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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 (p = 0.0113). Conclusions: We conclude that the 55GC is a useful and reproducible grading system for stage II CC recurrence risk stratification.

SUMMARYBackgroundWhile mRNA vaccines against SARS-CoV-2 have been exceedingly effective in preventing symptomatic viral infection, the features of immune response remain to be clarified.MethodsIn the present prospective observational study, 225 healthy individuals in Kumamoto General Hospital, Japan, who received two BNT162b2 doses in February 2021, were enrolled. Correlates of BNT162b2-elicited SARS-CoV-2-neutralizing activity (50% neutralization titer: NT50; assessed using infectious virions and live target cells) with SARS-CoV-2-S1-binding-IgG and -IgM levels, adverse effects (AEs), ages, and genders were examined. The average half-life of neutralizing activity and the average time length for the loss of detectable neutralizing activity were determined and the potency of serums against variants of concerns was also determined.FindingsSignificant rise in NT50s was seen in serums on day 28 post-1st dose. A moderate inverse correlation was seen between NT50s and ages, but no correlation was seen between NT50s and AEs. NT50s and IgG levels on day 28 post-1st dose and pain scores following the 2nd shot were greater in women than in men. The average half-life of neutralizing activity in the vaccinees was approximately 67.8 days and the average time length for their serums to lose the detectable neutralizing activity was 198.3 days. While serums from elite-responders (NT50s>1,500-fold: the top 4% among all participants’ NT50s) potently to moderately blocked the infectivity of variants of concerns, some serums with moderate NT50s failed to block the infectivity of a beta strain.InterpretationBNT162b2-elicited immune response has no significant association with AEs. BNT162b2-efficacy is likely diminished to under detection limit by 6-7 months post-1st shot. High-level neutralizing antibody-containing serums potently to moderately block the infection of SARS-CoV-2 variants; however, a few moderate-level neutralizing antibody-containing serums failed to do so. If BNT162b2-elicited immunity memory is short, an additional vaccine or other protective measures would be needed.Research in contextEvidence before this studyWhile mRNA vaccines against SARS-CoV-2 have been exceedingly effective in preventing symptomatic viral infection, the salient features of immune response including the persistence of protection remain to be clarified. There is a report that anti-SARS-CoV-2 antibodies persist through 6 months after the second dose of mRNA-1273 vaccine (Doria-Roseet al. N Engl J Med. 2021;384:2259-2261); however, more definite immune kinetics following mRNA-vaccine-elicited protection have to be clarified. The mRNA-vaccine-elicited protection against SARS-CoV-2 variants are also to be determined.Added value of this studyIn the present prospective study, 225 twice-BNT162b2-dose-receiving individuals in Japan were enrolled. No significant correlation was seen between 50% neutralizing titers (NT50s), determined by using infectious SARS-CoV-2 virions and live target cells, and adverse effects. Largely, NT50s and IgG levels were greater in women than in men. Following 28 days post-2ndshot, significant reduction was seen in NT50s, IgG, and IgM levels. The average half-life of NT50s was ∼68 days and the average time-length for participants’ serums to lose the detectable activity was ∼198 days. Although serums from elite-responders potently to moderately blocked the infectivity of variants of concerns, some serums with moderate NT50s failed to block the infectivity of a beta strain.Implications of all the available evidenceBNT162b2 efficacy is likely to be diminished to under detection limit by 6-7 months post-1stshot on average. Individuals with moderate NT50s may fail to block beta variants. If BNT162b2-elicited immune memory is lost soon, additional vaccine(s) or other protective means would be needed.

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).

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 Two hundred six 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.

