Proof of concept that melanoma nuclear count compares favourably with the benchmark histological prognostic feature, Breslow thickness.

AIMS: Breslow thickness (BT) is the most important histological prognostic feature for melanoma prognosis, but it only captures tumour size in one dimension. Adding a further measurement in a different axis has been shown to improve prognostic value. It seems reasonable that further prognostic value could be obtained by estimating the number of invasive melanoma cells using nuclear count. The aim of this study was to show proof of concept that nuclear count has prognostic value independent of BT. METHODS AND RESULTS: Melanoma cell nuclei were labelled with SRY-related HMG-box 10 (SOX10) protein, the sections scanned and StarDist machine-learning algorithm used to count nuclei in 102 cases of primary cutaneous melanoma. Prognostic value was assessed using survival analyses. Nuclear count correlated strongly with T category, BT and calculated tumour area (each P < 0.001), suggesting that it was a valid marker of melanoma burden. Nuclear count was a predictor for overall survival in univariable analysis [hazard ratio (HR) = 2.25, confidence interval (CI) = 1.66-3.06, P < 0.001] and multivariable analysis (HR = 2.60, CI = 1.59-4.24, P < 0.001). BT and ulceration were significant in univariable analyses, but not in multivariable models with nuclear count. Models containing nuclear count showed the best fit. Similar results were seen for melanoma-specific and metastasis-free survival. Nuclear count was able to stratify melanomas within a given T stage. CONCLUSIONS: This study demonstrated proof of concept that counting melanoma nuclei may be an improved measure of invasive tumour burden compared to BT. Future studies will need to refine methods of nuclear detection and also to confirm its prognostic value.

Comparison of the prognostic value of microscopically measured invasive width versus macroscopic width in cutaneous melanoma shows the superiority of microscopic invasive width measurement.

BACKGROUND: Invasive width, the distance between the most peripheral invasive melanoma cells on the section where Breslow thickness (BT) was measured, was recently identified as a prognostic feature. It is unclear whether a routine measurement is justified, given that macroscopic width is already included in many melanoma histopathology reports and may itself be a prognostic feature. This study sought to investigate this. METHODS: A retrospective cohort of 718 melanoma patients in which macroscopic width had been stated in the original histopathology report was used. Survival analysis was performed. RESULTS: Macroscopic and invasive widths were positively correlated (p < 0.001). Invasive width was typically smaller than the paired macroscopic width (median difference 3.7 mm, p < 0.001), a difference seen across all T groups. Both macroscopic and invasive widths were significantly associated with melanoma survival in Kaplan-Meier analysis, including overall survival, but invasive width survival curves were more widely separated. Both were significantly associated with outcome after correction for BT in Cox proportional hazards regression, but the models containing invasive width had a substantially better fit. CONCLUSIONS: This study shows that both macroscopic and invasive widths have prognostic values, but confirms that the latter is superior. It supports further investigation of this feature's prognostic value.

GSK137, a potent small-molecule BCL6 inhibitor with in vivo activity, suppresses antibody responses in mice.

B-cell lymphoma 6 (BCL6) is a zinc finger transcriptional repressor possessing a BTB-POZ (BR-C, ttk, and bab for BTB; pox virus and zinc finger for POZ) domain, which is required for homodimerization and association with corepressors. BCL6 has multiple roles in normal immunity, autoimmunity, and some types of lymphoma. Mice bearing disrupted BCL6 loci demonstrate suppressed high-affinity antibody responses to T-dependent antigens. The corepressor binding groove in the BTB-POZ domain is a potential target for small compound-mediated therapy. Several inhibitors targeting this binding groove have been described, but these compounds have limited or absent in vivo activity. Biophysical studies of a novel compound, GSK137, showed an in vitro pIC50 of 8 and a cellular pIC50 of 7.3 for blocking binding of a peptide derived from the corepressor silencing mediator for retinoid or thyroid hormone receptors to the BCL6 BTB-POZ domain. The compound has good solubility (128 µg/ml) and permeability (86 nM/s). GSK137 caused little change in cell viability or proliferation in four BCL6-expressing B-cell lymphoma lines, although there was modest dose-dependent accumulation of G1 phase cells. Pharmacokinetic studies in mice showed a profile compatible with achieving good levels of target engagement. GSK137, administered orally, suppressed immunoglobulin G responses and reduced numbers of germinal centers and germinal center B cells following immunization of mice with the hapten trinitrophenol. Overall, we report a novel small-molecule BCL6 inhibitor with in vivo activity that inhibits the T-dependent antigen immune response.

