Cell Cycle Progression Score, but Not Phosphatase and Tensin Homolog Loss, Is an Independent Prognostic Factor for Metastasis in Intermediate- and High-risk Prostate Cancer in Men Treated With and Without Salvage Radiotherapy.

PURPOSE: The prognostic value for metastasis of the cell-cycle progression score and phosphatase and tensin homolog haven't been evaluated jointly in contemporary men with exclusively intermediate- or high-risk prostate cancer. We evaluated associations of cell-cycle progression and phosphatase and tensin homolog with metastasis-free survival in contemporary intermediate/high-risk prostate cancer patients overall, and intermediate/high-risk men receiving salvage radiotherapy. MATERIALS AND METHODS: In a case-cohort of 209 prostatectomy patients with intermediate/high-risk prostate cancer, and a cohort of 172 such men who received salvage radiotherapy, cell-cycle progression score was calculated from RNA expression, and phosphatase and tensin homolog was analyzed by immunohistochemistry. Proportional hazards regression, weighted for case-cohort design or unweighted for the salvage radiotherapy cohort, was used to evaluate associations of cell-cycle progression, phosphatase and tensin homolog with metastasis-free survival. Improvement in model discrimination was evaluated with the concordance index. RESULTS: In the case-cohort 41 men had metastasis, and 17 developed metastasis in the salvage radiotherapy cohort, at median follow-up of 3 and 4 years, respectively. For both case-cohort and salvage radiotherapy cohort, cell-cycle progression was independently associated with metastasis-free survival after adjustment for Cancer of the Prostate Risk Assessment Post-Surgical: hazard ratio (95% confidence interval) = 3.11 (1.70-5.69) and 1.85 (1.19-2.85), respectively. Adding cell-cycle progression to Cancer of the Prostate Risk Assessment Post-Surgical increased the concordance index from 0.861 to 0.899 (case-cohort), and 0.745 to 0.819 (salvage radiotherapy cohort). Although statistically significant in univariate analyses, phosphatase and tensin homolog was no longer significant after adjustment for Cancer of the Prostate Risk Assessment Post-Surgical. Analysis of interaction with National Comprehensive Cancer Network risk group showed that cell-cycle progression had the strongest effect among unfavorable intermediate-risk men. CONCLUSIONS: In the first study to evaluate metastasis risk associated with cell-cycle progression and phosphatase and tensin homolog in exclusively intermediate/high-risk prostate cancer, and in such men with salvage radiotherapy, cell-cycle progression but not phosphatase and tensin homolog was associated with significantly increased 2- to 3-fold risk of metastasis after Cancer of the Prostate Risk Assessment Post-Surgical adjustment.

Comparison of the Prognostic Utility of the Cell Cycle Progression Score for Predicting Clinical Outcomes in African American and Non-African American Men with Localized Prostate Cancer.

BACKGROUND: Better prostate cancer risk stratification is necessary to inform medical management, especially for African American (AA) men, for whom outcomes are particularly uncertain. OBJECTIVE: To evaluate the utility of both a cell cycle progression (CCP) score and a clinical cell-cycle risk (CCR) score to predict clinical outcomes in a large cohort of men with prostate cancer highly enriched in an AA patient population. DESIGN, SETTING, AND PARTICIPANTS: Patients were diagnosed with clinically localized adenocarcinoma of the prostate and treated at The Ochsner Clinic (New Orleans, LA, USA) from January 2006 to December 2011. CCP scores were derived from archival formalin-fixed, paraffin-embedded biopsy tissue. CCR scores were calculated as the combination of molecular (CCP score) and clinical (Cancer of the Prostate Risk Assessment [CAPRA] score) components. INTERVENTION: Active treatment (radical prostatectomy, radiation therapy alone, or radiation and hormone therapy) or watchful waiting. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The primary outcome was progression to metastatic disease. Association with outcomes was evaluated via Cox proportional hazards survival analysis and likelihood ratio tests. RESULTS AND LIMITATIONS: The final cohort included 767 men, of whom 281 (36.6%) were AA. After accounting for ancestry, treatment, and CAPRA in multivariable analysis, the CCP score remained a significant predictor of metastatic disease (hazard ratio [HR] 2.04; p<0.001), and there was no interaction with ancestry (p=0.20) or treatment (p=0.09). The CCR score was highly prognostic (HR 3.86; p<0.001), and as with the CCP score, there was no interaction with ancestry (p=0.24) or treatment (p=0.32). Limitations include the retrospective study design and the use of self-reported ancestry information. CONCLUSIONS: A CCR score provided significant prognostic information regardless of ancestry. The findings demonstrate that AA men in this study cohort appear to have similar prostate cancer outcomes to non-AA patients after accounting for all available molecular and clinicopathologic variables. PATIENT SUMMARY: In this study we evaluated the ability of a combined molecular and clinical score to predict the progression of localized prostate cancer. We found that the combined molecular and clinical score predicted progression to metastasis regardless of patient ancestry or treatment. This suggests that the combined molecular and clinical score may be a valuable tool for determining the risk of metastasis in men with newly diagnosed prostate cancer in order to make appropriate treatment decisions.

