Enhanced Clinical Utility of Molecular Budding Signature as a Recurrence Risk Determinant in Stage II and III Colon Cancer Patients.

BACKGROUND: A molecular budding signature (MBS), which consists of seven tumor budding-related genes, was recently presented as a prominent prognostic indicator in colon cancer (CC) using microarray data acquired from frozen specimens. This study aimed to confirm the predictive power of MBS for recurrence risk based on formalin-fixed, paraffin-embedded (FFPE) materials. METHODS: This research utilized the same microarray data from a prior multicenter study using FFPE whole tissue sections, which retrospectively reviewed 232 stage II CC patients without adjuvant chemotherapy and 302 stage III CC patients with adjuvant chemotherapy. All patients underwent upfront curative surgery without neoadjuvant therapy between 2009 and 2012. An MBS score was calculated using the mean of log2 [each signal] of seven genes (MSLN, SLC4A11, WNT11, SCEL, RUNX2, MGAT3, and FOXC1) as described before. RESULTS: The MBS-low group exhibited a better relapse-free survival (RFS) than the MBS-high group in stage II (P = 0.0077) and in stage III CC patients (P = 0.0003). Multivariate analyses revealed that the MBS score was an independent prognostic factor in both stage II (P = 0.0257) and stage III patients (P = 0.0022). Especially among T4, N2, or both (high-risk) stage III patients, the MBS-low group demonstrated markedly better RFS compared with the MBS-high group (P = 0.0013). CONCLUSIONS: This study confirmed the predictive power of the MBS for recurrence risk by employing FFPE materials in stage II/III CC patients.

A 95-gene signature stratifies recurrence risk of invasive disease in ER-positive, HER2-negative, node-negative breast cancer with intermediate 21-gene signature recurrence scores.

PURPOSE: A subset of patients with intermediate 21-gene signature assay recurrence score may benefit from adjuvant chemoendocrine therapy, but a predictive strategy is needed to identify such patients. The 95-gene signature assay was tested to stratify patients with intermediate RS into high (95GC-H) and low (95GC-L) groups that were associated with invasive recurrence risk. METHODS: Patients with ER-positive, HER2-negative, node-negative breast cancer and RS 11-25 who underwent definitive surgery and adjuvant endocrine therapy without any cytotoxic agents were included. RNA was extracted from archived formalin-fixed, paraffin-embedded samples, and 95-gene signature was calculated. RESULTS: 206 patients had RS of 11-25 (95GC-L, N = 163; 95GC-H, N = 43). In Cox proportional hazards model, 95GC-H was significantly associated with shorter time to recurrence than was 95GC-L (HR 5.94; 95%CI 1.81-19.53; P = 0.005). The correlation between 95-gene signature and 21-gene signature assay scores was not strong (correlation coefficient r = 0.27), which might suggest that 95-gene signature reflects biological characteristics differing from what 21-gene signature shows. CONCLUSIONS: The 95-gene signature stratifies patients with ER-positive, HER2-negative, node-negative invasive breast cancer and intermediate RS of 11-25 into high and low groups that are associated with recurrence risk of invasive disease. Further retrospective analysis in the prospectively accrued TAILORx population is warranted to confirm that 95-gene signature can identify patients who would benefit from adjuvant chemoendocrine therapy.

Evaluation of a 55-gene classifier as a prognostic biomarker for adjuvant chemotherapy in stage III colon cancer patients.