3597 Background: Patient prognosis can be predicted based on cancer subtypes classified according to DNA microarray results. The most robust classification system involves the consensus molecular subtypes, which uses over 600 genes for classification. To simplify this classification, we recently constructed a 55-gene classifier (55GC) to classify colon cancer (CC) into three subtypes with different recurrence rates: “microsatellite instability (MSI)-like,” “chromosomal instability (CIN)-like,” and “stromal” colon cancers. The 55GC has been reported to be a useful and reproducible grading system for stage II CC recurrence risk stratification. This study aimed to explore the usefulness of 55GC for classifying stage III CC patients. Methods: We retrospectively identified stage III CC patients aged 20-79 years who underwent curative surgery and received adjuvant chemotherapy with or without oxaliplatin (OX) between 2009 and 2012 from 15 institutions. Propensity score matching was used to adjust for the number of lymph node metastases, tumor location, sex, and age. Results: Among 938 eligible patients, 203 and 201 cases involving adjuvant chemotherapy with and without OX were selected, respectively, using propensity score matching. Ninety-five cases each from groups were analyzed after exclusion of cases involving low-quality specimens and those involving chemotherapy for < 3 months. The 5-year relapse-free survival (RFS) in patients receiving adjuvant chemotherapy with and without OX were 77.1% and 73.7%, respectively. Classification of the stage III CC according to 55GC and related 5-year RFS rates were as follows: stromal (N = 60), 66.6%; CIN-like (N = 78), 80.5%; and MSI-like (N = 52), 78.4%. The HRs for 5-year RFS for adjuvant chemotherapy with and without OX in each subtype were as follows: stromal, HR = 0.791 (95% CI = 0.329-1.901); CIN-like, HR = 1.241 (95% CI = 0.465-3.308); and MSI-like, HR = 0.495 (95% CI = 0.145-1.692). Conclusions: The stromal subtype showed poor prognosis in stage III as well as stage II patients. Oxaliplatin had a good additive effect in adjuvant chemotherapy for MSI-like subtype. The 55GC is useful for predicting the efficacy of adjuvant chemotherapy for CC.

542 Background: The TAILORx trial demonstrated that adjuvant endocrine and chemoendocrine therapies had similar efficacy in patients with hormone receptor-positive, HER2-negative, node-negative breast cancer with an Oncotype DX recurrence score (RS) of 11-25. However, a predictive strategy is needed to identify patients with intermediate RS who may benefit from adjuvant chemoendocrine therapy. Curebest 95GC Breast (95GC) is a 95-gene signature that can stratify patients into two groups with high (95GC-H) and low (95GC-L) groups to predict the risk of recurrence. Our primary objective was to show that 95GC can classify patients with intermediate RS into binary recurrence risk groups. Methods: Patients with ER-positive, HER2-negative, node-negative invasive breast cancer and RS 11-30 who underwent definitive surgery and adjuvant endocrine therapy were included. RNA was derived from archived formalin-fixed, paraffin-embedded samples, and 95GC was calculated as reported previously. The Fisher exact and Brunner-Munzel tests were used to compare variables between 95GC groups. A Kaplan-Meier estimate with a log-rank test was used for recurrence-free survival (RFS) analysis. Results: The analysis included 178 patients from five institutions. The 5-year RFS rate in patients with RS 18-30 was higher in the 95GC-L group (n = 129, 96.3%) than in the 95GC-H group (n = 49, 90.9%; p = 0.002), which was consistent with results in an independent Japanese population (n = 224; p < 0.001). RFS rates significantly differed between the groups among patients with RS 11-25 as well (95GC-L, 97.4%; 95GC-H, 87.1%; p = 0.001). RFS rates did not differ between patients with RS 18-25 (94.8%) and those with RS 26-30 (93.8%; p = 0.33). Conclusions: 95GC can predict recurrence risk in patients with ER-positive, HER2-negative, node-negative invasive breast cancer and intermediate RS. Further prospective retrospective studies in the TAILORx population are warranted to confirm that 95GC can identify patients who may benefit from adjuvant chemoendocrine therapy.

Taxanes are among the drugs most commonly used for preoperative chemotherapy for breast cancer. Taxanes induce mitotic arrest and subsequent apoptosis. The spindle-assembly checkpoint (SAC) is known to be activated during mitosis, along with cyclin-dependent kinase-1 (CDK1), and is required for taxane-induced cell death. We hypothesized that CDK1 activity predicts response to taxane-containing chemotherapy. This study included breast cancer patients who received preoperative chemotherapy- taxane-containing treatment followed by anthracycline-based treatment-and then underwent surgery. Before starting taxane-containing chemotherapy, patients underwent fine-needle aspiration biopsy, and the biopsy samples were incubated in paclitaxel solution to measure CDK activity. Clinical were evaluated after taxane therapy, and pathological resposes were evaluated after completion of all preoperative chemotherapy. Thirty five patients were eligible for analysis of clinical response to taxane-containing therapy. Twenty-six patients had taxane-sensitive and 9 taxane-resistant tumors. Using a cut-off of CDK activity determined by the ROC analysis, patients were classified into SAC function and dysfunction groups. Univariate logistic regression analysis with clinicopathologic parameters showed that only CDK-based SAC functionality was significantly correlated with clinical response (P =0.017). No significant correlation was observed between SAC functionality and pathologic response. CDK-based SAC functionality significantly predicted clinical response (P =.0072, overall agreement = 71.4%), and this is a unique mechanism-based marker for predicting taxane chemosensitivity. Further, large prospective study is needed to determine CDK-based SAC functionality could be developed as a predictive biomarker.