Comparison of cell cycle progression score with two immunohistochemical markers (PTEN and Ki-67) for predicting outcome in prostate cancer after radical prostatectomy.

PURPOSE: Previous studies of the cell cycle progression (CCP) score in surgical specimens of prostate cancer (PCa) in patients treated by radical prostatectomy (RP) demonstrated significant association with time to biochemical recurrence (BCR). In this study, we compared the ability of the CCP score and the expression of PTEN or Ki-67 to predict BCR in a cohort of patients treated by RP. Finally, we constructed the best predictive model for BCR, incorporating biomarkers and relevant clinical variables. MATERIALS AND METHODS: The study population consisted of 652 PCa patients enrolled in a retrospective cohort and who had RP surgery in French urological centers from 2000 to 2007. RESULTS: Among the 652 patients with CCP scores and complete clinical data, BCR events occurred in 41%, and the median time from surgery to the last follow-up among BCR-free patients was 72 months. In univariate Cox analysis, the continuous CCP score and positive Ki-67 predicted recurrence with a HR of 1.44 (95% CI 1.17-1.75; p = 5.3 × 10-4) and 1.89 (95% CI 1.38-2.57; p = 1.6 × 10-4), respectively. In contrast, PTEN expression was not associated with BCR risk. Of the three biomarkers, only the CCP score remained significantly associated in a multivariable Cox model (p = 0.026). The best model incorporated CAPRA-S and CCP scores as predictors, with HRs of 1.32 and 1.24, respectively. CONCLUSION: The CCP score was superior to the two IHC markers (PTEN and Ki-67) for predicting outcome in PCa after RP.

Homologous recombination deficiency (HRD) status predicts response to standard neoadjuvant chemotherapy in patients with triple-negative or BRCA1/2 mutation-associated breast cancer.

PURPOSE: Defects in the homologous recombination (HR) DNA repair pathway sensitize tumors to therapeutics that target this pathway. A significant proportion of triple-negative breast cancers (TNBC) carry HR defects. The HRD assay is highly associated with sensitivity to neoadjuvant platinum-based chemotherapy in TNBC. Standard chemotherapy consists of some combination of an anthracycline, cyclophosphamide, and taxane. This study assesses the association of HR deficiency status with response to standard neoadjuvant chemotherapy in TNBC or BRCA1/2 mutation-associated breast cancer. METHODS: Tumor samples were retrospectively obtained from 45 TNBC patients and 2 BRCA1/2 mutant, hormone receptor-positive/HER2-negative breast cancer patients who received anthracycline- and/or taxane-based neoadjuvant chemotherapy at Stanford University or Cedars-Sinai Medical Centers. The HRD score and tumor BRCA1/2 mutation status were determined from baseline tumor biopsies. HR deficient tumors were those with a HRD score of ≥ 42 or a tumor BRCA1/2 mutation. Response was categorized by the residual cancer burden (RCB) index. RESULTS: HR deficient patients were more likely to achieve a pathologic complete response (pCR) compared with non-deficient patients (OR 13.06, CI 1.52-11.241, p = 0.0028). Among BRCA1/2 mutation wild-type patients, HR deficient patients were more likely to achieve a pCR (OR 16, 95% CI 1.65-160.41, p = 0.0041) compared with HR non-deficient patients. Further, HRD scores were highly concordant pre- and post-therapy (Spearman correlation > 99%). CONCLUSIONS: HR deficiency status is significantly associated with response to standard neoadjuvant chemotherapy in TNBC. This observation is consistent with the mechanisms of action of doxorubicin and cyclophosphamide as DNA damaging agents.

