Evaluation of early treatment with intravenous idursulfase and intrathecal idursulfase-IT on cognitive function in siblings with neuronopathic mucopolysaccharidosis II.

Mucopolysaccharidosis II (MPS II; Hunter syndrome; OMIM 309900) is a rare, X-linked, heterogeneous lysosomal storage disease. Approximately two-thirds of patients develop cognitive impairment, which is difficult to assess in clinical trials, partly owing to the variable nature of cognitive impairment. Analyzing data from siblings can help to minimize this heterogeneity. We report analyses of cognitive function from siblings with MPS II enrolled in clinical trials: a natural history study (NCT01822184), a randomized, open-label, phase 2/3 study of intravenous (IV) idursulfase with or without intrathecal idursulfase (idursulfase-IT; NCT02055118), and its extension (NCT2412787). Cognitive function was assessed using Differential Abilities Scales, Second Edition General Conceptual Ability (DAS-II GCA) scores; Bayley Scales of Infant and Toddler Development, Third Edition; and Vineland Adaptive Behavior Scales, Second Edition Adaptive Behavior Composite (VABS-II ABC). Seven sets of siblings (six pairs and one set of three) were included. All patients received IV idursulfase and 10 received subsequent idursulfase-IT. Younger siblings initiated IV idursulfase at an earlier age than their older sibling(s) in six of the sets; the younger sibling started treatment before 1 year of age in three sets. Monthly idursulfase-IT was generally associated with a stabilization of cognitive function: DAS-II GCA and VABS-II ABC scores were higher at age-matched assessments in the majority of those who either received idursulfase-IT earlier than their sibling or who received idursulfase-IT versus no idursulfase-IT. These data suggest that early initiation of intrathecal enzyme replacement therapy may stabilize or slow cognitive decline in some patients with neuronopathic MPS II.

Diagnostic and Prognostic Markers for Pancreatitis and Pancreatic Ductal Adenocarcinoma.

Diagnostic markers are desperately needed for the early detection of pancreatic ductal adenocarcinoma (PDA). We describe sets of markers expressed in temporal order in mouse models during pancreatitis, PDA initiation and progression. Cell type specificity and the differential expression of PDA markers were identified by screening single cell (sc) RNAseq from tumor samples of a mouse model for PDA (KIC) at early and late stages of PDA progression compared to that of a normal pancreas. Candidate genes were identified from three sources: (1) an unsupervised screening of the genes preferentially expressed in mouse PDA tumors; (2) signaling pathways that drive PDA, including the Ras pathway, calcium signaling, and known cancer genes, or genes encoding proteins that were identified by differential mass spectrometry (MS) of mouse tumors and conditioned media from human cancer cell lines; and (3) genes whose expression is associated with poor or better prognoses (PAAD, oncolnc.org). The developmental progression of PDA was detected in the temporal order of gene expression in the cancer cells of the KIC mice. The earliest diagnostic markers were expressed in epithelial cancer cells in early-stage, but not late-stage, PDA tumors. Other early markers were expressed in the epithelium of both early- and late-state PDA tumors. Markers that were expressed somewhat later were first elevated in the epithelial cancer cells of the late-stage tumors, then in both epithelial and mesenchymal cells, or only in mesenchymal cells. Stromal markers were differentially expressed in early- and/or late-stage PDA neoplasia in fibroblast and hematopoietic cells (lymphocytes and/or macrophages) or broadly expressed in cancer and many stromal cell types. Pancreatitis is a risk factor for PDA in humans. Mouse models of pancreatitis, including caerulein treatment and the acinar-specific homozygous deletion of differentiation transcription factors (dTFs), were screened for the early expression of all PDA markers identified in the KIC neoplasia. Prognostic markers associated with a more rapid decline were identified and showed differential and cell-type-specific expression in PDA, predominately in late-stage epithelial and/or mesenchymal cancer cells. Select markers were validated by immunohistochemistry in mouse and human samples of a normal pancreas and those with early- and late-stage PDA. In total, we present 2165 individual diagnostic and prognostic markers for disease progression to be tested in humans from pancreatitis to late-stage PDA.