Background We established the cell cycle profiling (C2P) assay for specific activity (SA; activity/expression) of cyclin-dependent kinases (CDKs). C2P risk score (C2P-RS) based on CDK1 and CDK2 SAs was significantly associated with relapse in breast cancer (BC). This study was conducted to investigate the predictive value of C2P-RS for neoadjuvant chemotherapy (NAC). Patients and methods Among 124 eligible patients, 122 were treated with weekly paclitaxel followed by 5-fluorouracil, epirubicin and cyclophosphamide (P-FEC) and 2 were treated with paclitaxel monotherapy. C2P-RS was determined via C2P using frozen biopsy samples before NAC. Results Negative estrogen receptor (ER), negative progesterone receptor (PR), positive human epidermal growth factor receptor 2 (HER2), high Ki-67 expression and intermediate + high C2P-RS were significantly associated with high pathological complete response (pCR) rates compared with positive ER (30% versus 9%), positive PR (25% versus 6%), negative HER2 (34% versus 11%), low Ki-67 expression (24% versus 7%) or low C2P-RS (24% versus 9%), respectively. The combination of C2P-RS and Ki-67 had a stronger impact on pCR than each parameter alone, and a multivariate analysis showed that the combination was an independent predictor of pCR (odds ratio 3.3, 95% confidence interval 1.1–9.5). Conclusions C2P-RS was significantly associated with pCR after P-FEC and may be a useful predictor for chemotherapy in BCs.

[Background] C2P is an assay measuring specific activities (SA; kinase activity compensated by its protein expression) of cyclin-dependent kinases CDK1 and CDK2 by examining a small piece of fresh-frozen tumor tissue. We reported previously that the C2P risk score given by CDK1SA and CDK2SA is a potent prognostic factor in node-negative breast cancer patients. Likewise, a uPA/PAI-1 ELISA test (FEMTELLE®, American Diagnostica Inc. Stamford, CT) quantitatively determines uPA (urokinase-type plasminogen activator) and PAI-1 (plasminogen activator inhibitor type-1) antigen levels in tumor tissue extracts, to identify patients with high or low risk of disease recurrence of node-negative breast cancer patients. These cancer biomarkers are recommended by the ASCO at the highest level of evidence (LOE-1) for therapy decision making in node-negative breast cancer patients. From the biological point of view, the above two assays can be placed into two categories: tumor cell proliferation for C2P and tumor cell invasion for uPA/PAI-1. This fact led us to examine the concept of combination of the two assays to better select breast cancer patients at risk. [Results] Fifty-nine cases of frozen primary breast cancer tissues were subjected to the C2P assay in a blinded manner. Twenty-one cases (40%) were categorized into “high risk”, 7 cases (14%) into “intermediate risk”, and 24 cases (46%) into “low risk”. Seven cases were judged as “not informative”. The uPA/PAI-1 results and clinical information (26: recurrent cases, 30: non-recurrent cases, 1: stage IV, 2: unknown) were provided by the TUM tumor bank. uPA/PAI-1 risk categories of 36 cases (61%) were classified “high” and 23 cases (39%) categorized “low”. C2P and uPA/PAI-1 showed statistically significant correlation to histological grading (Pearson correlation coefficient; 0.45 and 0.40, respectively). No significant correlation was observed between C2P and uPA/PAI-1. By Kaplan-Meier analysis for disease-free survival, in cases treated with endocrine therapy only, both C2P and uPA/PAI-1 showed a reproducible trend to the respective claimed performances as prognostic factors. In the combination analysis of the two parameters, where low/low was judged as “low” and the others as “high”, 11 cases (24%) were categorized into “low” and 34 cases (76%) into “high”. The sensitivity and negative predictive value for disease recurrence were 90% (19/20) and 91% (10/11), respectively. Strong statistical significance was observed between the risk categories by the log-rank test; p=0.0089, and also by Cox proportional hazards regression analysis; HR=9.18, p=0.032. By multivariate analysis, also including tumor size and nodal status, the CP2 uPA/PAI-1 combination evolved as a significant, statistically independent parameter (HR: 6.51, p Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-11-08.

