Zaina Sangale

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.

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.

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.

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

IntroductionHomologous 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.Methods215 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.ResultsBRCA1/2 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).ConclusionsThe 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.

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.

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

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.

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 [Directors 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 Directors 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 Directors 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. Clin Cancer Res; 19(22); 6261–71. ©2013 AACR.

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.

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 Harrells 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.

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.