A Multigene Signature Based on Cell Cycle Proliferation Improves Prediction of Mortality Within 5 Yr of Radical Nephrectomy for Renal Cell Carcinoma.

BACKGROUND: There is a critical need for improved prognostic discrimination in patients with renal cell carcinoma (RCC) given the increasing awareness that some patients may be managed with active surveillance, while others with higher-risk disease might benefit from adjuvant therapy following surgery. OBJECTIVE: To determine whether a multigene proliferation signature predicts long-term oncologic outcomes in surgically resected RCC. DESIGN, SETTING, AND PARTICIPANTS: The cell cycle proliferation (CCP) score was determined after radical nephrectomy for localized clear cell, papillary, or chromophobe RCC in 565 patients. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The primary end point was disease-specific mortality (DSM), and disease recurrence was a secondary end point. Association with outcomes was evaluated by Cox proportional hazards survival analysis. The CCP score was compared with the Karakiewicz nomogram, and a composite (R-CCP) score was developed. RESULTS AND LIMITATIONS: A total of 68 patients (12%) recurred and 32 (6%) died of disease within 5 yr of nephrectomy. The CCP score was an independent predictor of recurrence (hazard ratio [HR] 1.50, 95% confidence interval [CI] 1.07-2.09) and DSM (HR 2.49, 95% CI 1.53-4.04) after adjusting for clinical variables using the baseline nomogram. The composite R-CCP score gave a Harrell's concordance index of 0.87 and stratified patients into low- (n=338) and high-risk (n=202) categories with 99% and 84% cancer-specific survival probabilities, respectively (p<0.001). CONCLUSIONS: The CCP score is a significant, independent predictor of long-term oncologic outcomes in patients who have undergone nephrectomy for RCC. Combining the molecular classifier with baseline clinical variables allows for accurate, patient-specific risk assessment for use in guiding clinical management. PATIENT SUMMARY: In this study, we sought to understand how well gene expression information from individual kidney tumors can predict cancer recurrence and death following surgical removal. We found that the combination of the gene expression test and clinical characteristics provides an accurate prognostic assessment to help inform clinical decisions.

Prediction of distant recurrence in resected stage I and II lung adenocarcinoma.

OBJECTIVES: Optimal procedures for adjuvant treatment and post-surgical surveillance of resected non-small-cell lung cancer remain under discussion. Pathological features are the main determinant of follow-up therapy but have limited ability to identify patients at risk of recurrence. Increasingly, molecular markers are incorporated into clinical decision-making, including measures of tumor growth. The CCP score is a quantitative, molecular measure of proliferation derived from the RNA expression of 31 cell cycle genes and a component of the molecular prognostic score (mPS). The mPS score is a linear combination of CCP score and pathological stage. CCP score and mPS are independent predictors of survival in resected lung adenocarcinoma. MATERIALS AND METHODS: CCP scores were determined by RT-qPCR for 318 patients diagnosed with stage I-II lung adenocarcinoma. Association of mPS and CCP score with distant recurrence and lung-cancer specific survival was assessed in Cox proportional hazards regression models adjusted for age, gender, tumor size, pathological stage and pleural invasion. Distant recurrence-free survival and lung-cancer specific survival by mPS risk group were calculated by Kaplan-Meier survival analysis. RESULTS: CCP scores were obtained for 205 stage I and 84 stage II patients. CCP score and mPS were independent markers of distant recurrence (CCP: HR 1.62, 95%CI 1.15-2.29, p=0.0055; mPS: HR 2.22, 95%CI 1.11-4.44, p=0.023). Patients with low mPS tumors were at significantly reduced risk of distant recurrence (log-rank p=4.2×10-5). Among stage I patients, stratification by mPS identified a patient group with increased risk of distant recurrence (36%, 95%CI 28-46%, log-rank p=0.0011) CONCLUSIONS: The molecular prognostic score stratifies early-stage, resected lung cancer patients for risk of distant recurrence and could be useful to inform treatment and surveillance decisions.

