Data_Sheet_1_Pharmacokinetics and tolerability of the dual TORC1/2 inhibitor sapanisertib in combination with the MEK inhibitor trametinib in dogs.docx

Activation of one or both the Ras/MAPK and PI3K/Akt/mTOR signal transduction pathways are known to mediate oncogenicity of several canine and human cancers, including mucosal melanomas. Reciprocal cross activation between the two pathways can be a source of drug resistance. Consequently, oral dosing for plasma pharmacokinetic (PK) analysis and tolerability to a combination of sapanisertib, a dual TORC1/2 inhibitor, and trametinib, a MEK inhibitor, was evaluated in nontumor-bearing laboratory dogs for its potential application in parallel pathway targeting. Twelve dogs, divided into three equal cohorts, received either the combination or single agents. Animals were monitored for PK following single dose and 17-day repeat dosing, and by clinical observations, hematology, serum biochemistry, coagulation studies and urinalyses. A single trametinib dose (0.025 mg/kg), sulfated as dimethyl sulfoxide which enhanced its absorption, reached mean maximum concentration (Cmax) 0.64 ng/mL [18% coefficient of variation (CV)] at a median time to maximum concentration (Tmax) of 1.5 h (hr), and mean area under the concentration-time curve (AUC) 16.8 hr*ng/mL (14%CV), which were similar when given alone or in combination with sapanisertib. A prolonged half-life afforded 3–4-fold plasma accumulation of trametinib with daily dosing, analogous to humans. Trametinib PK mirrored previous regulatory data in dogs, while exposure approximated some published human values but generally not all patients. Sapanisertib-alone in canine plasma following single 0.1 mg/kg dose [mean Cmax 26.3 ng/mL (21%CV), median Tmax 2.0 hr, and mean AUC 248 hr*ng/mL (41%CV)] resembled levels in human therapeutic trials; whereas canine sapanisertib exposure was reduced when combined with trametinib, a known cytochrome P450 CYP3A4 inducer. Sex differences were not observed for either drug. Side effects upon repeat dosing with either or both drugs may include body weight loss, maldigestion, and cutaneous discoloration. The combination was tolerated without dose limiting toxicity, although clinical laboratory analyses revealed drug-induced acute-phase inflammation, proteinuria, and decreased blood reticulocytes, mild changes not necessitating intervention. Short-term results in dogs with this combination would appear to hold translational promise for clinical trial evaluation to target canine and possibly human melanoma, as well as other cancers having one or both signal transduction pathway activations.</p

Data_Sheet_2_Pharmacokinetics and tolerability of the dual TORC1/2 inhibitor sapanisertib in combination with the MEK inhibitor trametinib in dogs.docx

Activation of one or both the Ras/MAPK and PI3K/Akt/mTOR signal transduction pathways are known to mediate oncogenicity of several canine and human cancers, including mucosal melanomas. Reciprocal cross activation between the two pathways can be a source of drug resistance. Consequently, oral dosing for plasma pharmacokinetic (PK) analysis and tolerability to a combination of sapanisertib, a dual TORC1/2 inhibitor, and trametinib, a MEK inhibitor, was evaluated in nontumor-bearing laboratory dogs for its potential application in parallel pathway targeting. Twelve dogs, divided into three equal cohorts, received either the combination or single agents. Animals were monitored for PK following single dose and 17-day repeat dosing, and by clinical observations, hematology, serum biochemistry, coagulation studies and urinalyses. A single trametinib dose (0.025 mg/kg), sulfated as dimethyl sulfoxide which enhanced its absorption, reached mean maximum concentration (Cmax) 0.64 ng/mL [18% coefficient of variation (CV)] at a median time to maximum concentration (Tmax) of 1.5 h (hr), and mean area under the concentration-time curve (AUC) 16.8 hr*ng/mL (14%CV), which were similar when given alone or in combination with sapanisertib. A prolonged half-life afforded 3–4-fold plasma accumulation of trametinib with daily dosing, analogous to humans. Trametinib PK mirrored previous regulatory data in dogs, while exposure approximated some published human values but generally not all patients. Sapanisertib-alone in canine plasma following single 0.1 mg/kg dose [mean Cmax 26.3 ng/mL (21%CV), median Tmax 2.0 hr, and mean AUC 248 hr*ng/mL (41%CV)] resembled levels in human therapeutic trials; whereas canine sapanisertib exposure was reduced when combined with trametinib, a known cytochrome P450 CYP3A4 inducer. Sex differences were not observed for either drug. Side effects upon repeat dosing with either or both drugs may include body weight loss, maldigestion, and cutaneous discoloration. The combination was tolerated without dose limiting toxicity, although clinical laboratory analyses revealed drug-induced acute-phase inflammation, proteinuria, and decreased blood reticulocytes, mild changes not necessitating intervention. Short-term results in dogs with this combination would appear to hold translational promise for clinical trial evaluation to target canine and possibly human melanoma, as well as other cancers having one or both signal transduction pathway activations.</p

