Michael E. Ray

Cloning a novel member of the human interferon-inducible gene family associated with control of tumorigenicity in a model of human melanoma

Chromosome 6-mediated suppression of tumorigenicity in malignant melanoma cell lines provides a model system to identify genes associated with the reversion of the tumorigenic phenotype. Using subtractive cDNA selection, we recently identified a series of novel genes which are differentially expressed in association with chromosome 6-mediated suppression. We now report the molecular characterization of a novel gene termed AIM2 for (Absent In Melanoma), which represents a 1485 bp cDNA. An open reading frame of 1032 base pairs, corresponding to 344 amino acid residues, is predicted. The predicted protein shares a conserved sequence domain of approximately 200 amino acids with known interferon-inducible genes of both human and mouse. We demonstrate that the AIM2 gene encodes a transcript of approximately 2 kb which is expressed in spleen, small intestine, and peripheral blood leukocytes. In addition, we have localized AIM2 to the long arm of human chromosome 1 (band q22) in a highly conserved region which also contains the known interferon-inducible genes IFI16 and MNDA. We have also demonstrated that, like IFI16 and MNDA, AIM2 is induced in HL60 cells by interferon gamma. Our findings support the existence of a family of genes in this region similar to the well-characterized mouse Ifi200 gene family.

Genomic and Expression Analysis of the 8p11–12 Amplicon in Human Breast Cancer Cell Lines

Gene amplification is an important mechanism of oncogene activation in breast and other cancers. Characterization of amplified regions of the genome in breast cancer has led to the identification of important oncogenes including erbB-2/HER-2, C-MYC, and fibroblast growth factor receptor (FGFR) 2. Chromosome 8p11-p12 is amplified in 10–15% of human breast cancers. The putative oncogene FGFR1 localizes to this region; however, we show evidence that FGFR inhibition fails to slow growth of three breast cancer cell lines with 8p11-p12 amplification. We present a detailed analysis of this amplicon in three human breast cancer cell lines using comparative genomic hybridization, traditional Southern and Northern analysis, and chromosome 8 cDNA microarray expression profiling. This study has identified new candidate oncogenes within the 8p11-p12 region, supporting the hypothesis that genes other than FGFR1 may contribute to oncogenesis in breast cancers with proximal 8p amplification.

Multiple Interacting Oncogenes on the 8p11-p12 Amplicon in Human Breast Cancer

The 8p11-p12 genomic region is amplified in 15% of breast cancers and harbors several candidate oncogenes. However, functional evidence for a transforming role for these genes is lacking. We identified 21 genes from this region as potential oncogenes based on statistical association between copy number and expression. We further showed that three of these genes (LSM1, BAG4, and C8orf4) induce transformed phenotypes when overexpressed in MCF-10A cells, and overexpression of these genes in combination influences the growth factor independence phenotype and the ability of the cells to grow under anchorage-independent conditions. Thus, LSM1, BAG4, and C8orf4 are breast cancer oncogenes that can work in combination to influence the transformed phenotype in human mammary epithelial cells.

No Effect of Statins on Biochemical Outcomes After Radiotherapy for Localized Prostate Cancer

OBJECTIVES To examine the effect of concurrent statin use during definitive radiotherapy (RT) on the biochemical outcomes for localized prostate cancer. METHODS A total of 968 patients treated with RT had information about medication use available. Of these, 23% had been taking using statins during RT. Progression-free survival (PFS) was determined by a biochemical failure definition of prostate-specific antigen nadir plus 2 ng/mL, clinical failure, start of androgen deprivation therapy, or death. RESULTS The mean patient age was 68 years. The median radiation dose was 76 Gy. Of the patients, 29% underwent androgen deprivation therapy. The 5-year overall survival rate was 83%. The median PFS time was 7.8 years versus 6.4 years, and the 5-year PFS rate was 70% versus 59% in favor of the statin users (P = 0.03). The analysis by risk group demonstrated no significant statin effect in any of the three risk strata. Stratification by hydrophilic versus hydrophobic statin agents revealed similar results. Multivariate analysis revealed that T stage (P <0.0001), pretreatment prostate-specific antigen level (P <0.0001), and Gleason score (P = 0.0026) were significant predictors of PFS; however, statin use (P = 0.48), androgen deprivation therapy (P = 0.95), pelvic RT (P = 0.96), radiation dose (P = 0.13), age (P = 0.19), and year of treatment (P = 0.07) were not. CONCLUSIONS Statin use did not affect PFS after adjusting for differences in treatment year and multiple prognostic factors. However, T stage, baseline prostate-specific antigen level, and Gleason score were critical determinants of prostate-specific antigen failure. These results did not differ when hydrophilic pravastatin was excluded.