BACKGROUND: Adjuvant chemotherapy reduces the risk of recurrence of stage III colon cancer (CC). However, more effective prognostic and predictive biomarkers are needed for better treatment stratification of affected patients. Here, we constructed a 55-gene classifier (55GC) and investigated its utility for classifying patients with stage III CC. METHODS: We retrospectively identified patients aged 20-79 years, with stage III CC, who received adjuvant chemotherapy with or without oxaliplatin, between the years 2009 and 2012. RESULTS: Among 938 eligible patients, 203 and 201 patients who received adjuvant chemotherapy with and without oxaliplatin, respectively, were selected by propensity score matching. Of these, 95 patients from each group were analyzed, and their 5-year relapse-free survival (RFS) rates with and without oxaliplatin were 73.7 and 77.1%, respectively. The hazard ratios for 5-year RFS following adjuvant chemotherapy (fluoropyrimidine), with and without oxaliplatin, were 1.241 (95% CI, 0.465-3.308; P = 0.67) and 0.791 (95% CI, 0.329-1.901; P = 0.60), respectively. Stratification using the 55GC revealed that 52 (27.3%), 78 (41.1%), and 60 (31.6%) patients had microsatellite instability (MSI)-like, chromosomal instability (CIN)-like, and stromal subtypes, respectively. The 5-year RFS rates were 84.3 and 72.0% in patients treated with and without oxaliplatin, respectively, for the MSI-like subtype (HR, 0.495; 95% CI, 0.145-1.692; P = 0.25). No differences in RFS rates were noted in the CIN-like or stromal subtypes. Stratification by cancer sidedness for each subtype showed improved RFS only in patients with left-sided primary cancer treated with oxaliplatin for the MSI-like subtype (P = 0.007). The 5-year RFS rates of the MSI-like subtype in left-sided cancer patients were 100 and 53.9% with and without oxaliplatin, respectively. CONCLUSIONS: Subclassification using 55GC and tumor sidedness revealed increased RFS in patients within the MSI-like subtype with stage III left-sided CC treated with fluoropyrimidine and oxaliplatin compared to those treated without oxaliplatin. However, the predictive power of 55GC subtyping alone did not reach statistical significance in this cohort, warranting larger prospective studies. TRIAL REGISTRATION: The study protocol was registered in the University Hospital Medical Education Network (UMIN) clinical trial registry (UMIN study ID: 000023879 ).

A Validation Study for Recurrence Risk Stratification of Stage II Colon Cancer Using the 55-Gene Classifier.

INTRODUCTION: DNA microarrays, such as the consensus molecular subtype (CMS) classification using >600 genes, are used to predict cancer patient prognosis. We recently constructed a simple 55-gene classifier (55GC) system to risk stratify colon cancer (CC). OBJECTIVE: Here, we validate the 55GC specifically for stage II CC and compare it with CMS categories. METHODS: Tissue sections from 232 stage II CC patients who underwent curative surgery without adjuvant chemotherapy between 2009 and 2012 were subjected to DNA microarray analysis. RESULTS: Based on the 55GC, patients were classified into microsatellite instability-like (27%), chromosomal instability-like (41%), and stromal (32%) subtypes with 5-year relapse-free survival (RFS) rates of 88.5, 83.3, and 71.2%, respectively (stromal vs. others: p = 0.0049). Multivariate analysis by Cox's proportional hazard model revealed that the stromal subtype, pT4, and the number of lymph nodes examined (<12) were independent poor prognostic factors. The overall concordance rate between 55GC and CMS was 72%, and 5-year RFS rates of patients with CMS1, CMS2, CMS3, and CMS4 cancers were 100, 85.5, 92.3, and 73.0%, respectively (p = 0.0113). CONCLUSIONS: We conclude that the 55GC is a useful and reproducible grading system for stage II CC recurrence risk stratification.

Cyclin-dependent kinase-specific activity predicts the prognosis of stage I and stage II non-small cell lung cancer.