Long-term open-label extension study of the safety and efficacy of intrathecal idursulfase-IT in patients with neuronopathic mucopolysaccharidosis II.

Enzyme replacement therapy with weekly infused intravenous (IV) idursulfase is effective in treating somatic symptoms of mucopolysaccharidosis II (MPS II; Hunter syndrome). A formulation of idursulfase for intrathecal administration (idursulfase-IT) is under investigation for the treatment of neuronopathic MPS II. Here, we report 36-month data from the open-label extension (NCT02412787) of a phase 2/3, randomized, controlled study (HGT-HIT-094; NCT02055118) that assessed the safety and efficacy of monthly idursulfase-IT 10 mg in addition to weekly IV idursulfase on cognitive function in children older than 3 years with MPS II and mild-to-moderate cognitive impairment. Participants were also enrolled in this extension from a linked non-randomized sub-study of children younger than 3 years at the start of idursulfase-IT therapy. The extension safety population comprised 56 patients who received idursulfase-IT 10 mg once a month (or age-adjusted dose for sub-study patients) plus IV idursulfase (0.5 mg/kg) once a week. Idursulfase-IT was generally well tolerated over the cumulative treatment period of up to 36 months. Overall, 25.0% of patients had at least one adverse event (AE) related to idursulfase-IT; most treatment-emergent AEs were mild in severity. Of serious AEs (reported by 76.8% patients), none were considered related to idursulfase-IT treatment. There were no deaths or discontinuations owing to AEs. Secondary efficacy analyses (in patients younger than 6 years at phase 2/3 study baseline; n = 40) indicated a trend for improved Differential Ability Scale-II (DAS-II) General Conceptual Ability (GCA) scores in the early idursulfase-IT versus delayed idursulfase-IT group (treatment difference over 36 months from phase 2/3 study baseline: least-squares mean, 6.8 [90% confidence interval: -2.1, 15.8; p = 0.2064]). Post hoc analyses of DAS-II GCA scores by genotype revealed a clinically meaningful treatment effect in patients younger than 6 years with missense variants of the iduronate-2-sulfatase gene (IDS) (least-squares mean [standard error] treatment difference over 36 months, 12.3 [7.24]). These long-term data further suggest the benefits of idursulfase-IT in the treatment of neurocognitive dysfunction in some patients with MPS II. After many years of extensive review and regulatory discussions, the data were found to be insufficient to meet the evidentiary standard to support regulatory filings.

Intrathecal idursulfase-IT in patients with neuronopathic mucopolysaccharidosis II: Results from a phase 2/3 randomized study.

Two-thirds of patients with mucopolysaccharidosis II (MPS II; Hunter syndrome) have cognitive impairment. This phase 2/3, randomized, controlled, open-label, multicenter study (NCT02055118) investigated the effects of intrathecally administered idursulfase-IT on cognitive function in patients with MPS II. Children older than 3 years with MPS II and mild-to-moderate cognitive impairment (assessed by Differential Ability Scales-II [DAS-II], General Conceptual Ability [GCA] score) who had tolerated intravenous idursulfase for at least 4 months were randomly assigned (2:1) to monthly idursulfase-IT 10 mg (n = 34) via an intrathecal drug delivery device (IDDD; or by lumbar puncture) or no idursulfase-IT treatment (n = 15) for 52 weeks. All patients continued to receive weekly intravenous idursulfase 0.5 mg/kg as standard of care. Of 49 randomized patients, 47 completed the study (two patients receiving idursulfase-IT discontinued). The primary endpoint (change from baseline in DAS-II GCA score at week 52 in a linear mixed-effects model for repeated measures analysis) was not met: although there was a smaller decrease in DAS-II GCA scores with idursulfase-IT than with no idursulfase-IT at week 52, this was not significant (least-squares mean treatment difference [95% confidence interval], 3.0 [-7.3, 13.3]; p = 0.5669). Changes from baseline in Vineland Adaptive Behavioral Scales-II Adaptive Behavior Composite scores at week 52 (key secondary endpoint) were similar in the idursulfase-IT (n = 31) and no idursulfase-IT (n = 14) groups. There were trends towards a potential positive effect of idursulfase-IT across DAS-II composite, cluster, and subtest scores, notably in patients younger than 6 years at baseline. In a post hoc analysis, there was a significant (p = 0.0174), clinically meaningful difference in change from baseline in DAS-II GCA scores at week 52 with idursulfase-IT (n = 13) versus no idursulfase-IT (n = 6) among those younger than 6 years with missense iduronate-2-sulfatase gene variants. Overall, idursulfase-IT reduced cerebrospinal glycosaminoglycan levels from baseline by 72.0% at week 52. Idursulfase-IT was generally well tolerated. These data suggest potential benefits of idursulfase-IT in the treatment of cognitive impairment in some patients with neuronopathic MPS II. After many years of extensive review and regulatory discussions, the data were found to be insufficient to meet the evidentiary standard to support regulatory filings.

