Eliso Tikishvili

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.

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.

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.

Abstract P6-05-10: Association between BRCA1/2 status and DNA-based assays for homologous recombination deficiency in breast cancer

Homologous recombination (HR) repair defects are of potential therapeutic relevance in a variety of different cancers. Numerous studies have investigated the rate of BRCA1/2 mutations in triple negative breast cancer, and current clinical studies are investigating the efficacy of agents targeting HR deficiency in this breast cancer subtype. A more comprehensive assay for HR defects might expand the number of patients likely to benefit from these therapies, and may expand their utility to other breast cancer subtypes. Recently three DNA-based measures of HR deficiency (HRD) have been developed based on whole genome tumor LOH profiles, telomeric allelic imbalance, or large-scale state transitions. These will be referred herein as HRD-LOH, HRD-TAI and HRD-LST respectively. All 3 scores are highly correlated with defects in BRCA1/2 and other pathway genes in breast or ovarian cancer, and are associated with sensitivity to platinum agents. 213 invasive breast tumor samples and matched normal tissue blocks were obtained from 3 commercial vendors. The samples were selected to contain approximately equal numbers of all subtypes of breast cancer as defined by IHC analysis of ER, PR, and HER2. BRCA1/2 mutation screening and BRCA1 promoter methylation analysis was performed, and genome wide SNP profiles were generated. These data were used to calculate HRD-LOH, HRD-TAI, and HRD-LST scores. Somatic and germline BRCA1/2 mutations were detected in all subtypes of breast cancer at significant levels with the total mutation frequency ranging from 7.8 – 16.4% depending on subtype. In contrast BRCA1 promoter methylation was confined almost exclusively to triple negative tumors (19.7%). Overall BRCA1/2 deficiency ranged from approximately 10% in ER+/Her2- tumors up to approximately 36% in triple negative tumors. HRD-LOH, HRD-TAI, and HRD- LST scores have previously been shown to be highly significantly associated with BRCA1/2 status in both breast and ovarian cancer. In this dataset all 3 scores showed significant association with BRCA1/2 status for the entire dataset, in addition significant association was observed between the scores and BRCA1/2 status in each of the individual tumor subtypes. The 3 scores were found to be highly correlated with one another, but all 3 were still significant in multivariate analysis. This dataset is not of sufficient size to determine which of these scores is best able to identify BRCA1/2 deficient tumors. It is likely that a combination of the 3 scores will prove to be the most robust predictor of HR deficiency. This study has demonstrated significant levels of BRCA1/2 deficiency across all subtypes of breast cancer. All 3 HR deficiency assays showed significant association with BRCA1/2 deficiency regardless of breast cancer subtype. The 3 scores are highly correlated, but also additive and a combination of all 3 is likely to provide the best predictor of HR deficiency. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-05-10.

Abstract 1201: PTEN null expression is concordant in colorectal cancer primaries and metastases and associates with poor survival.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a negative regulator of the phosphoinositide-3-kinase (PI3K) survival pathway. In colorectal cancer, the method of detection is relevant in determining the frequency of loss of PTEN and the association of PTEN status with outcome. Earlier studies reported discordant PTEN expression in an average of 1 in 3 matched colorectal primaries and metastases. Ours is the first investigation of PTEN concordance using a standardized immunohistochemistry assay. Objectives: Our primary aim was to determine the level of concordant PTEN expression in matched human colorectal primaries and liver metastases using a robust assay. Secondary aims were to relate PTEN status to oncogenic mutations in the RAS and PI3K pathways, and to overall survival. Experimental Design: We used a standardized immunohistochemistry assay and a reproducible method of interpretation to characterize PTEN expression in paired colorectal primaries and liver metastases collected from 70 patients. Mutational hotspots in KRAS, NRAS, BRAF and PIK3CA were sequenced. Results: 7 patients were excluded due to insufficient primary tumor. PTEN null expression was found in 7 of 62 assessable colorectal primaries (12.3%) and 6 of 58 assessable liver metastases (10.3%). PTEN expression (positive or null) was concordant in 98% of matched primaries and liver metastases. There was no significant association between PTEN status and RAS family or PIK3CA mutations. The median overall survival of patients with PTEN null tumors was 9 months, compared to 49 months with PTEN expressing tumors (HR=6.25, 95% CI (1.98, 15.42), p = 0.0017). The association of increased risk of death with PTEN null expression remained significant in multivariate analysis (HR=6.31, 95% CI (2.03, 17.93), p = 0.0023). Conclusion: PTEN status was highly concordant in matched colorectal primaries and liver metastases. Loss of PTEN expression predicted poor overall survival. | | | | PRIMARIES | | | | |:----------- | ----------------- | ---------------------------- | ---------------- | ------- | ------------------ | -------- | | | | PTEN | | PIK3CA | | RAS/BRAF | | | | (−),(+),equivocal | | WT, Mut | | WT, Mut | | METASTASES | PTEN null (−) | 6, 0, 0 | PIK3CA wild-type | 54, 0 | RAS/BRAF wild-type | 29, 1 | | | PTEN positive (+) | 1, 43, 5 | PIK3CA mutant | 2, 5 | RAS/BRAF mutant | 0, 31 | | | PTEN equivocal | 0, 2, 0 | | | | | | CONCORDANCE | | 98%(86% including equivocal) | | 96.7% | | 98.4% | Concordance of PTEN, PIK3CA, RAS, and BRAF in paired colorectal primaries and liver metastases Citation Format: Chloe E. Atreya, Zaina Sangale, Nafei Xu, Mary R. Matli, Eliso Tikishvili, William Welbourn, Steve Stone, Kevan M. Shokat, Robert S. Warren. PTEN null expression is concordant in colorectal cancer primaries and metastases and associates with poor survival. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1201. doi:10.1158/1538-7445.AM2013-1201