Background: We have reported that breast tumors with a high ratio of cyclin-dependent kinase (CDK) 2 to CDK 1 are associated with high tumor cell proliferation and poor clinical outcome in Japanese. In addition, we have developed risk score based on CDK2/1 ratio (CDK-RS), and have demonstrated a high risk group showed a significantly poor prognosis in Hollanders (van Nes, et al: Br J C 2009: 100: 494). The aim of the present study is to evaluate the correlation of CDK-RS with response to neoadjuvant paclitaxel followed by fluorouracil + epirubicin + cyclophosphamide (FEC) in breast cancers. Material and Methods: Of 131 primary breast cancer patients (age: 25-73y, mean: 51.7y), 126 were treated with paclitaxel (80 mg/m2, weekly) for 12 cycles followed by FEC (500/75/500 mg/m2, q3w) for four cycles and 5 were treated with paclitaxel-monotherapy (5 — 29 cycles) in the NAC or primary chemotherapy setting. Frozen tumor tissues were obtained from core needle biopsy before NAC, and CDK-RS was determined by the Cell Cycle Profiling (C2P) assay as previously reported. Clinical response was evaluated with MRI before NAC and after paclitaxel and FEC. Patients were classified into responders showing ≥80 % in reduction rates and non-responders showing < 80 %. Pathological CR (pCR) was defined as no residual invasive foci and no axillary lymph node metastasis. Results: Patients characteristics were as follows: menopausal status: pre-47%, post-53%; Stage: II 68%, III 27%, IV 7%; Tumor size (cm): ≥5 cm 73%, > 5 cm 27%; histologic grade (HG): I 16%, II 60%, III 24%; ER: (+) 56%, (-) 44%; PR: (+) 39%, (-) 61%; HER2: (+) 28%, (-) 72%. Of 131 patients, 22 (18%) attained pCR but 100 (82%) did not pCR (9 were excluded from evaluation of pathologic response because of stage IV diseases, no operations, and not available pathologic examination). In CDK-RS, 47% of 131 tumors were classified into high, 17% intermediate, and 37% low. In combination with high + intermediate risk score groups, that group had a tendency to show high HG (grade 2 or 3) (87% vs 78%), ER negativity (49% vs 35%), and PR negativity (65% vs 54%) compared with a low risk group, but their differences were not statistically significant. Tumors in the high + intermediate group were significantly more likely to show clinical response after the completion of not only paclitaxel (52% vs 27%, p=0.006) but also FEC (75% vs 52%, p=0.011) as well as to show pCR (24% vs 9%, p=0.037) compared with a low risk score group. In univariate analysis, CDK-RS showed a significant correlation with pCR (high + intermediate vs low, Odds ratio 4.03, 95%CI 1.03 — 10.3, p=0.045). Discussion: CDK-RS in tumor samples before NAC is significantly associated with clinical and pathological response to paclitaxel followed by FEC. Thus CDK-RS seems to be a novel and useful predictive factor for paclitaxel — FEC in breast cancers. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-09-29.

3526Background: Cancer subtypes classified by DNA microarray data have shown excellent abilities in predicting patient prognosis; in particular, consensus molecular subtypes (CMSs) are regarded as ...

In a Japanese study, cyclin-dependent kinase (CDK) based risk determined by CDK 1 and 2 activities was associated with risk of distance recurrence in early breast cancer patients. The aim of our study was to validate this risk categorization in European early breast cancer patients. We retrospectively analyzed frozen breast cancer specimens of 352 Dutch patients with histologically confirmed primary invasive early breast cancer. CDK-based risk was determined in tumour tissues by calculating a risk score (RS) according to kinases activity and protein mass concentration assay without the knowledge of outcome. Determination of CDK-based risk was feasible in 184 out of 352 (52%) tumours. Median follow-up of these patients was 15 years. In patients not receiving systemic treatment, the proportions of risk categories were 44% low, 16% intermediate, and 40% high CDK-based risk. These groups remained significant after univariate and multivariate Cox-regression analysis. Factors associated with a shorter distant recurrence-free period were positive lymph nodes, mastectomy with radiotherapy, and high CDK-based risk. There was no significant correlation with overall survival (OS). CDK-based risk is a prognostic marker of distance recurrence of patients with early breast cancer. More validation would be warranted to use of CDK-based risk into clinical practice.