The Width of Invasion in Malignant Melanoma Is a Novel Prognostic Feature That Accounts for Outcome Better Than Breslow Thickness.

Breslow thickness (BT) is the cornerstone of malignant melanoma staging. However, to our knowledge no-one has ever assessed the horizontal width of invasion, measured microscopically, as a prognostic feature. This was investigated as a prognostic feature in this study. A retrospective cohort of 1329 melanoma cases was collected from patients presenting to a UK teaching hospital from January 1, 2004, to December 31, 2014. The main outcome was overall survival (OS). We found that width was associated with OS in multivariable analysis (hazard ratio=1.05, 95% confidence interval: 1.03-1.07, P<0.001) and was similarly significant for melanoma-specific survival and metastasis-free survival. Its presence rendered BT nonsignificant. The width was significantly associated with OS after adjustment for American Joint Committee on Cancer (AJCC), version 8 clinical tumor stage (hazard ratio=1.05, 95% confidence interval: 1.03-1.07, P<0.001), and bootstrap validation showed only slight model optimism. Similar associations were seen for melanoma-specific survival and metastasis-free survival. However, the combination of invasive width and BT did not account for the outcome as well as another novel histologic feature, tumor area, which was measured using the calculated tumor area method. In conclusion, this study is the first investigation of a novel histologic feature, invasive melanoma width, and demonstrates its strong independent association with outcome.

Global and mitosis-specific interobserver variation in mitotic count scoring and implications for malignant melanoma staging.

AIMS: Staging is the gold standard for predicting malignant melanoma outcome but changes in its criteria over time indicate ongoing evolution. One notable recent change from the 8th edition of the American Joint Committee on Cancer (AJCC) staging manual was removal of mitotic count. We explore the extent to which this feature is limited by interobserver error in order to find ways to improve its fitness for use should it be revisited in future staging versions. METHODS AND RESULTS: In a cohort of 476 patients with melanoma ≤1.0 mm, a mitotic count of 0 versus 1 was significant for metastasis-free survival, but not melanoma-specific or overall survival. In 10 melanomas that were 0.9-1.0 mm thick, the mitotic count intraclass correlation coefficient for histopathologists was 0.58 (moderate agreement). Uniquely, we also assessed agreement for specific putative mitotic figures, identifying precise reasons why specific mitotic figures qualified for scoring or elimination. A kappa score was 0.54 (moderate agreement). We also gathered data on other staging features. Breslow thickness had an intraclass correlation coefficient of 0.41 (moderate agreement) and there was a systematic difference between histopathologists among cases (P = 0.04). Every case had a range that crossed the AJCC8 0.8-mm pT1a/pT1b staging boundary. Ulceration was only identified in two of the 10 cases. For ulceration, kappa agreement score was 0.31 (fair). CONCLUSION: This study supports the removal of mitotic count from staging, but shows that its scoring is substantially affected by interobserver variation, suggesting that more prescriptive guidelines might have a beneficial impact on its prognostic value.

Identification, preclinical profile, and clinical proof of concept of an orally bioavailable pro-drug of miridesap.