Use of the cell cycle progression (CCP) score for predicting systemic disease and response to radiation of biochemical recurrence.

BACKGROUND: Determining the optimal treatment for biochemical recurrence (BCR) after radical prostatectomy (RP) is challenging. OBJECTIVE: We evaluated the ability of CCP score (a prognostic RNA expression signature) to discriminate between systemic disease and local recurrence in patients with BCR after RP. METHODS: Sixty patients with BCR after RP were selected for analysis based on: 1) metastatic disease, 2) non-response to salvage external beam radiotherapy (EBRT), and 3) durable response to salvage EBRT. CCP scores were generated from the RNA expression of 46 genes. Logistic regression assessed the association between CCP score and patient group. RESULTS: Passing CCP scores were generated for 47 patients with complete clinical and pathologic data. CCP score predicted clinical status when comparing patients with metastatic disease or non-responders to salvage therapy to patients with durable response (p = 0.006). CCP score remained significantly predictive of clinical status after accounting for time to BCR, PSA level at BCR, and Gleason score (p = 0.0031). CONCLUSIONS: Elevated CCP score was associated with increased risk of systemic disease, indicating that CCP score may be useful in identifying patients with BCR who are most likely to benefit from salvage radiation therapy.

Homologous Recombination Deficiency (HRD) Score Predicts Response to Platinum-Containing Neoadjuvant Chemotherapy in Patients with Triple-Negative Breast Cancer.

PURPOSE: BRCA1/2-mutated and some sporadic triple-negative breast cancers (TNBC) have DNA repair defects and are sensitive to DNA-damaging therapeutics. Recently, three independent DNA-based measures of genomic instability were developed on the basis of loss of heterozygosity (LOH), telomeric allelic imbalance (TAI), and large-scale state transitions (LST). EXPERIMENTAL DESIGN: We assessed a combined homologous recombination deficiency (HRD) score, an unweighted sum of LOH, TAI, and LST scores, in three neoadjuvant TNBC trials of platinum-containing therapy. We then tested the association of HR deficiency, defined as HRD score ≥42 or BRCA1/2 mutation, with response to platinum-based therapy. RESULTS: In a trial of neoadjuvant platinum, gemcitabine, and iniparib, HR deficiency predicted residual cancer burden score of 0 or I (RCB 0/I) and pathologic complete response (pCR; OR = 4.96, P = 0.0036; OR = 6.52, P = 0.0058). HR deficiency remained a significant predictor of RCB 0/I when adjusted for clinical variables (OR = 5.86, P = 0.012). In two other trials of neoadjuvant cisplatin therapy, HR deficiency predicted RCB 0/I and pCR (OR = 10.18, P = 0.0011; OR = 17.00, P = 0.0066). In a multivariable model of RCB 0/I, HR deficiency retained significance when clinical variables were included (OR = 12.08, P = 0.0017). When restricted to BRCA1/2 nonmutated tumors, response was higher in patients with high HRD scores: RCB 0/I P = 0.062, pCR P = 0.063 in the neoadjuvant platinum, gemcitabine, and iniparib trial; RCB 0/I P = 0.0039, pCR P = 0.018 in the neoadjuvant cisplatin trials. CONCLUSIONS: HR deficiency identifies TNBC tumors, including BRCA1/2 nonmutated tumors more likely to respond to platinum-containing therapy. Clin Cancer Res; 22(15); 3764-73. ©2016 AACR.