Digital scanning and diagnostic scoring of kidney biopsies from children with steroid resistant nephritic syndrome

<p><strong>Background:</strong> Digital pathology is an attractive new tool for multicenter research. Practical improvements on conventional pathology include parallel evaluation by several experts in distant locations and reproducible identification of individual lesions. Also, digital image analysis allows more objective measures of cell staining and 3D reconstruction of glomeruli. </p> <p> </p> <p><strong>Design:</strong> We are establishing a digital pathology archive within WP2 of EURenOmics. Kidney biopsies from children with steroid resistant nephrotic syndrome (SRNS) enrolled in the PodoNet registry are collected. Analysis will be harmonized with digital pathology scoring developed by the NEPTUNE study (Nephrotic Syndrome Study Network) in the US.</p> <p> </p> <p><strong>Methods:</strong> The material is scanned at high resolution in Heidelberg at the Hamamatsu Tissue Image and AnalysisCenter using a Hamamatsu Nanozoomer. Digital images of existing electron microscopy and immunofluorescence scans and original pathology reports are also collected. After anonymization and manual quality controls (e.g. checking correct focus in all areas) data is stored centrally for remote review.</p> <p>Digital images are first annotated, i.e. each glomerulus is given a number which can be tracked across different section levels. Histopathological scoring of each glomerulus in each section level will then be performed in an unbiased fashion by independent blinded pathologists. These scores will be correlated to clinical and genetic information, conventional histopathological diagnoses and molecular profiles obtained by multi-omics profiling. </p> <p> </p> <p><strong>Progress:</strong> So far 83 kidney biopsies have been collected and at least another 180 are expected. Test runs of different stain types have produced good quality images. Bulk scanning and annotations are commencing, so that pathology reviews will start in spring 2014. </p> <p> </p> <p><strong>Outlook:</strong> The new digital pathology archive for children with SRNS will enable standardized review and objective scoring of histopathological findings. We hope that this will improve correlations of histological findings with clinical outcomes and biomarkers. Setting up the relevant infrastructure will allow extension of the project to other registries within WP2.</p

Morphometry Predicts Early GFR Change in Primary Proteinuric Glomerulopathies: A Longitudinal Cohort Study Using Generalized Estimating Equations

<div><p>Objective</p><p>Most predictive models of kidney disease progression have not incorporated structural data. If structural variables have been used in models, they have generally been only semi-quantitative.</p><p>Methods</p><p>We examined the predictive utility of quantitative structural parameters measured on the digital images of baseline kidney biopsies from the NEPTUNE study of primary proteinuric glomerulopathies. These variables were included in longitudinal statistical models predicting the change in estimated glomerular filtration rate (eGFR) over up to 55 months of follow-up.</p><p>Results</p><p>The participants were fifty-six pediatric and adult subjects from the NEPTUNE longitudinal cohort study who had measurements made on their digital biopsy images; 25% were African-American, 70% were male and 39% were children; 25 had focal segmental glomerular sclerosis, 19 had minimal change disease, and 12 had membranous nephropathy. We considered four different sets of candidate predictors, each including four quantitative structural variables (for example, mean glomerular tuft area, cortical density of patent glomeruli and two of the principal components from the correlation matrix of six fractional cortical areas–interstitium, atrophic tubule, intact tubule, blood vessel, sclerotic glomerulus, and patent glomerulus) along with 13 potentially confounding demographic and clinical variables (such as race, age, diagnosis, and baseline eGFR, quantitative proteinuria and BMI). We used longitudinal linear models based on these 17 variables to predict the change in eGFR over up to 55 months. All 4 models had a leave-one-out cross-validated R² of about 62%.</p><p>Conclusions</p><p>Several combinations of quantitative structural variables were significantly and strongly associated with changes in eGFR. The structural variables were generally stronger than any of the confounding variables, other than baseline eGFR. Our findings suggest that quantitative assessment of diagnostic renal biopsies may play a role in estimating the baseline risk of succeeding loss of renal function in future clinical studies, and possibly in clinical practice.</p></div

Observed eGFR progression patterns over follow-up stratified by quartiles of FATA.

