these receptors is unresolved, but Ang IV inhibits the aminopeptidase activity. Ang IV improves cognition in animal models thus there is a desire to develop metabolically stable analogues for further development. METHODS: Peptide analogues of Ang IV were obtained commercially or synthesised. Each peptide was tested in vitro for its ability to inhibit the aminopeptidase activity (IRAP) of mouse brain homogenates and for its effects on isolated rat uterine smooth muscle. KEY FINDINGS: [Des-Val(1) ]-Ang IV, acetylated-Ang IV-amide, Ang IV-amide and [des-His(4) ]-Ang IV all inhibited IRAP. [Sar(1) , Ile(8) ]-Angiotensin II (10 µm) had an effect greater than that of Ang IV or any of the other analogues studied. In isolated uterine smooth muscle, angiotensins II and IV induced contractions, which could be antagonised by an AT(1) -receptor antagonist. None of the novel peptides induced uterine smooth muscle contractions, but [Sar(1) , des Arg(2) -Gly(8) ]-angiotensin II showed significant antagonism of the contractile effects of angiotensin II and carboxyamide-terminated Ang IV-NH(2) showed antagonism of Ang IV-induced contractions. CONCLUSIONS: This study provides five novel inhibitors of IRAP worthy of assessment in behavioural models of learning and memory. The analogues are devoid of AT(1) receptor agonist properties, and the carboxyamide analogue presents an opportunity to elucidate the mechanism of action of Ang IV as, like Ang IV, it inhibits IRAP, but antagonises the effects of Ang IV on isolated smooth muscle.

Strain differences in the effects of angiotensin IV on mouse cognition.

Angiotensin IV has been shown to improve learning and memory in rodents. Strain dependent variation in murine behaviour, aminopeptidase activity and inhibitory effect of Angiotensin IV, structural variation in insulin regulated aminopeptidase (IRAP) and aminopeptidase N (ApN) and expression of the encoding genes were explored. Strain differences in the behavioural response to Angiotensin IV were observed, where CD mice were refractory. All strains showed inhibition of aminopeptidase activity by Angiotensin IV but CD mice displayed reduced endogenous aminopeptidase activity. No differences in the coding sequence of IRAP or ApN were found. RT-PCR analysis showed no difference in IRAP expression between strains but an increased expression of ApN was observed in CD mice. The lack of cognitive response of CD mice to Angiotensin IV cannot be explained through variation within IRAP sequence nor expression but the results highlight a potential role for ApN in the effects of Angiotensin IV.