Background: We recently established a novel assay for specific activity (SA) of cyclin-dependent kinases (CDKs) using small tumor samples (>8 mm 3 ). The aim of this study was to investigate the prognostic significance of CDK1 SA and CDK2SA in human breast cancer. Methods: CDK1 SA and CDK2SA were determined in 284 breast cancer patients and their prognostic significance was investigated. Results: Tumors with high CDK1 SA and high CDK2SA showed significantly poorer 5-year relapse-free survival than those with low CDK1 SA and low CDK2SA, respectively (66.9% vs 84.2% for CDK1 SA; 43.6% vs 83.6% for CDK2SA). Moreover, combined analysis of CDK1 SA and CDK2SA enabled the classification of breast tumors into high-risk and low-risk groups, where tumors in the high-risk group were strongly associated with unfavorable prognosis (5-year relapse-free survival 69.4% for the high-risk group and 91.5% for the low-risk group). Multivariate analysis showed that the risk determined by combined analysis of CDK1SA and CDK2SA is a significant (hazard ratio 3.09, P < 0.001) prognostic indicator for relapse, especially in node-negative patients (hazard ratio 6.73, P < 0.001). Conclusion: Determination of CDK1 SA and CDK2SA may be useful in the prediction of outcomes in breast cancer patients and has potential for use as a routine laboratory test.

Receptor binding properties and antinociceptive activities of chimeric peptides linked by spacers were investigated. The peptides consisted of the μ-opioid receptor ligand dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH_2) or its analog YRFB (Tyr-D-Arg-Phe-βAla-NH_2) linked to the ORL-1 receptor ligand Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-NH_2 (Ac-RYYRIK-NH_2). All chimeric peptides were found to possess high receptor binding affinities for both μ-opioid and ORL-1 receptors in mouse brain membranes. In particular, chimeric peptide 2, which consists of dermorphin and Ac-RYYRIK-NH_2 connected by a long spacer, had high binding affinities toward both receptors. In the tail-flick test following intracerebroventricular (i.c.v.) administration of chimeric peptides containing the YRFB sequence (3 and 4) unexpectedly showed much less potent antinociceptive activity (ED_>100 pmole/mouse). These results suggest the involvement of nociceptin-like effects of the Ac-RYYRIK pharmacophore in the peptides, and the regulation of μ-opioid receptor-mediated antinociception in brain. The present chimeric peptides (2, 3 and 4) may be useful as pharmacological tools for studies on μ-opioid receptor/ORL-1 receptor heterodimers.

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.

In recent years, lithium secondary batteries have been widely utilized as a power source for mobile electronics, but modifications are required to apply this type of battery to motor vehicles or energy storage purposes. Although the excellent cycle characteristics of lithium titanate have been verified for small cells, the high rate characteristics of this material should be improved if it is used as an active material in large batteries. The authors prepared very fine particulates of lithium titanate having an average size of 0.7 μm. The prepared lithium titanate shows superior high rate characteristics, as well as excellent cycle characteristics. A high capacity maintenance ratio of 99% was achieved at 1 C and 25 °C after the 100th cycle. Taking the discharge capacity at 0.15 C, that at 10 C was 86% at 25 °C, while that at 50 °C was 96%.

The estimation of recurrence risk remains a critical issue in relation to gastrointestinal stromal tumors (GISTs) treated with adjuvant therapy. The accuracy of the commonly used risk stratifications is not always adequate. For this study, data were prospectively collected from 68 patients with GISTs who underwent R0 surgery between 2004 and 2009. The results from this analysis cohort were evaluated using the data obtained from an additional 40 patients in the validation cohort. Cyclin-dependent kinase 1 (CDK1)- and CDK2-specific activities were measured using a non-RI kinase assay system. The specific activities of CDK1 and CDK2, but not their expression, significantly correlated with recurrence. The specific activities of both CDK1 and CDK2 were independently correlated with mitosis and significantly correlated with recurrence-free survival (RFS). In the multivariate analysis, CDK2-specific activity (P = 0.0006), tumor size (P = 0.0347), and KIT deletion mutations (P = 0.0006) were significantly correlated with RFS in the analysis cohort. In the validation cohort, CDK2-specific activity (P = 0.0368) was identified as an independent prognostic factor for tumor recurrences with tumor location (P = 0.0442). The results suggest that the specific activities of CDK1 and CDK2 may reflect the proliferative activity of GISTs and that CDK2-specific activity is a good prognostic factor predicting recurrence after macroscopic complete resection of GISTs.

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. Electronic supplementary material The online version of this article (doi:10.1186/1471-2407-14-755) contains supplementary material, which is available to authorized users.

