COGNITION: a prospective precision oncology trial for patients with early breast cancer at high risk following neoadjuvant chemotherapy.

BACKGROUND: COGNITION (Comprehensive assessment of clinical features, genomics and further molecular markers to identify patients with early breast cancer for enrolment on marker driven trials) is a diagnostic registry trial that employs genomic and transcriptomic profiling to identify biomarkers in patients with early breast cancer with a high risk for relapse after standard neoadjuvant chemotherapy (NACT) to guide genomics-driven targeted post-neoadjuvant therapy. PATIENTS AND METHODS: At National Center for Tumor Diseases Heidelberg patients were biopsied before starting NACT, and for patients with residual tumors after NACT additional biopsy material was collected. Whole-genome/exome and transcriptome sequencing were applied on tumor and corresponding blood samples. RESULTS: In the pilot phase 255 patients were enrolled, among which 213 were assessable: thereof 48.8% were identified to be at a high risk for relapse following NACT; 86.4% of 81 patients discussed in the molecular tumor board were eligible for a targeted therapy within the interventional multiarm phase II trial COGNITION-GUIDE (Genomics-guided targeted post neoadjuvant therapy in patients with early breast cancer) starting enrolment in Q4/2022. An in-depth longitudinal analysis at baseline and in residual tumor tissue of 16 patients revealed some cases with clonal evolution but largely stable genetic alterations, suggesting restricted selective pressure of broad-acting cytotoxic neoadjuvant chemotherapies. CONCLUSIONS: While most precision oncology initiatives focus on metastatic disease, the presented concept offers the opportunity to empower novel therapy options for patients with high-risk early breast cancer in the post-neoadjuvant setting within a biomarker-driven trial and provides the basis to test the value of precision oncology in a curative setting with the overarching goal to increase cure rates.

Effect of low-molecular-weight dextran on proteolytic and nonproteolytic lysosomal enzymes in isolated segments of rat proximal tubule.

Dextrans undergo glomerular filtration and are taken up by endocytosis into lysosomes of proximal tubule cells. We measured the activities of the intralysosomal proteolytic enzymes cathepsin B and L and the nonproteolytic acid phosphatase in microdissected segments of rat proximal tubules by means of fluorescent ultramicroassays. Cathepsin activities were increased following dextran injection in a dose-dependent manner. Cathepsin stimulation was not dextran-specific since enhanced proximal albumin uptake also increased cathepsin activities. Acid phosphatase remained almost unchanged. We conclude that increased proximal tubule uptake of macromolecules is accompanied by a rather specific stimulation of proteolytic enzymes in the lysosomes. For proteins this constitutes an adequate response to increased protein uptake to maintain the steady state between uptake and catabolism of proteins in lysosomes. For the nondigestible dextran the cathepsin induction is an inadequate response. Since lysosomal cathepsins are involved in breakdown of intracellular proteins, it is conceivable that the increased activities reflect increased cellular autophagy leading to proximal tubule damage.

Alterations in lysosomal enzymes of the proximal tubule in gentamicin nephrotoxicity.

Gentamicin accumulates in proximal tubule lysosomes, increases their number, and changes their structure. An important lysosomal function is degradation of intracellular proteins. To evaluate the effect of gentamicin on this lysosomal function, we measured the activity of the key lysosomal proteinases, cathepsin B and L, in microdissected S1, S2, and S3 segments of rat proximal tubules by means of a fluorometric microassay. The cathepsin activities were decreased in S1 and S2 following one and four gentamicin injections of 100 mg/kg body weight. The lysosomal enzyme, acid phosphatase, was also measured and was not decreased by gentamicin. The urine excretion of cathepsins B and L was decreased after gentamicin. This excludes an increase in urinary loss of cathepsins as the cause of decreased tubule activity. Structural changes of the lysosomes per se were excluded as the factor responsible for the reduced cathepsin activity by demonstrating increased cathepsin B and L activity in proximal tubule segments from rats injected with dextran, since dextran induces an increase in number and size of proximal tubule lysosomes. In vitro incubation of urine and tubule segments with gentamicin demonstrated a concentration-dependent reversible inhibition of cathepsin B and L. We conclude that gentamicin per se decreased cathepsin B and L activities in proximal tubule segments as early as 24 hours following one injection due to either enzyme inhibition or reduced generation of active intralysosomal cathepsin B and L. Gentamicin may, therefore, reduce renal protein catabolism by decreasing the activity of the key proteolytic enzymes, cathepsin B and L. Since cathepsin B and L are proteolytic activators of other lysosomal enzymes, their reduced activity may also decrease the activities of other lysosomal enzymes.

Alterations in proteolytic enzymes of the proximal tubule in a rat model of cyclosporine nephrotoxicity.

The presence of autophagolysosomes in proximal tubule cells and the increased urine excretion of the lysosomal enzyme N-acetyl-glucosaminidase following administration of cyclosporine suggests involvement of the lysosomes in tubular toxicity of CsA. To evaluate the effect of CsA on lysosomal function, the activity of the lysosomal proteinases cathepsin B and L was measured in microdissected segments of the proximal tubule by means of a fluorometric microassay. Rats received oral doses of 30 mg/kg CsA for eight weeks. Controls received olive oil. CsA reduced renal blood flow, glomerular filtration rate, and kidney weight. Hence, a second control group was included where the left renal artery was clipped to reduce RBF and GFR. CsA administration was accompanied by a 130% increase in cathepsin activities in the S1 segments of the proximal convoluted tubule. The activity remained unchanged in the pars recta. Enzyme activities in convoluted proximal tubules and pars recta from the control groups were not increased irrespective of reduced RBF, decreased GFR, and decreased KW. Hence, cathepsin B and L stimulation was induced by CsA per se. Since lysosomes are involved in cellular protein catabolism, the increased cathepsin activities may reflect an increased rate of protein breakdown. The tubular atrophy induced by CsA may be related to increased intracellular protein degradation.

Effect of low molecular weight proteins and dextran on renal cathepsin B and L activity.

Renal extraction of low molecular weight proteins (LMWP) accounts for 30% to 80% of their total metabolic clearance. Extraction includes glomerular filtration, proximal tubular uptake, and intralysosomal proteolysis. To characterize the anatomic sites and enzymes involved in digestion of reabsorbed LMWP, the lysosomal proteases, cathepsin B and L, were measured by ultramicroassay in isolated S1, S2 and S3 segments of the proximal tubule of proteinuric rats. Increased glomerular filtration and tubular uptake of LMWP were induced by i.v. and i.p. injections of myoglobin and cationic and anionic lysozyme. Both cationic lysozyme and myoglobin increased cathepsin B and L activities in the proximal tubule, while anionic lysozyme had no effect. Morphologic examination of kidney tissue suggested that proximal tubular uptake of anionic lysozyme was negligible in comparison with the cationic form. Hence, only LMWP absorbed by the proximal tubule cells stimulated cathepsin B and L activities. Proximal tubular uptake of cationic lysozyme was determined by measurement of lysozyme activities in S1, S2, and S3. S1 segments contained the highest lysozyme activity, while S2 and S3 had much lower activities, and cathepsin B and L activity following cationic lysozyme injection was stimulated only in S1 segments. These results suggest that cathepsin B and L participate in lysosomal digestion of certain LMWP. Furthermore, the activities of cathepsin B and L adapt to increased uptake of LMWP. To gain additional insight into the mechanism of cathepsin adaptation, the cathepsin B and L activities were measured following injection of dextran with a similar low molecular weight.(ABSTRACT TRUNCATED AT 250 WORDS)