Cell cycle progression score predicts metastatic progression of clear cell renal cell carcinoma after resection.

BACKGROUND: The outcome of surgically resected, apparently localized, clear cell renal carcinoma (ccRCC) is uncertain. OBJECTIVE: To evaluate if cell cycle progression (CCP) gene expression can predict future metastasis. METHODS: Pathologic T2a-T3b tumors at University of Iowa were reviewed. Patients with known or suspected metastasis, lymph node involvement or who received neoadjuvant or adjuvant radiation, chemotherapy or immunotherapy were excluded. Case and control cohorts were defined as those who did or did not develop metastatic disease within 5 years. Measured levels of 31 cell cycle genes and 15 control genes from the tumor were calculated as a CCP score. Additionally, gene expression data for a separate ccRCC cohort was downloaded from The Cancer Genome Atlas (TCGA). RESULTS: Univariate analysis of 26 cases and 38 controls revealed that the CCP score predicted progression to metastasis (OR 2.65, p = 0.0091). In multivariate logistic regression modeling, CCP expression remained a significant independent predictor for progression (p = 0.026). The CCP score was also significantly associated with distant metastasis in the TCGA renal cancer cohort in both univariate (p = 1.0 × 10-9) and multivariate (p = 5.6 × 10-3) analysis. CONCLUSION: The CCP score has prognostic value in predicting metastatic progression after resection of organ-confined ccRCC.

Phase II neoadjuvant clinical trial of carboplatin and eribulin in women with triple negative early-stage breast cancer (NCT01372579).

The purpose of this study is to evaluate the efficacy and safety of neoadjuvant treatment with carboplatin and eribulin in patients with early-stage triple negative breast cancer (TNBC), and to explore biomarkers based on DNA and protein expression profiles as predictors of response. Patients with histologically confirmed early-stage TNBC received carboplatin AUC 6 iv every 21 days, and eribulin 1.4 mg/m(2) day 1 and day 8 every 21 days for four cycles. The primary endpoint of the study was pathologic complete response (pCR), with secondary endpoints including clinical response and safety of the combination. Exploratory studies assessed DNA-based biomarkers [homologous recombination deficiency (HRD) score, and HR deficiency status (HRD score + BRCA1/BRCA2 mutation status)], protein-based biomarkers (Ki67, TP53, androgen receptor, Cyclin E, CDK2, Cyclin D, CDK4, Pin1 and Smad3), and clinical pretreatment factors as predictors of pCR. 13/30 (43.3 %) patients enrolled in the study achieved pCR. 24 (80.0 %) had a clinical complete or partial response. The combination was safe with mostly grade 1 and 2 toxicities. HRD score (P = 0.0024) and HR deficiency status (P = 0.0012) significantly predicted pCR. Pretreatment cytoplasmic CDK2 was also associated with pCR (P = 0.021). Significant differences in pre- versus post-treatment expression levels of nuclear Cyclin D (P = 0.020), nuclear CDK4 (P = 0.0030), and nuclear Smad3 (P = 0.015) were detected. The combination of carboplatin and eribulin is safe and efficacious in the treatment of early-stage TNBC. HRD score, HR deficiency status, and cytoplasmic CDK2 predicted pCR in this patient population.

Association of BRCA1/2 defects with genomic scores predictive of DNA damage repair deficiency among breast cancer subtypes.