Background In renal cell carcinoma (RCC), the prediction of metastasis via tumor prognostic markers remains a major problem. The objective of our study was to evaluate the efficacy of cyclin-dependent kinase (CDK)1 and CDK2 activity as a prognostic marker in human RCC. Methods Surgical specimens were obtained from 125 patients with RCC without metastasis. Protein expression and kinase activity of CDKs were analyzed using a newly developed assay system named C2P (Sysmex, Kobe, Japan). We then examined the specific activities (SAs) of CDK1 and CDK2 and calculated CDK2SA-CDK1SA ratio in RCC. Also, risk score (RS) was examined. Results A total of 125 cases were tested, though 34 cases were excluded because of low sample quality (25 cases) and assay failure (9 cases). In total, 91 cases were analyzed. They included 68 male and 23 female patients, ranging in age from 19 to 83 years. At a median follow-up of 36 months (1–109M), tumor with low CDK2SA-CDK1SA ratio showed significantly better 5-year recurrence-free survival than those with high CDK2SA-CDK1SA ratio (88.7% vs. 54.7%, P = 0.00141). Also, RS enabled the classification of RCCs into high-risk and low-risk groups, and patients with tumors classified as low RS showed better recurrence-free survival than patients with tumors with high RS (88.7% vs. 54.7%, P = 0.0141). Conclusion CDK1SA of tumors and the CDK2SA are both associated with recurrence and prognosis. Impact CDK-based risk demonstrated is strongly associated with clinical outcome. CDK-based risk should be an accurate system for predicting recurrence and survival for planning follow-up.

Increasing ability of early breast cancer (BC) diagnosis leading to more early stage detection, better survival, and low relapse marks one of the milestones achieved over the decades. Foregoing poses a challenge for clinicians regarding optimal treatment, in which over- and under-treatment should be avoided. Classical prognostic and predictive factors fall short for individualized adjuvant therapy selection in this patient group. The key to better characterization may be found in the biology underlying individual tumors. We hypothesized that markers related to cellular proliferation and apoptosis and the balance between these two processes in tumor development will be predictive for clinical outcome. Our study population (N = 822) consisted of all early stage BC patients primarily treated with surgery in our center between 1985 and 1996. Sections of available tumor tissue (87 %, 714/822) were immunohistochemically stained for expression of p53, active-caspase-3, and Ki67. In 43 % (304/714) and 18 % (126/714) of this cohort, respectively, a biochemical C2P(®) risk prediction and caspase-3 assay were performed. Expression data of the mentioned markers, single, or combined, were analyzed. Results showed that both the single and combined markers, whether of apoptotic or proliferative origin had associations with clinical outcome. An additive effect was seen for the hazard ratios when data on p53, active caspase-3, and Ki67 status were combined. The assembled prognostic apoptotic-proliferative subtype showed significant association for both the overall survival (p = 0.024) and relapse-free period (p = 0.001) in the multivariate analyses of grade I breast tumors. Combined markers of tumor cell apoptosis and proliferation represent tumor aggressiveness. The apoptotic-proliferative subtypes that we present in this study represent a clinical prognostic profile with solid underlying biological rationale and pose a promising method for accurate identification of grade I BC patients in need of an aggressive therapeutic approach, thus contributing to precision medicine in BC disease.

Background: There are no established biomarkers to identify tumour recurrence in stage II colon cancer. As shown previously, the enzymatic activity of the cyclin-dependent kinases 1 and 2 (CDK1 and CDK2) predicts outcome in breast cancer. Therefore, we investigated whether CDK activity identifies tumour recurrence in colon cancer. Methods: In all, 254 patients with completely resected (R0) UICC stage II colon cancer were analysed retrospectively from two independent cohorts from Munich (Germany) and Leiden (Netherlands). None of the patients received adjuvant treatment. Development of distant metastasis was observed in 27 patients (median follow-up: 86 months). Protein expression and activity of CDKs were measured on fresh-frozen tumour samples. Results: Specific activity (SA) of CDK1 (CDK1SA), but not CDK2, significantly predicted distant metastasis (concordance index=0.69, 95% confidence interval (CI): 0.55–0.79, P=0.036). Cutoff derivation by maximum log-rank statistics yielded a threshold of CDK1SA at 11 (SA units, P=0.029). Accordingly, 59% of patients were classified as high-risk (CDK1SA ⩾11). Cox proportional hazard analysis revealed CDK1SA as independent prognostic variable (hazard ratio=6.2, 95% CI: 1.44–26.9, P=0.012). Moreover, CKD1SA was significantly elevated in microsatellite-stable tumours. Conclusion: Specific activity of CDK1 is a promising biomarker for metastasis risk in stage II colon cancer.

