Novel neoadjuvant therapy paradigms for bladder cancer: results from the National Cancer Center Institute Forum.

OBJECTIVE: To bridge gaps in translational science and develop the concepts for 2 novel biomarker-driven clinical trials: one in the presurgical setting and the other in the setting of bladder preservation with chemoradiation. METHODS AND MATERIALS: The National Cancer Institute sponsored a forum, "Novel Neoadjuvant Therapy for Bladder Cancer," which brought leading clinical and laboratory-based scientists together with the advocacy community. RESULTS: The group designed a neoadjuvant clinical trial to compare the clinical efficacy of the two frontline chemotherapy regimens (gemcitabine plus cisplatin versus MVAC) and the ability of a gene expression profiling-based algorithm (CoXEN) to predict complete pathological response. The trial was recently opened under the leadership of the Southwest Oncology Group (SWOG, S1314), receiving support for the biomarker studies from the NCI's BISQFP resource. A second clinical trial was planned that will examine the relationship between expression of the DNA repair protein MRE11 and complete response in patients treated with concurrent 5-fluorouracil/mitomycin C plus radiation. CONCLUSION: The meeting provided a unique opportunity to launch a collective effort to establish molecular-based therapies for muscle-invasive urothelial cancer. The goal is to use this framework to develop comparable trials with immunotherapy in non-muscle invasive cancers and to exploit the neoadjuvant platform to develop targeted therapy in muscle-invasive disease.

An optimized five-gene multi-platform predictor of hormone receptor negative and triple negative breast cancer metastatic risk.

INTRODUCTION: Outcome predictors in use today are prognostic only for hormone receptor-positive (HRpos) breast cancer. Although microarray-derived multigene predictors of hormone receptor-negative (HRneg) and/or triple negative (Tneg) breast cancer recurrence risk are emerging, to date none have been transferred to clinically suitable assay platforms (for example, RT-PCR) or validated against formalin-fixed paraffin-embedded (FFPE) HRneg/Tneg samples. METHODS: Multiplexed RT-PCR was used to assay two microarray-derived HRneg/Tneg prognostic signatures IR-7 and Buck-4) in a pooled FFPE collection of 139 chemotherapy-naïve HRneg breast cancers. The prognostic value of the RTPCR measured gene signatures were evaluated as continuous and dichotomous variables, and in conditional risk models incorporating clinical parameters. An optimized five-gene index was derived by evaluating gene combinations from both signatures. RESULTS: RT-PCR measured IR-7 and Buck-4 signatures proved prognostic as continuous variables; and conditional risk modeling chose nodal status, the IR-7 signature, and tumor grade as significant predictors of distant recurrence (DR). From the Buck-4 and IR-7 signatures, an optimized five-gene (TNFRSF17, CLIC5, HLA-F, CXCL13, XCL2) predictor was generated, referred to as the Integrated Cytokine Score (ICS) based on its functional pathway linkage through interferon-γ and IL-10. Across all FFPE cases, the ICS was prognostic as either a continuous or dichotomous variable, and conditional risk modeling selected nodal status and ICS as DR predictors. Further dichotomization of node-negative/ICS-low FFPE cases identified a subset of low-grade HRneg tumors with <10% 5-year DR risk. The prognostic value of ICS was reaffirmed in two previously studied microarray assayed cohorts containing 274 node-negative and chemotherapy naive HRneg breast cancers, including 95 Tneg cases where it proved prognostically independent of Tneg molecular subtyping. In additional HRneg/Tneg microarray assayed cohorts, the five-gene ICS also proved prognostic irrespective of primary tumor nodal status and adjuvant chemotherapy intervention. CONCLUSION: We advanced the measurement of two previously reported microarray-derived HRneg/Tneg breast cancer prognostic signatures for use in FFPE samples, and derived an optimized five-gene Integrated Cytokine Score (ICS) with multi-platform capability of predicting metastatic outcome from primary HRneg/Tneg tumors independent of nodal status, adjuvant chemotherapy use, and Tneg molecular subtype.

Genomic aberrations in normal tissue adjacent to HER2-amplified breast cancers: field cancerization or contaminating tumor cells?