BACKGROUND AND PURPOSE: Miridesap, a depleter of serum amyloid P component (SAP), forms an essential component of a novel approach to remove systemic amyloid deposits; low oral bioavailability necessitates that it is given parenterally. We sought to identify and clinically characterise a pro-drug that preserves the pharmacological properties of miridesap while having adequate oral bioavailability and physical stability. EXPERIMENTAL APPROACH: We utilised a preclinical screening cascade focused on appropriate physicochemical properties, physical and gut stability, and conversion to miridesap in liver microsomes and blood. GSK3039294 (GSK294) had the desired in vitro profile and progressed to preclinical in vivo pharmacokinetic and safety assessments. Based on a favourable profile, it was tested in healthy participants after single and repeat dosing. KEY RESULTS: GSK294 was highly soluble and stable in simulated gastric and intestinal fluids, stable in intestinal microsomes, and permeable in Madine Darby Canine Kidney type II cells. GSK294 was rapidly hydrolysed to miridesap and its mono pro-drug ester in blood and liver microsomes. GSK294 showed good oral bioavailability of miridesap in rats and dogs. Following administration of GSK294 600 mg QD for 7 days in humans, pharmacodynamically active concentrations of miridesap were achieved with substantial and sustained depletion of plasma SAP. The study was terminated due to observations of arrhythmia, the relation of which to GSK294 remains unclear. CONCLUSION AND IMPLICATIONS: Using a preclinical screening cascade, we identified a pro-drug for a palindromic molecule with unique pharmacology (miridesap). The pro-drug depleted circulating SAP with a time course and extent similar to that of parenterally administered miridesap.

Development and Initial Validation of Calculated Tumor Area as a Prognostic Tool in Cutaneous Malignant Melanoma.

IMPORTANCE: Breslow thickness is a 1-dimensional surrogate prognostic feature for tumor size, yet tissue sections have 2 dimensions. Therefore, a 2-dimensional feature, calculated tumor area (CTA), was devised. OBJECTIVE: To determine CTA precision and prognostic value. DESIGN, SETTING, AND PARTICIPANTS: This retrospective cohort of patients with cutaneous melanoma presented to the Leicester and Nottingham National Health Service hospital trusts in the United Kingdom. Eligible patients in the Leicester development sample had available primary tumor tissue; a diagnosis from January 1, 2004, through December 31, 2011; invasive disease; and Leicestershire residency. Patients in the Nottingham validation sample had an anonymized spreadsheet with primary melanoma diagnosed from January 1, 2003, through December 31, 2005, or from January 1, 2008, through December 31, 2010. From a starting population of 1463 patients in both data sets, a total of 224 (15.3%) were excluded to yield a study population of 1239. Data were analyzed from April 30, 2018, through January 10, 2019. INTERVENTION: An observational analysis of the prognostic value of CTA in patients with cutaneous melanoma. MAIN OUTCOMES AND MEASURES: Independent association of CTA with melanoma-specific survival and confounding effect of CTA on Breslow thickness in survival analysis. RESULTS: A total of 1239 patients with melanoma were assessed, including 649 (52.4%) women, with a median age of 60 years (interquartile range, 47-71 years). An intraclass correlation coefficient for CTA on 13 cases was 0.99. In 918 patients in the Leicester cohort, CTA was an independent prognostic factor in Cox proportional hazards regression models after adjusting for Breslow thickness, age, sex, ulcer, mitotic rate, and microsatellites (hazard ratio [HR], 1.87; 95% CI, 1.49-2.34; P < .001). Validation in 321 patients in the Nottingham cohort showed an HR of 1.55 (95% CI, 1.15-2.09; P = .005) and in the combined 1239 cases, an HR of 1.70 (95% CI, 1.43-2.03; P < .001). Breslow thickness was significant in multivariable analysis only when CTA was not in the model. The relative importance of CTA was shown by its retention in all 100 bootstrap multivariable models with backward selection, whereas Breslow thickness was retained in only 53. Melanomas stratified by CTA showed wider separation of survival curves than those stratified by Breslow thickness using the American Joint Committee on Cancer Staging Manual, 8th Edition (HRs, 1.00 to 41.46 vs 1.00 to 36.95, respectively), and the model with CTA categories had a Bayesian information criterion difference of 13.9 compared with T category, indicating substantially better fit. This model had a Harrell C index of 83.7%, and bootstrap analysis showed little evidence of model optimism, with a corrected calibration slope of 0.99. CONCLUSIONS AND RELEVANCE: This study provides a novel microscopic feature, CTA, with evidence of its independent prognostic value. This evidence suggests that CTA should be a priority for further study.