Introduction Paclitaxel is used widely in the treatment of breast cancer. Not all tumors respond to this drug, however, and the characteristics that distinguish resistant tumors from sensitive tumors are not well defined. Activation of the spindle assembly checkpoint is required for paclitaxel-induced cell death. We hypothesized that cyclin-dependent kinase (CDK) 1 activity and CDK2 activity in cancer cells, which reflect the activation state of the spindle assembly checkpoint and the growth state, respectively, predict sensitivity to paclitaxel. Methods Cell viability assays and DNA and chromatin morphology analyses were performed in human breast cancer cell lines to evaluate sensitivity to paclitaxel and the cell cycle response to paclitaxel. We then examined the specific activities of CDK1 and CDK2 in these cell lines and in xenograft models of human breast cancer before and after paclitaxel treatment. Protein expression and kinase activity of CDKs and cyclins were analyzed using a newly developed assay system. Results In the cell lines, biological response to paclitaxel in vitro did not accurately predict sensitivity to paclitaxel in vivo. Among the breast cancer xenograft tumors, however, tumors with significantly increased CDK1 specific activity after paclitaxel treatment were sensitive to paclitaxel in vivo, whereas tumors without such an increase were resistant to paclitaxel in vivo. Baseline CDK2 specific activity was higher in tumors that were sensitive to paclitaxel than in tumors that were resistant to paclitaxel. Conclusions The change in CDK1 specific activity of xenograft tumors after paclitaxel treatment and the CDK2 specific activity before paclitaxel treatment are both associated with the drug sensitivity in vivo. Analysis of cyclin-dependent kinase activity in the clinical setting could be a powerful approach for predicting paclitaxel sensitivity.

Receptor binding properties and antinociceptive activities of chimeric peptides linked by spacers were investigated. The peptides consisted of the micro-opioid receptor ligand dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH(2)) or its analog YRFB (Tyr-D-Arg-Phe-betaAla-NH(2)) linked to the ORL1 receptor ligand Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-NH(2) (Ac-RYYRIK-NH(2)). All chimeric peptides were found to possess high receptor binding affinities for both micro-opioid and ORL1 receptors in mouse brain membranes although their binding affinities for both receptors in spinal membranes were significantly lower. Among them, chimeric peptide 2, which consists of dermorphin and Ac-RYYRIK-NH(2) connected by a long spacer, had the highest binding affinity towards both receptors. In the tail-flick test following intrathecal (i.t.) administration to mice, all chimeric peptides showed potent and dose-dependent antinociceptive activities with an ED(50) of 1.34-4.51 (pmol/mouse), nearly comparable to dermorphin alone (ED(50); 1.08 pmol/mouse). In contrast to their micro-opioid receptor binding profiles, intracerebroventricular (i.c.v.) administration of the chimeric peptides resulted in much less potent antinociceptive activity (ED(50) 5.55-100pmol/mouse) than when administered i.t. (ED(50): 1.34-4.51 pmol/mouse). These results suggest the involvement of nociceptin-like agonistic effects of the Ac-RYYRIK pharmacophore in the peptides, and the regulation of mu-opioid receptor-mediated antinociception in brain. The present chimeric peptides may be useful as pharmacological tools for studies on micro-opioid receptor/ORL1 receptor heterodimers.

e20501 Background: Size, mitosis and locus are important for risk stratification of gastrointestinal stromal tumors (GIST). Genotype and cell cycle are suggested related to aggressiveness and progression, and considered to have prognostic impact, although most reports have analyzed expression of proteins or mRNAs. In this study, we examined the biological and prognostic values of CDK activities in primary GIST with R0 surgery. Methods: Histologic Dx was done with HE and IH. Using lysate of frozen materials from 76 pts with GIST (n=70), leiomyoma (2), schwannoma (3) and abdominal sarcoma (1), CDK-specific activities were measured by the C2P system (activities of CDK1 & CDK2). The median age was 64 yrs and 33 (45%) pts were male. The median follow-up was 37 months. Results: KIT and PDGFRA mutations were found in 57 and 7 GIST pts, respectively, and deletion of codon 557-558 in 16 pts. Two pediatric GISTs and one with NF-1 had no mutation in both genes. CDK activities were measurable in 63 (86%) pts, and med...

e14586 Background: In recent years, the proportion of elderly patients has increased to more than 20% of the population in Japan. Three previous Japanese phase III trials on advanced or recurrent g...