BACKGROUND: Lung cancer is one of the leading causes of cancer death worldwide. Even with complete resection, the prognosis of early-stage non-small cell lung cancer is poor due to local and distant recurrence, and it remains unclear which biomarkers are clinically useful for predicting recurrence or for determining the efficacy of chemotherapy. Recently, several lines of evidence have indicated that the enzymatic activity of cyclin-dependent kinases could be a clinically relevant prognostic marker for some cancers. We investigated whether the specific activity of cyclin-dependent kinases 1 and 2 could predict recurrence or death in early non-small cell lung cancer patients. METHODS: Patients with newly diagnosed, pathologically confirmed non-small cell lung cancer were entered into this blinded cohort study. The activity of cyclin-dependent kinases was determined in 171 samples by the C2P® assay, and the results were subjected to statistical analysis with recurrence or death as a clinical outcome. RESULTS: The Cox proportional hazards model revealed that the activity of cyclin-dependent kinase 1, but not 2, was a predictor of recurrence, independent of sex, age, and stage. By contrast, cyclin-dependent kinase 2 activity was a predictor of death, independent of sex and stage. CONCLUSION: This study suggested the possible clinical use of cyclin-dependent kinase 1 as a predictor of recurrence and cyclin-dependent kinase 2 as a predictor of overall survival in early-stage non-small cell lung cancer. Thus, a combination of activity of cyclin-dependent kinases 1 and 2 is useful in decision-making regarding treatment strategies for non-small cell lung cancer after surgery.

Species differences between human and rat in the substrate specificity of cathepsin K.

Cathepsin K is known to play an important role in bone resorption, and it has the P2 specificity for proline. Rat cathepsin K has 88% identity with the human enzyme. However, it has been reported that its enzymatic activity for a Cbz-Leu-Arg-MCA substrate is lower than that of human cathepsin K, and that the rat enzyme is not well inhibited by human cathepsin K inhibitors. For this study, we prepared recombinant enzyme to investigate the substrate specificity of rat cathepsin K. Cleavage experiments using the fragment of type I collagen and peptidic libraries demonstrated that rat cathepsin K preferentially hydrolyses the substrates at the P2 Hyp position. Comparison of the S2 site between rat and human cathepsin K sequences indicated that two S2 residues at Ser134 and Val160 in rat are varied to Ala and Leu, respectively, in the human enzyme. Cleavage experiments using two single mutants, S134A and V160L, and one double mutant, S134A/V160L, of rat cathepsin K showed that all the rat mutants lost the P2 Hyp specificity. The information obtained from our comparative studies on rat and human cathepsin K should make a significant impact on developing specific inhibitors of human cathepsin K since rat is usually used as test species.

New chemotypes for cathepsin K inhibitors.

Cyano pyrimidine acetylene and cyano pyrimidine t-amine, which belong to a new chemical class, were prepared and tested for inhibitory activities against cathepsin K and the highly homologous cathepsins L and S. The use of novel chemotypes in the development of cathepsin K inhibitors has been demonstrated by derivatives of compounds 1 and 8.

A new cancer diagnostic system based on a CDK profiling technology.

A series of molecular pathological investigations of the molecules that stimulate the cyclin dependent kinases (CDK1, 2, 4, and 6) have led to enormous accumulation of knowledge of the clinical significance of these molecules for cancer diagnosis. However, the molecules have yet to be applied to clinical cancer diagnosis, as there is no available technology for application of the knowledge in a clinical setting. We hypothesized that the direct measurement of CDK activities and expressions (CDK profiling) might produce clinically relevant values for the diagnosis. This study investigated the clinical relevance of CDK profiling in gastrointestinal carcinoma tissues by using originally developed expression and activity analysis methods. We have established novel methods and an apparatus for analyzing the expression and activities of the CDK molecules in lysate of tumor tissue in a clinical setting, and examined 30 surgically dissected gastrointestinal carcinomas and corresponding normal mucosal specimens. We demonstrate here that remarkably elevated CDK2 activity is evident in more than 70% of carcinoma tissues. Moreover, a G1-CDK activity profiling accurately mirrored the differences in proliferation between tumor and normal colonic tissues. Our results suggest that CDK profiling is a potent molecular-clinical approach to complement the conventional pathological diagnosis, and to further assist in the individualized medications.

Novel scaffold for cathepsin K inhibitors.

Pyrrolopyrimidine, a novel scaffold, allows to adjust interactions within the S3 subsite of cathepsin K. The core intermediate 10 facilitated the P3 optimization and identified highly potent and selective cathepsin K inhibitors 11-20.