Sézary Syndrome Presenting With Renal Involvement.

Sézary syndrome is a rare aggressive leukemic variant of primary cutaneous T-cell lymphoma, typically presenting with erythroderma, lymphadenopathy, and an atypical clonal T-cell population. Though it often involves the spleen and liver, we report a case of Sézary syndrome with renal involvement that was treated successfully. Visceral involvement confers a poor prognosis requiring systemic treatment. The patient we describe was a 66-year-old man who was referred from Dermatology services for deteriorating kidney function. Polymerase chain reaction of genomic DNA from skin and kidney biopsies confirmed a clonal T-cell population matching a population isolated in peripheral blood. The patient was treated initially with alemtuzumab, which led to a significant improvement in kidney function, and he has subsequently received a successful allogeneic stem cell transplant. This case represents a rare cause of decreased kidney function and highlights the role of biopsy in patients with suspected Sézary syndrome.

Breslow Density Is a Novel Prognostic Feature That Adds Value to Melanoma Staging.

Histomorphologic prognostic biomarkers that can be measured using only an hematoxylin and eosin stain are very attractive because they are simple and cheap. We conceived an entirely novel biomarker of this type, the Breslow density (BD), which measures invasive melanoma cell density at the site where Breslow thickness (BT) is measured. This study assessed BD's prognostic value. In this study, BD was measured in 1329 melanoma patients. Measurement accuracy and precision was assessed using intraclass correlation coefficient (ICC). Survival was assessed with a primary end-point of melanoma-specific survival (MSS) and also overall survival and metastasis-free survival. We found that BD measurement was accurate compared with gold standard image analysis (ICC, 0.84). Precision was excellent for 3 observers with different experience (ICC, 0.93) and for an observer using only written instructions (ICC, 0.93). BD was a highly significant predictor in multivariable analysis for overall survival, MSS, and metastasis-free survival (each, P<0.001) and it explained MSS better than BT, but BT and BD together had best explanatory capability. A BD cut point of ≥65% was trained in 970 melanomas and validated in 359. This cut point showed promise as a novel way to upstage melanoma from T stage "a" to "b." BD was combined with BT to create a targeted burden score. This was a validated as an adjunct to American Joint Committee on Cancer stage. In summary, BD can be measured accurately and precisely. It demonstrated independent prognostic value and explained MSS better than BT alone. Notably, we demonstrated ways that BD could be used with American Joint Committee on Cancer version 8 staging.

In search of a small molecule agonist of the relaxin receptor RXFP1 for the treatment of liver fibrosis.

The peptide hormone human relaxin-2 (H2-RLX) has emerged as a potential therapy for cardiovascular and fibrotic diseases, but its short in vivo half-life is an obstacle to long-term administration. The discovery of ML290 demonstrated that it is possible to identify small molecule agonists of the cognate G-protein coupled receptor for H2-RLX (relaxin family peptide receptor-1 (RXFP1)). In our efforts to generate a new medicine for liver fibrosis, we sought to identify improved small molecule functional mimetics of H2-RLX with selective, full agonist or positive allosteric modulator activity against RXFP1. First, we confirmed expression of RXFP1 in human diseased liver. We developed a robust cellular cAMP reporter assay of RXFP1 signaling in HEK293 cells transiently expressing RXFP1. A high-throughput screen did not identify further specific agonists or positive allosteric modulators of RXFP1, affirming the low druggability of this receptor. As an alternative approach, we generated novel ML290 analogues and tested their activity in the HEK293-RXFP1 cAMP assay and the human hepatic cell line LX-2. Differences in activity of compounds on cAMP activation compared with changes in expression of fibrotic markers indicate the need to better understand cell- and tissue-specific signaling mechanisms and their disease-relevant phenotypes in order to enable drug discovery.