Background: We established original methods enabling simultaneous analysis of protein expressions and kinase activities of the CDK (cyclin-dependent kinase) molecules in lysate of tumor tissue in a clinical setting (C2P technology, Ishihara et al: Biochim Biophys Acta. 1741; 226-233, 2005). The clinical utility of the technology was first evaluated in breast cancer, and combination analysis of CDK1 and CDK2 activity was shown to be a significant prognostic indicator for relapse (Kim et al: Ann Oncol. 19; 68-72, 2009). The objective of our study is to evaluate the efficacy of CDK1 and CDK2 activity as a prognostic marker in human renal cell carcinoma (RCC).Methods: Surgical specimens were obtained from 125 patients with RCC without metastasis. These patients were selected randomly for this study. Protein expression and kinase activity of CDKs and cyclins were analyzed using a newly developed assay system. The system to measure the CDK specific activity (SA) is named C2Ps (Sysmex, Kobe, Japan). We then examined the specific activities of CDK1 and CDK2 and calculated CDK2/CDK1 ratio in RCC. Also, risk score (RS) was examined as described in previous study (JGH van Nes et al: Br J Cancer. 100; 494-500, 2009). Cut off value was calculated by ROC analysis.Results: 125 cases were tested, though 34 cases were excluded of low sample quality (25 cases) and of assay failure (9 cases). 91 cases were analyzed. They included 68 male and 23 female patients, ranging in age from 19 to 83 years. At a median follow up of 36 months (1-109M), tumor with low CDK2/CDK1 ratio showed significantly better 5-year progression free survival (PFS) than those with high CDK2/CDK1 ratio (88.7% vs 54.7%, P=0.00141). Also, RS enabled the classification of RCCs into high-risk and low-risk groups, patients with tumors classified as low RS showed better PFS than patients with tumors with high RS (88.7% vs 54.7%, P=0.0141).Conclusion: CDK1 specific activity of tumors and the CDK2 specific activity are both associated with recurrence and prognosis. Analysis of cyclin-dependent kinase activity in the clinical setting could be a powerful approach for predicting cancer recurrence and prognosis in RCC after surgery and has potential for use as a routine laboratory test. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 299. doi:10.1158/1538-7445.AM2011-299

341 Background: We established original methods enabling simultaneous analysis of protein expressions and kinase activities of the CDK (cyclin-dependent kinase) molecules in lysate of tumor tissue in a clinical setting (C2P technology, Ishihara et al: Biochim Biophys Acta. 1741; 226-233, 2005). The clinical utility of the technology was first evaluated in breast cancer, and combination analysis of CDK1 and CDK2 activity was shown to be a significant prognostic indicator for relapse (Kim et al. Ann Oncol. 19;68-72,2009). The objective of our study is to evaluate the efficacy of CDK1 and CDK2 activity as a prognostic marker in human renal cell carcinoma (RCC). Methods: Surgical specimens were obtained from 115 patients with RCC without metastasis. These patients were selected randomly for this study. Protein expression and kinase activity of CDKs and cyclins were analyzed using a newly developed assay system. The system to measure the CDK specific activity (SA) is named C2Ps (Sysmex, Kobe, Japan). We then examined the specific activities of CDK1 and CDK2 and calculated CDK2/CDK1 ratio in RCC. Also, risk score (RS) was examined as described in previous study (JGH van Nes et al: Br J Cancer. 100; 494-500, 2009). Cut off value was calculated by ROC analysis. Results: 115 cases were tested, though 32 cases were excluded of low sample quality (30 cases) and of assay failure (2 cases). 83 cases were analyzed. They included 63 male and 20 female patients, ranging in age from 19 to 83 years. At a median follow up of 33 months (1-109M), tumor with low CDK2/CDK1 ratio showed significantly better 5-year progression-free survival (PFS) than those with high CDK2/CDK1 ratio (91.2% vs. 51.6%, p=0.0016). Also, RS enabled the classification of RCCs into high-risk and low-risk groups, patients with tumors classified as low RS showed better PFS than patients with tumors with high RS (88.9% vs. 63.9%, p=0.0488). Conclusions: CDK1 specific activity of tumors and the CDK2 specific activity are both associated with recurrence and prognosis. Analysis of cyclin-dependent kinase activity in the clinical setting could be a powerful approach for predicting cancer recurrence and prognosis in RCC after surgery and has potential for use as a routine laboratory test. No significant financial relationships to disclose.