INTRODUCTION: Homologous recombination (HR) DNA repair is of clinical relevance in breast cancer. Three DNA-based homologous recombination deficiency (HRD) scores (HRD-loss of heterozygosity score (LOH), HRD-telomeric allelic imbalance score (TAI), and HRD-large-scale state transition score (LST)) have been developed that are highly correlated with defects in BRCA1/2, and are associated with response to platinum therapy in triple negative breast and ovarian cancer. This study examines the frequency of BRCA1/2 defects among different breast cancer subtypes, and the ability of the HRD scores to identify breast tumors with defects in the homologous recombination DNA repair pathway. METHODS: 215 breast tumors representing all ER/HER2 subtypes were obtained from commercial vendors. Next-generation sequencing based assays were used to generate genome wide SNP profiles, BRCA1/2 mutation screening, and BRCA1 promoter methylation data. RESULTS: BRCA1/2 deleterious mutations were observed in all breast cancer subtypes. BRCA1 promoter methylation was observed almost exclusively in triple negative breast cancer. BRCA1/2 deficient tumors were identified with BRCA1/2 mutations, or BRCA1 promoter methylation, and loss of the second allele of the affected gene. All three HRD scores were highly associated with BRCA1/2 deficiency (HRD-LOH: P = 1.3 × 10(-17); HRD-TAI: P = 1.5 × 10(-19); HRD-LST: P = 3.5 × 10(-18)). A combined score (HRD-mean) was calculated using the arithmetic mean of the three scores. In multivariable analyses the HRD-mean score captured significant BRCA1/2 deficiency information not captured by the three individual scores, or by clinical variables (P values for HRD-Mean adjusted for HRD-LOH: P = 1.4 × 10(-8); HRD-TAI: P = 2.9 × 10(-7); HRD-LST: P = 2.8 × 10(-8); clinical variables: P = 1.2 × 10(-16)). CONCLUSIONS: The HRD scores showed strong correlation with BRCA1/2 deficiency regardless of breast cancer subtype. The frequency of elevated scores suggests that a significant proportion of all breast tumor subtypes may carry defects in the homologous recombination DNA repair pathway. The HRD scores can be combined to produce a more robust predictor of HRD. The combination of a robust score, and the FFPE compatible assay described in this study, may facilitate use of agents targeting homologous recombination DNA repair in the clinical setting.

PTEN loss in biopsy tissue predicts poor clinical outcomes in prostate cancer.

OBJECTIVES: To determine whether PTEN status in prostate biopsy represents a predictor of intermediate and long-term oncological outcomes after radical prostatectomy, and whether PTEN status predicts response to androgen deprivation therapy. METHODS: In a retrospective analysis of 77 men treated by radical prostatectomy who underwent diagnostic biopsy between 1992-2006, biopsy samples were stained for PTEN expression by the PREZEON assay with >10% staining reported as positive. Cox proportional hazards and log-rank models were used to assess the correlation between PTEN loss and clinical outcomes. RESULTS: During a median follow-up period after radical prostatectomy of 8.8 years, 39 men (51%) developed biochemical recurrence, four (5%) had castration-resistant prostate cancer, two (3%) had metastasis and two (3%) died from prostate cancer. PTEN loss was not significantly associated with biochemical recurrence (hazard ratio 2.1, 95% confidence interval 0.9-5.1, P = 0.10), but significantly predicted increased risk of castration-resistant prostate cancer, metastasis and prostate cancer-specific mortality (all log-rank, P < 0.0001), and time from androgen deprivation therapy to castration-resistant prostate cancer (log-rank, P = 0.003). No patient without PTEN loss developed metastases or died from prostate cancer. CONCLUSIONS: PTEN loss at the time of biopsy seems to predict time to development of metastasis, prostate cancer-specific mortality and, for the first time, castration-resistant prostate cancer and response to androgen deprivation therapy after radical prostatectomy. If confirmed by larger studies, this would support the use of PTEN loss as an early marker of aggressive prostate cancer.

Prognostic utility of the cell cycle progression score generated from biopsy in men treated with prostatectomy.