A Novel Numerical Scoring System for Melanoma Tumor-infiltrating Lymphocytes Has Better Prognostic Value Than Standard Scoring.

The purpose of this study was to evaluate the prognostic value of tumor-infiltrating lymphocytes (TILs) in melanoma and to determine whether a simpler numerical scoring system would be more effective. In total, 655 patients presenting to a UK teaching hospital with primary invasive melanoma were analyzed. TILs were rescored using the standard Clark's method and univariable and multivariable analyses of the effect of TILs on overall survival (OS), disease-specific survival (DSS), and metastasis-free survival (MFS) was assessed using Cox regression. In total, 30 (5%) melanomas showed absent, 464 (71%) nonbrisk, and 161 (24%) brisk TILs. There was a statistically significant relationship between TILs and Breslow thickness, age, melanoma type, mitotic rate, and histologic regression. TIL grade was a significant predictor of MFS in multivariable analysis (hazard ratio [HR], 0.44; 95% confidence interval [CI], 0.25-0.77) but was not significant for OS or DSS. By contrast, when a simple numerical TIL percentage score was used this was a strong predictor of OS (HR, 0.55; 95% CI, 0.38-0.78), DSS (HR, 0.25; 95% CI, 0.14-0.44), and MFS (HR, 0.32; 95% CI, 0.21-0.51) in multivariable analysis. The percentage TIL score was also significant when adjusted for the prognostic gold standard, American Joint Committee on Cancer stage: OS (HR, 0.66; 95% CI, 0.46-0.95), DSS (HR, 0.33; 95% CI, 0.19-0.60), and MFS (HR, 0.41; 95% CI, 0.26-0.65). The TIL percentage score was subsequently validated in new cases. In summary, this study strongly confirms that higher amounts of TILs are associated with better prognosis and in addition demonstrates the value of a simplified numerical TIL scoring system.

Breslow density is a novel prognostic feature in cutaneous malignant melanoma.

AIMS: In 1970, Breslow described his eponymously named thickness measurement. No-one has sought to enhance the Breslow thickness (BT). The aim of this study was to demonstrate a proof of concept that the density of melanoma cells at the position where the BT is measured is a morphological prognostic biomarker, which we name the Breslow density (BD). The hypothesis was that the BD has prognostic value for overall survival (OS) and is independent of the BT. METHODS AND RESULTS: We analysed 100 cutaneous melanomas, and followed REMARK guidelines. The BD was the estimated percentage dermal area occupied by melanoma cells in a specified location. The BT and BD had a strong correlation (P = 2.1 × 10-11 ) but, despite this, they were independent prognostic factors for OS in Cox regression [BD hazard ratio (HR) 1.03, P = 0.001849; and BT HR 1.09, P = 0.000146]. This was corroborated by an independent effect on melanoma-specific survival. We assessed whether the BT and BD could be combined into a Breslow score. A prognostic index based on Cox regression coefficients was used, and this showed a marginal improvement in predicted 5-year survival as compared with the BT alone (area under the curve of 94.8% versus 96.7%). CONCLUSIONS: We show a proof of concept that the BD represents a novel morphological prognostic biomarker that is independent of the BT, and that there is potential to combine these into a Breslow score. Larger studies are needed to validate the BD, but the simplicity of this biomarker makes it a strong candidate for translation to clinical practice.

5-Hydroxymethylcytosine is an independent predictor of survival in malignant melanoma.