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

47 Background: Accurate risk stratification improves decision making in localized prostate cancer. The CCP score, a prognostic RNA signature based on the average expression level of 31 cell cycle progression (CCP) genes, was developed to aid in this task. Previously, the CCP score was shown to be predictive of biochemical recurrence (BCR) after prostatectomy, and prostate cancer specific mortality in men undergoing observation. However, the value of CCP score in men undergoing primary electron beam radiation therapy (EBRT) was untested. METHODS The CCP score was derived retrospectively from the diagnostic biopsy of 141 patients treated with EBRT at the Durham VA medical Center. Inclusion criteria were disease diagnosis from 1991 to 2006 and available biopsy tissue. Approximately half of the cohort was African-American. Outcome was time from EBRT to BCR using Phoenix definition, and median follow-up for patients without BCR was 4.8 years. Association with outcome was evaluated by CoxPH survival analysis and likelihood ratio tests. RESULTS Patient data were censored at 5-years of follow-up and 19 patients (13%) had BCR. The median CCP score was 0.12(IQR -0.43 to 0.66). In univariable analysis, CCP score was a significant prognostic variable (p-value = 0.0017). The hazard ratio (HR) for BCR was 2.55 (95% CI (1.43, 4.55)) for a one-unit increase in CCP score (equivalent to a doubling of gene expression). In a predefined multivariable analysis that included Gleason score, PSA, and percent positive cores, the HR for CCP changed only marginally and remained significant (HR per CCP unit 2.09 (95% CI (1.05, 4.18), p-value = 0.035), indicating that CCP provides prognostic information that is not provided by standard clinical parameters. Further adjustment for hormonal therapy or radiation dose did not materially alter this association. The score was also associated with prostate cancer specific mortality. There was no evidence for interaction between CCP and any clinical variable, including ethnicity. CONCLUSIONS In a cohort of men treated with EBRT, the CCP score was significantly associated with outcome and provided prognostic information beyond what was available from clinical parameters. If validated in a larger cohort, CCP score could be used to select high-risk men undergoing EBRT who may need combination therapy for their clinically localized prostate cancer.

Abstract 1763: Frequency of homologous recombination repair defects across breast cancer subtypes.

Introduction: Defects in homologous recombination (HR) repair have potential therapeutic relevance, and current clinical studies are focused on examining the efficacy of agents that exploit HR deficiency in triple negative breast cancer. A homologous recombination deficiency (HRD) score based on whole genome tumor LOH profiles has been developed that is highly correlated with defects in BRCA1/2, and other HR pathway genes, in ovarian cancer(1), and which predicts response to platinum-based neoadjuvant therapy in triple negative breast cancer(2). This study examines the frequency of BRCA1/2 defects and elevated HRD score across breast cancer subtypes as defined by IHC hormone receptor status. Methods: A targeted custom hybridization panel was developed targeting BRCA1, BRCA2, and 50,000 selected SNPs across the entire human genome. This panel, in combination with sequencing on the Illumina HiSeq, was used to analyze approximately 50 randomly ascertained tumors from each of 4 breast cancer subtypes (triple negative, ER+/Her2-, ER-/Her2+, ER+/Her2+) for BRCA1/2 somatic and germline mutations, and SNP allele frequencies. HRD scores were calculated using LOH profiles reconstructed from the SNP analysis. A BRCA1 promoter methylation assay was also performed on all samples. Results: BRCA1/2 somatic and germline mutations were detected in all breast cancer subtypes. BRCA1/2 mutations were observed most frequently in triple negative and ER+/Her2+ tumors. BRCA1 promoter methylation was confined almost exclusively to triple negative tumors. Association between elevated HRD score and BRCA defects was observed regardless of tumor type, and BRCA1/2 intact tumors with elevated HRD scores were observed in all breast cancer subtypes. Conclusions: Elevated HRD score is significantly associated with BRCA1/2 defects in breast cancer. BRCA1/2 defects and elevated HRD scores were observed in all subtypes of breast cancer, suggesting the presence of HR defects in genes other than BRCA1/2 are present in all breast cancer subtypes. HRD score could potentially be used to facilitate the expansion of platinum or PARP inhibitor therapy beyond triple negative breast cancer into other subtypes. Citation Format: Kirsten M. Timms, Victor Abkevich, Chris Neff, Brian Morris, Jennifer Potter, Thanh V. Tran, Jian Chen, Zaina Sangale, Eliso Tikishvili, Andrey Zharkikh, Michael Perry, Alexander Gutin, Jerry Lanchbury. Frequency of homologous recombination repair defects across breast cancer subtypes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1763. doi:10.1158/1538-7445.AM2013-1763