Development and Validation of a Test to Monitor Endoscopic Activity in Patients With Crohn's Disease Based on Serum Levels of Proteins.

BACKGROUND & AIMS: Noninvasive tests to measure endoscopic activity in patients with Crohn's disease (CD) have limitations. We aimed to develop a test to identify patients in remission, based on endoscopic analysis, and monitor CD activity based on serum levels of proteins. METHODS: We developed a test to measure 13 proteins in blood (ANG1, ANG2, CRP, SAA1, IL7, EMMPRIN, MMP1, MMP2, MMP3, MMP9, TGFA, CEACAM1, and VCAM1), called the endoscopic healing index [EHI], using samples from 278 patients with CD from a multinational training cohort. We validated the test using 2 independent cohorts of patients with CD: 116 biologic-naive patients with early-stage CD (validation cohort 1) and 195 biologic-exposed patients with chronic CD (validation cohort 2). The ability of the test to identify patients with active disease vs patients in remission (defined as a simple endoscopic score for CD of ≤2 and ≤1 in each segment, or a total CD endoscopic index of severity score <3) was assessed by using area under receiver operating characteristic curve (AUROC) analysis. The diagnostic accuracy of the test was compared with that of measurement of serum C-reactive protein (CRP) and fecal calprotectin. RESULTS: The EHI scores range from 0 to 100 units; higher scores indicate more severe CD activity, based on endoscopy findings. The EHI identified patients in remission with an AUROC of 0.962 in validation cohort 1 (95% confidence interval, 0.942-0.982) and an AUROC of 0.693 in validation cohort 2 (95% confidence interval, 0.619-0.767), regardless of CD location or phenotype. A cutoff value of 20 points identified patients in remission with the highest level of sensitivity (97.1% in validation cohort 1 and 83.2% in validation cohort 2), with specificity values of 69.0% and 36.6%, respectively. A cutoff value of 50 points identified patients in remission with the highest level of specificity (100% in validation cohort 1 and 87.8% in validation cohort 2), with sensitivity values of 37.3% and 30.0%, respectively. The EHI identified patients in remission with a significantly higher AUROC value than the test for CRP (0.876, P < .001 in validation cohort 1 and 0.624, P = .109 in validation cohort 2). In analysis of patients with available FC measurements, the AUROC value for the EHI did not differ significantly from that of measurement of FC (AUROC, 0.950 for EHI vs AUROC, 0.923 for FC; P = .147 in validation cohort 1 and AUROC, 0.803 for EHI vs AUROC, 0.854 for FC; P = .298 in validation cohort 2). CONCLUSIONS: We developed an index called the EHI to identify patients with CD in endoscopic remission based on blood levels of 13 proteins. The EHI identified patients with resolution of endoscopic disease activity, with good overall accuracy, although with variation between the 2 cohorts assessed. The EHI AUROC values were comparable to measurement of FC and higher than measurement of serum CRP. The test might be used in practice to assess endoscopic activity in patients with CD.

Assessing goodness-of-fit for evaluation of dose-proportionality.

For the clinical development of a new drug, the determination of dose-proportionality is an essential part of the pharmacokinetic evaluations, which may provide early indications of non-linear pharmacokinetics and may help to identify sub-populations with divergent clearances. Prior to making any conclusions regarding dose-proportionality, the goodness-of-fit of the model must be assessed to evaluate the model performance. We propose the use of simulation-based visual predictive checks to improve the validity of dose-proportionality conclusions for complex designs. We provide an illustrative example and include a table to facilitate review by regulatory authorities.