PURPOSE: The cell cycle progression score is associated with prostate cancer outcomes in various clinical settings. However, previous studies of men treated with radical prostatectomy evaluated cell cycle progression scores generated from resected tumor tissue. We evaluated the prognostic usefulness of the score derived from biopsy specimens in men treated with radical prostatectomy. MATERIALS AND METHODS: We evaluated the cell cycle progression score in cohorts of patients from the Martini Clinic (283), Durham Veterans Affairs Medical Center (176) and Intermountain Healthcare (123). The score was derived from simulated biopsy (Martini Clinic) or diagnostic biopsy (Durham Veterans Affairs Medical Center and Intermountain Healthcare) and evaluated for an association with biochemical recurrence and metastatic disease. RESULTS: In all 3 cohorts the cell cycle progression score was associated with biochemical recurrence and metastatic disease. The association with biochemical recurrence remained significant after adjusting for other prognostic clinical variables. On combined analysis of all cohorts (total 582 patients) the score was a strong predictor of biochemical recurrence on univariate analysis (HR per score unit 1.60, 95% CI 1.35-1.90, p=2.4×10(-7)) and multivariate analysis (HR per score unit 1.47, 95% CI 1.23-1.76, p=4.7×10(-5)). Although there were few events (12), the cell cycle progression score was the strongest predictor of metastatic disease on univariate analysis (HR per score unit 5.35, 95% CI 2.89-9.92, p=2.1×10(-8)) and after adjusting for clinical variables (HR per score unit 4.19, 95% CI 2.08-8.45, p=8.2×10(-6)). CONCLUSIONS: The cell cycle progression score derived from a biopsy sample was associated with adverse outcomes after surgery. These results indicate that the score can be used at disease diagnosis to better define patient prognosis and enable more appropriate clinical care.

PTEN expression is consistent in colorectal cancer primaries and metastases and associates with patient survival.

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) negatively regulates the phosphoinositide-3-kinase (PI3K) signaling pathway. In colorectal cancer (CRC), observed frequencies of loss of PTEN expression, concordant expression in primary tumors and metastases, and the association of PTEN status with outcome vary markedly by detection method. We determined the degree to which PTEN expression is consistent in 70 matched human CRC primaries and liver metastases using a validated immunohistochemistry assay. We found loss of PTEN expression in 12.3% of assessable CRC primaries and 10.3% of assessable liver metastases. PTEN expression (positive or negative) was concordant in 98% of matched colorectal primaries and liver metastases. Next we related PTEN status to mutations in RAS and PI3K pathway genes (KRAS, NRAS, BRAF , and PIK3CA) and to overall survival (OS). PTEN expression was not significantly associated with the presence or absence of mutations in RAS or PI3K pathway genes. The median OS of patients whose tumors did not express PTEN was 9 months, compared to 49 months for patients whose tumors did express PTEN (HR = 6.25, 95% confidence intervals (CI) (1.98, 15.42), P = 0.0017). The association of absent PTEN expression with increased risk of death remained significant in multivariate analysis (HR = 6.31, 95% CI (2.03, 17.93), P = 0.0023). In summary, PTEN expression was consistent in matched CRC primaries and in liver metastases. Therefore, future investigations of PTEN in metastatic CRC can use primary tumor tissue. In patients with liver-only metastases, loss of PTEN expression predicted poor OS.

Validation of a proliferation-based expression signature as prognostic marker in early stage lung adenocarcinoma.

PURPOSE: New prognostic markers to guide treatment decisions in early stage non-small cell lung cancer are necessary to improve patient outcomes. In this report, we assess the utility of a predefined mRNA expression signature of cell-cycle progression genes (CCP score) to define 5-year risk of lung cancer-related death in patients with early stage lung adenocarcinoma. EXPERIMENTAL DESIGN: A CCP score was calculated from the mRNA expression levels of 31 proliferation genes in stage I and stage II tumor samples from two public microarray datasets [Director's Consortium (DC) and GSE31210]. The same gene set was tested by quantitative PCR in 381 formalin-fixed paraffin-embedded (FFPE) primary tumors. Association of the CCP score with outcome was assessed by Cox proportional hazards analysis. RESULTS: In univariate analysis, the CCP score was a strong predictor of cancer-specific survival in both the Director's Consortium cohort (P = 0.00014; HR = 2.08; 95% CI, 1.43-3.02) and GSE31210 (P = 0.0010; HR = 2.25; 95% CI, 1.42-3.56). In multivariate analysis, the CCP score remained the dominant prognostic marker in the presence of clinical variables (P = 0.0022; HR = 2.02; 95% CI, 1.29-3.17 in Director's Consortium, P = 0.0026; HR = 2.16; 95% CI, 1.32-3.53 in GSE31210). On a quantitative PCR platform, the CCP score maintained highly significant prognostic value in FFPE-derived mRNA from clinical samples in both univariate (P = 0.00033; HR = 2.10; 95% CI, 1.39-3.17) and multivariate analyses (P = 0.0071; HR = 1.92; 95% CI, 1.18-3.10). CONCLUSIONS: The CCP score is a significant predictor of lung cancer death in early stage lung adenocarcinoma treated with surgery and may be a valuable tool in selecting patients for adjuvant treatment.