Targeted and systemic radiotherapy in the treatment of bone metastasis

Cancer metastasis to the bone develops commonly in patients with a variety of malignancies, and is a major cause of morbidity and diminished quality of life in a significant proportion of cancer patients. The effective treatment of bone metastasis requires cooperation between medical, surgical and radiation oncologists. Radiotherapy, either in the form of targeted external beam radiation therapy, or systemic administration of radionuclides, plays a central role in treatment of symptomatic bone metastases. The appropriate external beam treatment techniques, dose and fractionation regimens for the treatment of symptomatic, localized bone metastasis have been established in prospective clinical trials. Large-field, hemi-body irradiation has been utilized for treatment of symptoms related to more widely disseminated bone metastases, but has been associated with substantial toxicity. Strontium-89 and Samarium-153 are widely available systemically administered radionuclides that are useful for the treatment of widely disseminated disease, and have largely supplanted the use of hemi-body irradiation. Combined with appropriate medical and surgical interventions, as well as the appropriate use of analgesics, radiotherapy is a well-tolerated and highly effective treatment for the palliation of symptomatic bone metastases.

Loss of Raf Kinase Inhibitory Protein Induces Radioresistance in Prostate Cancer

PURPOSE External beam radiotherapy (RT) is often used in an attempt to cure localized prostate cancer (PCa), but it is only palliative against disseminated disease. Raf kinase inhibitory protein (RKIP) is a metastasis suppressor whose expression is reduced in approximately 50% of localized PCa tissues and is absent in metastases. Chemotherapeutic agents have been shown to induce tumor apoptosis through induction of RKIP expression. Our goal was to test whether RT similarly induces apoptosis through induction of RKIP expression. METHODS AND MATERIALS The C4-2B PCa cell line was engineered to overexpress or underexpress RKIP. The engineered cells were tested for apoptosis in cell culture and tumor regression in mice after RT. RESULTS RT induced both RKIP expression and apoptosis of PCa cells. Overexpression of RKIP sensitized PCa cells to radiation-induced apoptosis. In contrast, short-hairpin targeting of RKIP, so that RT could not induce RKIP expression, protected cells from radiation-induced apoptosis. In a murine model, knockdown of RKIP in PCa cells diminished radiation-induced apoptosis. Molecular concept mapping of genes altered on manipulation of RKIP expression revealed an inverse correlation with the concept of genes altered by RT. CONCLUSION The data presented in this report indicate that the loss of RKIP, as seen in primary PCa tumors and metastases, confers protection against radiation-induced apoptosis. Therefore, it is conceivable that the loss of RKIP confers a growth advantage on PCa cells at distant sites, because the loss of RKIP would decrease apoptosis, favoring proliferation.

Randomized phase II trial of urethral sparing intensity modulated radiation therapy in low-risk prostate cancer: implications for focal therapy

BackgroundLow-risk prostate cancer (PCa) patients have excellent outcomes, with treatment modality often selected by perceived effects on quality of life. Acute urinary symptoms are common during external beam radiotherapy (EBRT), while chronic symptoms have been linked to urethral dose. Since most low-risk PCa occurs in the peripheral zone (PZ), we hypothesized that EBRT using urethral sparing intensity modulated radiation therapy (US-IMRT) could improve urinary health-related quality of life (HRQOL) while maintaining high rates of PCa control.MethodsPatients with National Comprehensive Cancer Network (NCCN) defined low-risk PCa with no visible lesion within 5 mm of the prostatic urethra on MRI were randomized to US-IMRT or standard (S-) IMRT. Prescription dose was 75.6 Gy in 41 fractions to the PZ + 3–5 mm for US-IMRT and to the prostate + 3 mm for S-IMRT. For US-IMRT, mean proximal and distal urethral doses were limited to 65 Gy and 74 Gy, respectively. HRQOL was assessed using the Expanded Prostate Cancer Index (EPIC) Quality of Life questionnaire. The primary endpoint was change in urinary HRQOL at 3 months.ResultsFrom June 2004 to November 2006, 16 patients were randomized, after which a futility analysis concluded that continued accrual was unlikely to demonstrate a difference in the primary endpoint. Mean change in EPIC urinary HRQOL at 3 months was −0.5 ± 11.2 in the US-IMRT arm and +3.9 ± 15.3 in the S-IMRT arm (p = 0.52). Median PSA nadir was higher in the US-IMRT arm (1.46 vs. 0.78, p = 0.05). At 4.7 years median follow-up, three US-IMRT and no S-IMRT patients experienced PSA failure (p = 0.06; HR 8.8, 95% CI 0.9–86). Two out of 3 patients with PSA failure had biopsy-proven local failure, both located contralateral to the original site of disease.ConclusionsCompared with S-IMRT, US-IMRT failed to improve urinary HRQOL and resulted in higher PSA nadir and inferior biochemical control. The high rate of PSA failure and contralateral local failures in US-IMRT patients, despite careful selection of MRI-screened low-risk patients, serve as a cautionary tale for focal PCa treatments.