Ex vivo mechanical testing of various suture patterns for use in tendon plating.

OBJECTIVE: To determine the influence of suture patterns on resistance to gap formation after tendon plating STUDY DESIGN: Ex vivo study SAMPLE POPULATION: Suspensory ligament and superficial and deep digital flexor tendons harvested from 16 neonatal cadaver foal limbs. METHODS: Each tendon/ligament from a given limb was randomly assigned to one of three treatment groups consisting of a 7-hole veterinary cuttable plate fixed with alternating simple interrupted, figure-8, or hybrid suture patterns. The constructs were distracted at a rate of 1.0 mm/s to failure, as determined by the formation of a 1 mm gap for initial failure and a 3 mm gap for ultimate failure. The mode of failure was also recorded. RESULTS: Constructs prepared with hybrid and figure-8 patterns sustained over 2× higher (p < .01) forces before gap formation compared to those prepared with a simple interrupted pattern. No difference was identified between tendons whose plate was secured with hybrid or figure-8 suture patterns. Mode of failure between groups did not differ. CONCLUSION: The figure-8 and hybrid patterns described herein improved the resistance to gap formation of plated tendons compared to an alternating simple interrupted pattern described previously. CLINICAL SIGNIFICANCE: Figure-8 and hybrid patterns should be considered over simple interrupted patterns to improve resistance to gap formation in horses undergoing tendon plating.

Malnutrition in Pancreatic Ductal Adenocarcinoma (PDA): Dietary Pancreatic Enzymes Improve Short-Term Health but Stimulate Tumor Growth.

Pancreatic ductal adenocarcinoma (PDA) is a deadly cancer that resists efforts to identify better chemotherapeutics. PDA is associated with chronic pancreatitis and acinar cell dedifferentiation. This reduces enzyme production by the exocrine pancreas, resulting in digestive insufficiencies. Malabsorption of partially digested food causes bloating, overfilled intestines, abdominal pain, excessive feces, steatorrhea, and malnutrition. These maladies affect quality of life and restrict treatment options for pancreatitis and PDA. Here, we characterize health benefits and risks of dietary pancreatic enzymes in three mouse models of PDA-KC, KCR8-16, and KIC. KC expresses oncogenic KrasG12D in pancreatic tissue whereas KCR8-16 also has deletions of the Rgs8 and Rgs16 genes. Rgs proteins inhibit the release of digestive enzymes evoked by G-protein-coupled-receptor agonists. KC and KCR8-16 mice developed dedifferentiated exocrine pancreata within 2 months of age and became malnourished, underweight, hypoglycemic, and hypothermic. KC mice adapted but KCR8-16 mice rapidly transitioned to starvation after mild metabolic challenges. Dietary pancreatic enzyme supplements reversed these symptoms in KC and KCR8-16 animals, and extended survival. Therefore, we tested the benefits of pancreatic enzymes in an aggressive mouse model of PDA (KIC). Median survival improved with dietary pancreatic enzyme supplements and was extended further when combined with warfarin and gemcitabine chemotherapy. However, dietary pancreatic enzymes stimulated tumor growth in the terminal stages of disease progression in KIC mice.

Application of tendon plating to manage failed calcaneal tendon repairs in a dog.

OBJECTIVE: To report the application of a plate to treat a complicated calcaneal tendon rupture and the resulting outcome. STUDY DESIGN: Clinical case report. ANIMALS: A 3-year-old Labrador retriever. METHODS: A 3-year-old Labrador retriever was treated for a partially ruptured calcaneal tendon. Two primary tendon repairs with sutures and external fixation had failed. A veterinary cuttable plate was placed to internally span the tendon over the primary repair. The external fixation that had been previously placed was maintained, and the dog was placed in a non-weight-bearing sling for 3 weeks after surgery. Adjunct therapy included an injection of platelet-rich plasma in the tendon and laser therapy. The external fixator was removed 4 weeks postoperatively, and the plate was removed 8 weeks postoperatively. RESULTS: A mature fibrous union bridged the site of anastomosis 8 weeks after surgery. The owners reported normal activity 6 months after surgery and symmetric hock angles and musculature 2 years after surgery. CONCLUSION: Adjunct plating of previously failed calcaneal tendon repairs in a dog resulted in an excellent long-term outcome. CLINICAL SIGNIFICANCE: Plating tendons can be considered as an additional method that may improve tendon apposition and decrease gap formation.