Field cancerization effects as well as isolated tumor cell foci extending well beyond the invasive tumor margin have been described previously to account for local recurrence rates following breast conserving surgery despite adequate surgical margins and breast radiotherapy. To look for evidence of possible tumor cell contamination or field cancerization by genetic effects, a pilot study (Study 1: 12 sample pairs) followed by a verification study (Study 2: 20 sample pairs) were performed on DNA extracted from HER2-positive breast tumors and matching normal adjacent mammary tissue samples excised 1-3 cm beyond the invasive tumor margin. High-resolution molecular inversion probe (MIP) arrays were used to compare genomic copy number variations, including increased HER2 gene copies, between the paired samples; as well, a detailed histologic and immunohistochemical (IHC) re-evaluation of all Study 2 samples was performed blinded to the genomic results to characterize the adjacent normal tissue composition bracketing the DNA-extracted samples. Overall, 14/32 (44 %) sample pairs from both studies produced genome-wide evidence of genetic aberrations including HER2 copy number gains within the adjacent normal tissue samples. The observed single-parental origin of monoallelic HER2 amplicon haplotypes shared by informative tumor-normal pairs, as well as commonly gained loci elsewhere on 17q, suggested the presence of contaminating tumor cells in the genomically aberrant normal samples. Histologic and IHC analyses identified occult 25-200 µm tumor cell clusters overexpressing HER2 scattered in more than half, but not all, of the genomically aberrant normal samples re-evaluated, but in none of the genomically normal samples. These genomic and microscopic findings support the conclusion that tumor cell contamination rather than genetic field cancerization represents the likeliest cause of local clinical recurrence rates following breast conserving surgery, and mandate caution in assuming the genomic normalcy of histologically benign appearing peritumor breast tissue.

A multigene predictor of metastatic outcome in early stage hormone receptor-negative and triple-negative breast cancer.

INTRODUCTION: Various multigene predictors of breast cancer clinical outcome have been commercialized, but proved to be prognostic only for hormone receptor (HR) subsets overexpressing estrogen or progesterone receptors. Hormone receptor negative (HRneg) breast cancers, particularly those lacking HER2/ErbB2 overexpression and known as triple-negative (Tneg) cases, are heterogeneous and generally aggressive breast cancer subsets in need of prognostic subclassification, since most early stage HRneg and Tneg breast cancer patients are cured with conservative treatment yet invariably receive aggressive adjuvant chemotherapy. METHODS: An unbiased search for genes predictive of distant metastatic relapse was undertaken using a training cohort of 199 node-negative, adjuvant treatment naive HRneg (including 154 Tneg) breast cancer cases curated from three public microarray datasets. Prognostic gene candidates were subsequently validated using a different cohort of 75 node-negative, adjuvant naive HRneg cases curated from three additional datasets. The HRneg/Tneg gene signature was prognostically compared with eight other previously reported gene signatures, and evaluated for cancer network associations by two commercial pathway analysis programs. RESULTS: A novel set of 14 prognostic gene candidates were identified as outcome predictors: CXCL13, CLIC5, RGS4, RPS28, RFX7, EXOC7, HAPLN1, ZNF3, SSX3, HRBL, PRRG3, ABO, PRTN3, MATN1. A composite HRneg/Tneg gene signature index proved more accurate than any individual candidate gene or other reported multigene predictors in identifying cases likely to remain free of metastatic relapse. Significant positive correlations between the HRneg/Tneg index and three independent immune-related signatures (STAT1, IFN, and IR) were observed, as were consistent negative associations between the three immune-related signatures and five other proliferation module-containing signatures (MS-14, ONCO-RS, GGI, CSR/wound and NKI-70). Network analysis identified 8 genes within the HRneg/Tneg signature as being functionally linked to immune/inflammatory chemokine regulation. CONCLUSIONS: A multigene HRneg/Tneg signature linked to immune/inflammatory cytokine regulation was identified from pooled expression microarray data and shown to be superior to other reported gene signatures in predicting the metastatic outcome of early stage and conservatively managed HRneg and Tneg breast cancer. Further validation of this prognostic signature may lead to new therapeutic insights and spare many newly diagnosed breast cancer patients the need for aggressive adjuvant chemotherapy.

Biomarker expression and risk of subsequent tumors after initial ductal carcinoma in situ diagnosis.