Potential Surrogate Endpoints for Prostate Cancer Survival: Analysis of a Phase III Randomized Trial

BACKGROUND The identification of surrogate endpoints for prostate cancer-specific survival may shorten the length of clinical trials for prostate cancer. We evaluated distant metastasis and general clinical treatment failure as potential surrogates for prostate cancer-specific survival by use of data from the Radiation Therapy and Oncology Group 92-02 randomized trial. METHODS Patients (n = 1554 randomly assigned and 1521 evaluable for this analysis) with locally advanced prostate cancer had been treated with 4 months of neoadjuvant and concurrent androgen deprivation therapy with external beam radiation therapy and then randomly assigned to no additional therapy (control arm) or 24 additional months of androgen deprivation therapy (experimental arm). Data from landmark analyses at 3 and 5 years for general clinical treatment failure (defined as documented local disease progression, regional or distant metastasis, initiation of androgen deprivation therapy, or a prostate-specific antigen level of 25 ng/mL or higher after radiation therapy) and/or distant metastasis were tested as surrogate endpoints for prostate cancer-specific survival at 10 years by use of Prentices four criteria. All statistical tests were two-sided. RESULTS At 3 years, 1364 patients were alive and contributed data for analysis. Both distant metastasis and general clinical treatment failure at 3 years were consistent with all four of Prentices criteria for being surrogate endpoints for prostate cancer-specific survival at 10 years. At 5 years, 1178 patients were alive and contributed data for analysis. Although prostate cancer-specific survival was not statistically significantly different between treatment arms at 5 years (P = .08), both endpoints were consistent with Prentices remaining criteria. CONCLUSIONS Distant metastasis and general clinical treatment failure at 3 years may be candidate surrogate endpoints for prostate cancer-specific survival at 10 years. These endpoints, however, must be validated in other datasets.

Primary versus Radiation-Associated Craniofacial Osteosarcoma Biologic and Clinicopathologic Comparisons

Craniofacial osteosarcoma differs from long bone osteosarcoma in that patients are older, tumors are often low grade, and prognosis is more favorable. Although most are sporadic, some tumors occur in association with prior radiation therapy. The purpose of the current study was to compare clinicopathologic and prognostic features of primary and radiation‐associated osteosarcoma.

Risk factors for local recurrence and metastasis in soft tissue sarcomas of the extremity.

ObjectivesWe reviewed our institutions experience in treating soft tissue sarcomas of the extremity to identify factors associated with local recurrence, metastasis, and overall survival, to identify patients who may benefit from intensification of therapy. MethodsA retrospective analysis was performed for patients who underwent both limb-sparing surgery and external beam radiotherapy for extremity sarcoma. Those who had gross residual disease or who presented with recurrent or metastatic disease were excluded. The Kaplan-Meier product limit and multivariate Cox regression were used to estimate local failure-free probability, distant failure-free probability, and overall survival along with associations with patient, tumor, and treatment characteristics. ResultsOne hundred eighty-eight patients were included in the analysis. Twenty-five (13%) and 46 (24%) experienced local and distant recurrence, respectively. Patients with high/intermediate-grade tumors [hazard ratio (HR)=5.63, 95% confidence interval (CI): 1.27-24.89, P=0.023] or with multifocally positive margins (HR=4.27, 95% CI: 1.20-15.24, P=0.026) were more likely to fail locally. Those with a preceding local recurrence (HR=8.58, 95% CI: 3.87-19.04, P<0.0001), high/intermediate-grade tumors (HR=5.68, 95% CI: 1.28-25.25, P=0.023), or no secondary reexcision (HR=2.5, 95% CI: 1.09-5.74, P=0.031) were more likely to develop metastasis. Patients with local recurrence (HR=3.6, 95% CI: 1.77-7.29, P<0.001), metastasis (HR=16.0, 95% CI: 7.93-32.31, P<0.0001), or without secondary reexcision (HR=3.2, 95% CI: 1.27-8.09, P=0.014) had decreased overall survival. ConclusionsPatients whose tumor grade or margin status put them at high risk for local failure should be considered for intensification of therapy. Those with a local recurrence should be considered for increased surveillance or systemic therapy, as local failure is associated with subsequent metastasis and decreased survival.