Dominant and context-specific control of endodermal organ allocation by Ptf1a.

The timing and gene regulatory logic of organ-fate commitment from within the posterior foregut of the mammalian endoderm is largely unexplored. Transient misexpression of a presumed pancreatic-commitment transcription factor, Ptf1a, in embryonic mouse endoderm (Ptf1a(EDD)) dramatically expanded the pancreatic gene regulatory network within the foregut. Ptf1a(EDD) temporarily suppressed Sox2 broadly over the anterior endoderm. Pancreas-proximal organ territories underwent full tissue conversion. Early-stage Ptf1a(EDD) rapidly expanded the endogenous endodermal Pdx1-positive domain and recruited other pancreas-fate-instructive genes, thereby spatially enlarging the potential for pancreatic multipotency. Early Ptf1a(EDD) converted essentially the entire glandular stomach, rostral duodenum and extrahepatic biliary system to pancreas, with formation of many endocrine cell clusters of the type found in normal islets of Langerhans. Sliding the Ptf1a(EDD) expression window through embryogenesis revealed differential temporal competencies for stomach-pancreas respecification. The response to later-stage Ptf1a(EDD) changed radically towards unipotent, acinar-restricted conversion. We provide strong evidence, beyond previous Ptf1a inactivation or misexpression experiments in frog embryos, for spatiotemporally context-dependent activity of Ptf1a as a potent gain-of-function trigger of pro-pancreatic commitment.

The nuclear hormone receptor family member NR5A2 controls aspects of multipotent progenitor cell formation and acinar differentiation during pancreatic organogenesis.

The orphan nuclear receptor NR5A2 is necessary for the stem-like properties of the epiblast of the pre-gastrulation embryo and for cellular and physiological homeostasis of endoderm-derived organs postnatally. Using conditional gene inactivation, we show that Nr5a2 also plays crucial regulatory roles during organogenesis. During the formation of the pancreas, Nr5a2 is necessary for the expansion of the nascent pancreatic epithelium, for the subsequent formation of the multipotent progenitor cell (MPC) population that gives rise to pre-acinar cells and bipotent cells with ductal and islet endocrine potential, and for the formation and differentiation of acinar cells. At birth, the NR5A2-deficient pancreas has defects in all three epithelial tissues: a partial loss of endocrine cells, a disrupted ductal tree and a >90% deficit of acini. The acinar defects are due to a combination of fewer MPCs, deficient allocation of those MPCs to pre-acinar fate, disruption of acinar morphogenesis and incomplete acinar cell differentiation. NR5A2 controls these developmental processes directly as well as through regulatory interactions with other pancreatic transcriptional regulators, including PTF1A, MYC, GATA4, FOXA2, RBPJL and MIST1 (BHLHA15). In particular, Nr5a2 and Ptf1a establish mutually reinforcing regulatory interactions and collaborate to control developmentally regulated pancreatic genes by binding to shared transcriptional regulatory regions. At the final stage of acinar cell development, the absence of NR5A2 affects the expression of Ptf1a and its acinar specific partner Rbpjl, so that the few acinar cells that form do not complete differentiation. Nr5a2 controls several temporally distinct stages of pancreatic development that involve regulatory mechanisms relevant to pancreatic oncogenesis and the maintenance of the exocrine phenotype.

Translating slow-binding inhibition kinetics into cellular and in vivo effects.