BACKGROUND: Studies have failed to identify characteristics of women who have been diagnosed with ductal carcinoma in situ (DCIS) and have a high or low risk of subsequent invasive cancer. METHODS: We conducted a nested case-control study in a population-based cohort of 1162 women who were diagnosed with DCIS and treated by lumpectomy alone from 1983 to 1994. We collected clinical characteristics and information on subsequent tumors, defined as invasive breast cancer or DCIS diagnosed in the ipsilateral breast containing the initial DCIS lesion or at a regional or distant site greater than 6 months after initial treatment of DCIS (N = 324). We also conducted standardized pathology reviews and immunohistochemical staining for the estrogen receptor (ER), progesterone receptor, Ki67 antigen, p53, p16, epidermal growth factor receptor-2 (ERBB2, HER2/neu oncoprotein), and cyclooxygenase-2 (COX-2) on the initial paraffin-embedded DCIS tissue. Competing risk models were used to determine factors associated with risk of subsequent invasive cancer vs DCIS, and cumulative incidence survival functions were used to estimate 8-year risk. RESULTS: Factors associated with subsequent invasive cancer differed from those associated with subsequent DCIS. Eight-year risk of subsequent invasive cancer was statistically significantly (P = .018) higher for women with initial DCIS lesions that were detected by palpation or that were p16, COX-2, and Ki67 triple positive (p16(+)COX-2(+)Ki67(+)) (19.6%, 95% confidence interval [CI] = 18.0% to 21.3%) than for women with initial lesions that were detected by mammography and were p16, COX-2, and Ki67 triple negative (p16(-)COX-2(-)Ki67(-)) (4.1%, 95% CI = 3.4% to 5.0%). In a multivariable model, DCIS lesions that were p16(+)COX-2(+)Ki67(+) or those detected by palpation were statistically significantly associated with subsequent invasive cancer, but nuclear grade was not. Eight-year risk of subsequent DCIS was highest for women with DCIS lesions that had disease-free margins of 1 mm or greater combined with either ER(-)ERBB2(+)Ki67(+) or p16(+)COX-2(-)Ki67(+) status (23.6%, 95% CI = 18.1% to 34.0%). CONCLUSION: Biomarkers can identify which women who were initially diagnosed with DCIS are at high or low risk of subsequent invasive cancer, whereas histopathology information cannot.

Predictive models in external beam radiotherapy for clinically localized prostate cancer.

Predictive models are being used increasingly in effort to allow physician and patient expectations to be aligned with outcomes that are based on available data. Most predictive models for men who receive external beam radiotherapy for clinically localized prostate cancer are based on Gleason score, clinical tumor classification, and prostate-specific antigen (PSA) levels. More sophisticated models also have been developed that incorporate treatment-related variables, such as the dose of radiation and the use of androgen-deprivation therapy. Most of the predictive models applied to prostate cancer were derived using PSA recurrence rates as the major endpoint, but clinical endpoints have been incorporated increasingly into predictive models. Biomarkers also are increasingly being added to predictive models in an effort to strengthen them. The Radiation Therapy Oncology Group (RTOG) has completed studies on a wide range of markers using tissue from 2 phase 3 trials (RTOG 8610 and 9202). To date, preliminary assessments of p53; DNA ploidy; p16/retinoblastoma 1 protein; Ki-67; mouse double-minute p53 binding protein homolog; Bcl-2/Bcl-2-associated X protein; cytosine, adenine, and guanine repeats; cyclooxygenase-2; signal transducer and activator of transcription 3; cytochrome P450 3A4; and protein kinase A have been completed. Although they are not ready for widespread, routine use, there are reasons to believe that future models will combine these markers with traditional pretreatment and treatment-related variables and will improve our ability to predict outcome and select the optimal treatment. Cancer 2009;115(13 suppl):3112-20. (c) 2009 American Cancer Society.

Risk estimation of distant metastasis in node-negative, estrogen receptor-positive breast cancer patients using an RT-PCR based prognostic expression signature.

BACKGROUND: Given the large number of genes purported to be prognostic for breast cancer, it would be optimal if the genes identified are not confounded by the continuously changing systemic therapies. The aim of this study was to discover and validate a breast cancer prognostic expression signature for distant metastasis in untreated, early stage, lymph node-negative (N-) estrogen receptor-positive (ER+) patients with extensive follow-up times. METHODS: 197 genes previously associated with metastasis and ER status were profiled from 142 untreated breast cancer subjects. A "metastasis score" (MS) representing fourteen differentially expressed genes was developed and evaluated for its association with distant-metastasis-free survival (DMFS). Categorical risk classification was established from the continuous MS and further evaluated on an independent set of 279 untreated subjects. A third set of 45 subjects was tested to determine the prognostic performance of the MS in tamoxifen-treated women. RESULTS: A 14-gene signature was found to be significantly associated (p < 0.05) with distant metastasis in a training set and subsequently in an independent validation set. In the validation set, the hazard ratios (HR) of the high risk compared to low risk groups were 4.02 (95% CI 1.91-8.44) for the endpoint of DMFS and 1.97 (95% CI 1.28 to 3.04) for overall survival after adjustment for age, tumor size and grade. The low and high MS risk groups had 10-year estimates (95% CI) of 96% (90-99%) and 72% (64-78%) respectively, for DMFS and 91% (84-95%) and 68% (61-75%), respectively for overall survival. Performance characteristics of the signature in the two sets were similar. Ki-67 labeling index (LI) was predictive for recurrent disease in the training set, but lost significance after adjustment for the expression signature. In a study of tamoxifen-treated patients, the HR for DMFS in high compared to low risk groups was 3.61 (95% CI 0.86-15.14). CONCLUSION: The 14-gene signature is significantly associated with risk of distant metastasis. The signature has a predominance of proliferation genes which have prognostic significance above that of Ki-67 LI and may aid in prioritizing future mechanistic studies and therapeutic interventions.