Prognostic utility of cell cycle progression score in men with prostate cancer after primary external beam radiation therapy.

PURPOSE: To evaluate the prognostic utility of the cell cycle progression (CCP) score, a RNA signature based on the average expression level of 31 CCP genes, for predicting biochemical recurrence (BCR) in men with prostate cancer treated with external beam radiation therapy (EBRT) as their primary curative therapy. METHODS AND MATERIALS: The CCP score was derived retrospectively from diagnostic biopsy specimens of men diagnosed with prostate cancer from 1991 to 2006 (n=141). All patients were treated with definitive EBRT; approximately half of the cohort was African American. Outcome was time from EBRT to BCR using the Phoenix definition. Median follow-up for patients without BCR was 4.8 years. Association with outcome was evaluated by Cox proportional hazards survival analysis and likelihood ratio tests. RESULTS: Of 141 patients, 19 (13%) had BCR. The median CCP score for patient samples was 0.12. In univariable analysis, CCP score significantly predicted BCR (P=.0017). The hazard ratio for BCR was 2.55 for 1-unit increase in CCP score (equivalent to a doubling of gene expression). In a multivariable analysis that included Gleason score, prostate-specific antigen, percent positive cores, and androgen deprivation therapy, the hazard ratio for CCP changed only marginally and remained significant (P=.034), indicating that CCP provides prognostic information that is not provided by standard clinical parameters. With 10-year censoring, the CCP score was associated with prostate cancer-specific mortality (P=.013). There was no evidence for interaction between CCP and any clinical variable, including ethnicity. CONCLUSIONS: Among men treated with EBRT, the CCP score significantly predicted outcome and provided greater prognostic information than was available with clinical parameters. If validated in a larger cohort, CCP score could identify high-risk men undergoing EBRT who may need more aggressive therapy.

A robust immunohistochemical assay for detecting PTEN expression in human tumors.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a negative regulator of the phosphoinositol-3-kinase (PI3K)/AKT signaling pathway that controls cell cycle progression, growth and inhibition of apoptosis. Loss of PTEN protein expression has been associated with tumorigenesis, cancer progression and drug resistance, but conflicting results exist which may be due in part to difficulties inherent in PTEN immunohistochemistry (IHC). We sought a robust PTEN IHC assay. Human tumor cell lines with PTEN status verified by copy number analysis were formalin fixed and paraffin embedded for use as positive and negative controls. PTEN antibodies were optimized on tumor cell lines. Five optimized antibodies were analyzed on 10 molecularly characterized endometrial carcinoma samples. Four antibodies (CST, Millipore, Abcam, Novus) stained 3/10 positive and 7/10 negative, however, all but CST exhibited nonspecific nucleolar staining of negative controls. One antibody (Dako) stained 5/10 positive and 5/10 negative but with areas (≤10%) of positivity. The 4 samples predicted to be negative by sequencing were negative with the CST antibody, however, one was positive with Dako; as a result we chose the CST antibody for our assay. The assay was validated on an automated platform using 50 formalin fixed and paraffin embedded colon, lung, prostate and breast adenocarcinoma cases. Tumor cell lines served as external controls; endothelial cells and peripheral nerves served as internal positive controls. Dichotomous scoring achieved 100% concordance between three independent pathologists. This reproducible PTEN assay (PREZEON) has been implemented in a CLIA certified laboratory.