Many drug candidates fail in clinical trials owing to a lack of efficacy from limited target engagement or an insufficient therapeutic index. Minimizing off-target effects while retaining the desired pharmacodynamic (PD) response can be achieved by reduced exposure for drugs that display kinetic selectivity in which the drug-target complex has a longer half-life than off-target-drug complexes. However, though slow-binding inhibition kinetics are a key feature of many marketed drugs, prospective tools that integrate drug-target residence time into predictions of drug efficacy are lacking, hindering the integration of drug-target kinetics into the drug discovery cascade. Here we describe a mechanistic PD model that includes drug-target kinetic parameters, including the on- and off-rates for the formation and breakdown of the drug-target complex. We demonstrate the utility of this model by using it to predict dose response curves for inhibitors of the LpxC enzyme from Pseudomonas aeruginosa in an animal model of infection.

Negishi cross-coupling enabled synthesis of novel NAD(+)-dependent DNA ligase inhibitors and SAR development.

Two novel compounds, pyridopyrimidines (1) and naphthyridines (2) were identified as potent inhibitors of bacterial NAD(+)-dependent DNA ligase (Lig) A in a fragment screening. SAR was guided by molecular modeling and X-ray crystallography. It was observed that the diaminonitrile pharmacophore made a key interaction with the ligase enzyme, specifically residues Glu114, Lys291, and Leu117. Synthetic challenges limited opportunities for diversification of the naphthyridine core, therefore most of the SAR was focused on a pyridopyrimidine scaffold. The initial diversification at R(1) improved both enzyme and cell potency. Further SAR developed at the R(2) position using the Negishi cross-coupling reaction provided several compounds, among these compounds 22g showed good enzyme potency and cellular potency.

Overexpression of Pseudomonas aeruginosa LpxC with its inhibitors in an acrB-deficient Escherichia coli strain.

In Gram-negative bacteria, the cell wall is surrounded by an outer membrane, the outer leaflet of which is comprised of charged lipopolysaccharide (LPS) molecules. Lipid A, a component of LPS, anchors this molecule to the outer membrane. UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a zinc-dependent metalloamidase that catalyzes the first committed step of biosynthesis of Lipid A, making it a promising target for antibiotic therapy. Formation of soluble aggregates of Pseudomonas aeruginosa LpxC protein when overexpressed in Escherichia coli has limited the availability of high quality protein for X-ray crystallography. Expression of LpxC in the presence of an inhibitor dramatically increased protein solubility, shortened crystallization time and led to a high-resolution crystal structure of LpxC bound to the inhibitor. However, this approach required large amounts of compound, restricting its use. To reduce the amount of compound needed, an overexpression strain of E. coli was created lacking acrB, a critical component of the major efflux pump. By overexpressing LpxC in the efflux deficient strain in the presence of LpxC inhibitors, several structures of P. aeruginosa LpxC in complex with different compounds were solved to accelerate structure-based drug design.

Machine Learning ADME Models in Practice: Four Guidelines from a Successful Lead Optimization Case Study.

Optimization of the ADME properties and pharmacokinetic (PK) profile of compounds is one of the critical activities in any medicinal chemistry campaign to discover a future clinical candidate. Finding ways to expedite the process to address ADME/PK shortcomings and reduce the number of compounds to synthesize is highly valuable. This article provides practical guidelines and a case study on the use of ML ADME models to guide compound design in small molecule lead optimization. These guidelines highlight that ML models cannot have an impact in a vacuum: they help advance a program when they have the trust of users, are tuned to the needs of the program, and are integrated into decision-making processes in a way that complements and augments the expertise of chemists.

The Pan-RAF-MEK Nondegrading Molecular Glue NST-628 Is a Potent and Brain-Penetrant Inhibitor of the RAS-MAPK Pathway with Activity across Diverse RAS- and RAF-Driven Cancers.