The International Bladder Cancer Bank: proposal for a new study concept.

At present, results of marker studies are often inconsistent and sometimes contradictory. Recognized problems include multiple different methods of performing the assays, different subsets of patients and different endpoints, leading to incompatible datasets. Although there has been discussion of establishing general methodological principles and guidelines (analogous to those for clinical trials) for design, conduct, analysis, and reporting of marker studies, these have not been widely implemented. There are no well-recognized prototypes or examples that the urologic researcher can use to model future marker studies. We will discuss our plans to establish a multi-institutional bladder cancer data base and virtual tumor bank as a resource for participating institutions to evaluate the biological and prognostic significance of potential markers for bladder cancer. Samples will be identified and stored at each participating institution and will be available for analysis. A standard, minimal set of patient and pathologic information will be collected. The use of common software, as part of this proposal will facilitate the data transfer of updated patient information to a central database. All contributing centers will have access to summarized information, also to simplify the process of finding collaborating partners. Prospectively collected, consistent datasets with available long-term follow-up, should provide information sooner than with a conventional prospective study. Furthermore, the quality of these data and samples may be superior to that of retrospectively collected data and samples. The proposed International Bladder Cancer Bank of specimens and data will be an effective tool during all phases of marker development.

Characteristics associated with recurrence among women with ductal carcinoma in situ treated by lumpectomy.

BACKGROUND: Clinical and histopathologic characteristics that may predict risks of recurrence in women with ductal carcinoma in situ (DCIS) have not been consistently identified. We identified factors associated with recurrence as DCIS versus invasive breast cancer and determined the 5-year absolute risks of recurrence as a function of these factors. METHODS: We conducted a population-based cohort study among 1036 women in the San Francisco Bay Area who were aged 40 years or older when diagnosed with DCIS and treated by lumpectomy alone from January 1983 through December 1994. Standardized pathology reviews were conducted to determine disease recurrence, defined as DCIS or invasive breast cancer diagnosed in the ipsilateral breast containing the initial DCIS lesion or at a distant site more than 6 months after the initial diagnosis and treatment of DCIS. Conditional logistic regression models were used to determine factors associated with recurrence. All statistical significance tests were two-sided. RESULTS: During a median follow-up of 77.9 months, 209 women (20.2%) experienced a recurrence. Overall, the 5-year risks of recurrence as invasive cancer and as DCIS were 8.2% (95% confidence interval [CI] = 6.6% to 9.8%) and 11.7% (95% CI = 9.9% to 13.3%), respectively. The 5-year risks of recurrence as invasive cancer and as DCIS were 4.8% (95% CI = 3.7% to 6.8%) and 4.8% (95% CI = 3.8% to 5.8%), respectively, for women with low-nuclear-grade DCIS; 11.8% (95% CI = 9.9% to 14.1%) and 17.1% (95% CI = 15.5% to 18.7%), respectively, for women with high-nuclear-grade DCIS; 11.6% (95% CI = 11.3% to 12.0%) and 8.6% (95% CI = 7.1% to 10.2%), respectively, for women whose initial DCIS lesion was detected by palpation; and 6.6% (95% CI = 6.2% to 7.1%) and 14.1% (95% CI = 11.4% to 17.8%), respectively, for women with DCIS detected by mammography alone. High- (versus low-) nuclear-grade DCIS lesions and detection of the initial DCIS lesion by palpation (versus mammography) were associated with recurrence as invasive cancer. High- (versus low-) nuclear-grade lesions; resection margins that were positive, uncertain, or less than 10 mm disease-free (versus > or = 10 mm disease-free); and age 40-49 years at diagnosis (versus > or =50 years) were associated with recurrence as DCIS. CONCLUSIONS: Nuclear grade is strongly associated with recurrence but not with the type of recurrence. Women with high-nuclear-grade DCIS or DCIS detected by palpation who are treated by lumpectomy alone are at relatively high risk of having an invasive breast cancer recurrence, compared with women with low-nuclear-grade or mammographically detected DCIS, and may be appropriate candidates for additional treatment.