<p>Linear fits to observed eGFR values stratified by FATA from lowest (red) to highest (purple). Initial eGFR is highest for the lowest FATA quartile and decreases with each quartile. Compared to the reference category of the lowest FATA quartile, testing for the differential eGFR slopes of the second, third and fourth quartiles yielded P values of 0.15, 0.25 and <0.001, respectively. The apparent more negative slope of the lowest quartile may be due to a smaller number of long follow-up points.</p

Illustration of the point counting method on a section of cortex (PAS stain).

<p>Illustration of the point counting principle for assessing cortical compartments using SlidePath software. The hand-written numbers (10, 11, 12, 13 and part of 20) were added by the slide annotator, to keep track of individual glomeruli. Cross points of the red grid box were used other than the upper and right-side lines (equivalently, using the bottom-left corner point of each grid sub-box). Starting at the top-left, the first point ‘hits’ an intact glomerulus (#12). Moving right, the second point hits an intact tubule. The third hits an intact glomerulus (#11). Twenty-five points per grid are evaluated.</p

Pearson correlations among fractional cortical areas.

<p>Pearson correlations among fractional cortical areas.</p

Synaptonemal Complex Protein 3 Is a Prognostic Marker in Cervical Cancer

<div><p>Synaptonemal complex protein 3 (SCP3), a member of Cor1 family, is up-regulated in various cancer cells; however, its oncogenic potential and clinical significance has not yet been characterized. In the present study, we investigated the oncogenic role of SCP3 and its relationship with phosphorylated AKT (pAKT) in cervical neoplasias. The functional role of SCP3 expression was investigated by overexpression or knockdown of SCP3 in murine cell line NIH3T3 and human cervical cancer cell lines CUMC6, SiHa, CaSki, and HeLa both <i>in vitro</i> and <i>in vivo</i>. Furthermore, we examined SCP3 expression in tumor specimens from 181 cervical cancer and 400 cervical intraepithelial neoplasia (CIN) patients by immunohistochemistry and analyzed the correlation between SCP3 expression and clinicopathologic factors or survival. Overexpression of SCP3 promoted AKT-mediated tumorigenesis both <i>in vitro</i> and <i>in vivo</i>. Functional studies using NIH3T3 cells demonstrated that the C-terminal region of human SCP3 is important for AKT activation and its oncogenic potential. High expression of SCP3 was significantly associated with tumor stage (<i>P</i> = 0.002) and tumor grade (<i>P</i><0.001), while SCP3 expression was positively associated with pAKT protein level in cervical neoplasias. Survival times for patients with cervical cancer overexpressing both SCP3 and pAKT (median, 134.0 months, <i>n</i> = 68) were significantly shorter than for patients with low expression of either SCP3 or pAKT (161.5 months, n = 108) as determined by multivariate analysis (<i>P</i> = 0.020). Our findings suggest that SCP3 plays an important role in the progression of cervical cancer through the AKT signaling pathway, supporting the possibility that SCP3 may be a promising novel cancer target for cervical cancer therapy.</p></div

hSCP3 increases tumorigenesis through AKT pathway.

<p>(A) Western blot analysis of levels of pAKT, pERK, and pp38 in NIH3T3 cells ectopically expressing hSCP3 or no insert. Numbers below western blots refer to the relative values of the intensity normalized to no insert control. (B) <i>In vitro</i> growth curves of NIH3T3/hSCP3 cells treated with DMSO or API2 (Akt inhibitor), PD98059 (Erk inhibitor), or SB203580 (p38 inhibitor). (C) Soft agar colony-forming capacity of NIH3T3/hSPC3 cells in the presence of API2, PD98059, or SB203580; (C, Right) Representative images of average colony size in each group; (C, Left) bar graph showing the number of colonies with diameters greater than 2 mm in soft agar (scale bar: 1 mm). (D) Western blot analysis of levels of pAKT in NIH3T3/hSCP3 cells transfected with a siRNA targeting GFP or AKT (siGFP or siAKT) to confirm the reduction of AKT protein level. Numbers below western blots refer to the relative values of the intensity normalized to no insert control. (E) <i>In vitro</i> growth curves of NIH3T3/hSCP3 cells transfected with siGFP or siAKT. (F) Soft agar colony-forming capacity of NIH3T3/hSPC3 cells treated with siGFP or siAKT; (Left) Representative images of average colony size in each group; (Right) Bar graph representing the number of colonies with diameters greater than 2 mm in soft agar (scale bar: 1 mm). Error bars represent the mean ± SD.</p

Association between SCP3 and pAKT expression in CIN and cervical cancer.

<p>CIN, cervical intraepithelial neoplasia.</p