Alterations in the RAS-MAPK signaling cascade are common across multiple solid tumor types and are a driver for many cancers. NST-628 is a potent pan-RAF-MEK molecular glue that prevents the phosphorylation and activation of MEK by RAF, overcoming the limitations of traditional RAS-MAPK inhibitors and leading to deep durable inhibition of the pathway. Cellular, biochemical, and structural analyses of RAF-MEK complexes show that NST-628 engages all isoforms of RAF and prevents the formation of BRAF-CRAF heterodimers, a differentiated mechanism from all current RAF inhibitors. With a potent and durable inhibition of the RAF-MEK signaling complex as well as high intrinsic permeability into the brain, NST-628 demonstrates broad efficacy in cellular and patient-derived tumor models harboring diverse MAPK pathway alterations, including orthotopic intracranial models. Given its functional and pharmacokinetic mechanisms that are differentiated from previous therapies, NST-628 is positioned to make an impact clinically in areas of unmet patient need. Significance: This study introduces NST-628, a molecular glue having differentiated mechanism and drug-like properties. NST-628 treatment leads to broad efficacy with high tolerability and central nervous system activity across multiple RAS- and RAF-driven tumor models. NST-628 has the potential to provide transformative clinical benefits as both monotherapy and vertical combination anchor.

Evaluation of CD3 and CD8 T-Cell Immunohistochemistry for Prognostication and Prediction of Benefit From Adjuvant Chemotherapy in Early-Stage Colorectal Cancer Within the QUASAR Trial.

PURPOSE: High densities of tumor infiltrating CD3 and CD8 T-cells are associated with superior prognosis in colorectal cancer (CRC). Their value as predictors of benefit from adjuvant chemotherapy is uncertain. PATIENTS AND METHODS: Tumor tissue from 868 patients in the QUASAR trial (adjuvant fluorouracil/folinic acid v observation in stage II/III CRC) was analyzed by CD3 and CD8 immunohistochemistry. Pathologists, assisted by artificial intelligence, calculated CD3 and CD8 cell densities (cells/mm2) in the core tumor (CT) and invasive margin (IM). Participants were randomly partitioned into training and validation sets. The primary outcome was recurrence-free interval (RFI), 2-year RFI for assessment of biomarker-treatment interactions. Maximum-likelihood methods identified optimal high-risk/low-risk group cutpoints in the training set. Prognostic analyses were repeated in the validation set. RESULTS: In the training set, the recurrence rate in the high-risk group was twice that in the low-risk group for all measures (CD3-CT: rate ratio [RR], 2.00, P = .0008; CD3-IM: 2.38, P < .00001; CD8-CT: 2.17, P = .0001; CD8-IM: 2.13, P = .0001). This was closely replicated in the validation set (RR, 1.96, 1.79, 1.72, 1.72, respectively). In multivariate analyses, prognostic effects were similar in colon and rectal cancers, and in stage II and III disease. Proportional reductions in recurrence with adjuvant chemotherapy were of similar magnitude in the high- and low-recurrence risk groups. Combining information from CD3-IM and CD3-CT (CD3 Score) generated high-, intermediate-, and low-risk groups with numbers needed to treat (NNTs) to prevent one disease recurrence being 11, 21, and 36, respectively. CONCLUSION: Recurrence rates in the high-risk CD3/CD8 groups are twice those in the low-risk groups. Proportional reductions with chemotherapy are similar, allowing NNTs derived in QUASAR to be updated using contemporary, nonrandomized data sets.

Beyond glass transitions: studying the highly viscous and elastic behavior of frozen protein formulations using low temperature rheology and its potential implications on protein stability.

PURPOSE: A novel application of oscillatory shear rheology was used to directly monitor global phase behavior of protein formulations in the frozen state and study its correlation with physical instability of frozen protein formulations. METHODS: Oscillatory rheology was used to measure changes in rheological parameters and to identify mechanical softening temperature (Ts*) and related properties of an IgG2 mAb formulation. Rheological measurements were compared to DSC/MDSC. Physical stability of IgG2 formulations was monitored by SE-HPLC. RESULTS: Rheological parameters and Ts* of an IgG2 formulation were sensitive to physical/morphological phase changes during freezing and thawing. Ts* of the frozen formulation was a function of concentration of protein and excipient. Complex modulus, G*, and phase angle, δ, for IgG2 at 70 mg/mL in a sucrose-containing formulation showed the system was not completely frozen at -10°C, which correlated to stability data consistent with ice-induced protein aggregation. CONCLUSIONS: We report the first application of oscillatory shear rheology to study phase behavior of IgG2 in a sucrose-containing formulation and its correspondence with physical stability not explained by glass transition (Tg'). We provide a mechanism and data suggesting that protein instability occurs at the ice/water interface.