Outcomes for melanoma patients vary within cancer stage. Prognostic biomarkers are potential adjuncts to provide more precise prognostic information. Simple, low-cost biomarker assays, such as those based on immunohistochemistry, have strong translational potential. 5-hydroxymethylcytosine (5 hmC) shows prognostic potential in melanoma but prior studies were small. We, therefore, analysed 5 hmC in a retrospective cohort to provide external validation of its prognostic value. Two hundred primary melanomas were evaluated for 5 hmC expression using immunohistochemistry. The primary objective was to assess the effect on overall survival while controlling for important confounders. Univariable and multivariable analyses were performed. REMARK guidelines were followed. The 5 hmC immunohistochemistry scoring showed very strong inter-observer agreement (ICC 0.88) and expression was significantly related to age, site, Breslow thickness, ulceration, mitotic rate, and stage. Kaplan-Meier analysis showed 5 hmC was associated with metastasis-free, melanoma-specific, and overall survival, P<0.0001 for each. In univariable Cox proportional hazards models, 5 hmC hazard ratios were significant and remained so in a multivariable model. A two-step cox model was created using stage and 5 hmC, as stage is the gold standard for clinical practice. The addition of 5 hmC produced significant improvement in the model and 5 hmC and stage were independent significant predictors. This is the largest study of the prognostic value of 5 hmC immunohistochemistry in melanoma. The 5 hmC scoring was easily and reproducibly performed and it was an independent predictor of metastasis-free survival, melanoma-specific survival, and overall survival. This work supports further development of 5 hmC as a prognostic biomarker and suggests that it could add more precision to American Joint Committee on Cancer staging.

Identification of clinical candidates from the benzazepine class of histamine H3 receptor antagonists.

This Letter describes the discovery of GSK189254 and GSK239512 that were progressed as clinical candidates to explore the potential of H3 receptor antagonists as novel therapies for the treatment of Alzheimer's disease and other dementias. By carefully controlling the physicochemical properties of the benzazepine series and through the implementation of an aggressive and innovative screening strategy that employed high throughput in vivo assays to efficiently triage compounds, the medicinal chemistry effort was able to rapidly progress the benzazepine class of H3 antagonists through to the identification of clinical candidates with robust in vivo efficacy and excellent developability properties.

The discovery of the benzazepine class of histamine H3 receptor antagonists.

This Letter describes the discovery of a novel series of H3 receptor antagonists. The initial medicinal chemistry strategy focused on deconstructing and simplifying an early screening hit which rapidly led to the discovery of a novel series of H3 receptor antagonists based on the benzazepine core. Employing an H3 driven pharmacodynamic model, the series was then further optimised through to a lead compound that showed robust in vivo functional activity and possessed overall excellent developability properties.

Discovery of biaryl inhibitors of H+/K+ ATPase.

We report the identification of a novel biaryl template for H(+)/K(+) ATPase inhibition. Evaluation of critical SAR features within the biaryl imidazole framework and the use of pharmacophore modelling against known imidazopyridine and azaindole templates suggested that the geometry of the molecule is key to achieving activity. Herein we present our work optimising the potency of the molecule through modifications and substitutions to each of the ring systems. In particular sub-micromolar potency is achieved with (4b) presumably through a proposed intramolecular hydrogen bond that ensures the required imidazole basic centre is appropriately located.

Evaluation of basic, heterocyclic ring systems as templates for use as potassium competitive acid blockers (pCABs).

A variety of basic, heterocyclic templates has been reported as potassium-competitive, acid pump antagonists. Herein, we report a comparison of potencies of these templates and others to establish which offers the best start point for further systematic optimisation. Modifications were carried out to improve the developability profile of the more potent 1H-pyrrolo[2,3-c]pyridine template, affording molecules with improved overall in vitro characteristics versus the reported clinical candidate AR-H047108, and comparable to the clinically efficacious AZD-0865.

Orally active C-6 heteroaryl- and heterocyclyl-substituted imidazo[1,2-a]pyridine acid pump antagonists (APAs).

Acid pump antagonists (APAs) such as the imidazo[1,2-a]pyridine AZD-0865 2 have proven efficacious at low oral doses in acid related gastric disorders. Herein we describe some of the broader SAR in this class of molecule and detail the discovery of an imidazo[1,2-a]pyridine 15 which has excellent efficacy in animal models of gastric acid secretion following oral administration, as well as a good overall developability profile. The discovery strategy focuses on use of heteroaryl and heterocyclic substituents at the C-6 position and optimization of developability characteristics through modulation of global physico-chemical properties.