Subtype-specific expression of MELK is partly due to copy number alterations in breast cancer.

Maternal embryonic leucine-zipper kinase (MELK) regulates cell cycle progression and is highly expressed in many cancers. The molecular mechanism of MELK dysregulation has not been determined in aggressive forms of breast cancer, such as triple negative breast cancer (TNBC). To evaluate molecular markers of MELK aberrations in aggressive breast cancer, we assessed MELK gene amplification and expression in breast tumors. MELK mRNA expression is highly up-regulated in basal-like breast cancer (BLBC), the major molecular subtype of TNBC, compared to luminal or other subtypes of breast tumors. MELK copy number (CN) gains are significantly associated with BLBC, whereas no significant association of CpG site methylation or histone modifications with breast cancer subtypes was observed. Accordingly, the CN gains appear to contribute to an increase in MELK expression, with a significant correlation between mRNA expression and CN in breast tumors and cell lines. Furthermore, immunohistochemistry (IHC) assays revealed that both nuclear and cytoplasmic staining scores of MELK were significantly higher in invasive ductal carcinoma (IDC) tumors compared to ductal carcinoma in situ (DCIS) and normal breast tissues. Our data showed that upregulation of MELK in BLBC may be in part driven by CN gains, rather than epigenetic modifications, indicating a potential for overexpression and CN gains of MELK to be developed as a diagnostic and prognostic marker to identify patients who have more aggressive breast cancer.

Effects of Slide Storage on Detection of Molecular Markers by IHC and FISH in Endometrial Cancer Tissues From a Clinical Trial: An NRG Oncology/GOG Pilot Study.

We performed a pilot study in anticipation of using long-aged precut formalin-fixed paraffin-embedded tissue sections stored in real-world conditions for translational biomarker studies of topoisomerase 2A (TOP2A), Ki67, and human epidermal growth factor receptor 2 (HER2) in endometrial cancer. Formalin-fixed paraffin-embedded tissue blocks or unstained slides or both from GOG-0177 were collected centrally (1999-2000) and stored at room temperature. During 2004 to 2011 specimens were stored at 4°C. Matched pairs of stored slides and freshly cut slides from stored blocks were analyzed for TOP2A (KiS1), Ki67 (MIB1), and HER2 (HercepTest) proteins. To assess DNA stability (HER2 PathVision), fluorescence in situ hybridization (FISH) was repeated on stored slides from 21 cases previously shown to be HER2 amplified. Immunohistochemistry (IHC) staining intensity and extent, mean FISH copies/cell, and copy number ratios were compared using the κ statistic for concordance or signed rank test for differences in old cut versus new cut slides. IHC results reflected some protein degradation in stored slides. The proportion of cells with TOP2A staining was lower on average by 12% in older sections (P=0.03). The proportion of Ki67-positive cells was lower in stored slides by an average of 10% (P<0.01). Too few cases in the IHC cohort were FISH positive for any conclusions. HER2 amplification by FISH was unaffected by slide storage. We conclude that use of aged stored slides for proliferation markers TOP2A and Ki67 is feasible but may modestly underestimate true values in endometrial cancer. Pilot studies for particular storage conditions/durations/antigens to be used in translational studies are warranted.

Advances in breast cancer: pathways to personalized medicine.

Breast cancer is a complex disease caused by the progressive accumulation of multiple gene mutations combined with epigenetic dysregulation of critical genes and protein pathways. There is substantial interindividual variability in both the age at diagnosis and phenotypic expression of the disease. With an estimated 1,152,161 new breast cancer cases diagnosed worldwide per year, cancer control efforts in the postgenome era should be focused at both population and individual levels to develop novel risk assessment and treatment strategies that will further reduce the morbidity and mortality associated with the disease. The discovery that mutations in the BRCA1 and BRCA2 genes increase the risk of breast and ovarian cancers has radically transformed our understanding of the genetic basis of breast cancer, leading to improved management of high-risk women. A better understanding of tumor host biology has led to improvements in the multidisciplinary management of breast cancer, and traditional pathologic evaluation is being complemented by more sophisticated genomic approaches. A number of genomic biomarkers have been developed for clinical use, and increasingly, pharmacogenetic end points are being incorporated into clinical trial design. For women diagnosed with breast cancer, prognostic or predictive information is most useful when coupled with targeted therapeutic approaches, very few of which exist for women with triple-negative breast cancer or those with tumors resistant to chemotherapy. The immediate challenge is to learn how to use the molecular characteristics of an individual and their tumor to improve detection and treatment, and ultimately to prevent the development of breast cancer. The five articles in this edition of CCR Focus highlight recent advances and future directions on the pathway to individualized approaches for the early detection, treatment, and prevention of breast cancer.

BRCA1 promoter methylation in sporadic breast cancer is associated with reduced BRCA1 copy number and chromosome 17 aneusomy.

To explore the molecular mechanisms for the similarities between inherited and noninherited forms of breast cancer, we tested the hypothesis that inactivation of BRCA1 by promoter hypermethylation is associated with reduced gene copy number and chromosome 17 aneusomy as observed in tumors from BRCA1 mutation carriers. Using a combination of methylation-specific PCR analysis and fluorescence in situ hybridization, we observed varying degrees of promoter methylation in 39 of 131 (29.8%) primary tumors. Despite significant tumor heterogeneity, mean copy numbers of BRCA1 and CEP17 per cell were lower in methylated cases compared with unmethylated cases [1.78 versus 2.30 (P = 0.001) and 1.85 versus 2.29 (P = 0.005), respectively]. Methylation was more frequently observed in younger women (P = 0.05) with high-grade (P = 0.001), estrogen receptor-negative (P = 0.04), and progesterone receptor-negative (P = 0.01) tumors. Moreover, methylation was associated with reduced or absent BRCA1 transcripts, which was reversible in the heavily BRCA1-methylated cell line UACC3199 following treatment with 5-aza-2'-deoxycytidine and trichostatin A. We identified five CpGs at positions -533, -355, -173, -21, and +44 as critical in the reexpression of BRCA1. We conclude that BRCA1 methylation contributes to a subset of sporadic breast cancers with the resulting molecular and clinicopathologic phenotype similar to that of hereditary BRCA1-associated breast cancers. Our data support a model of carcinogenesis in which BRCA1 promoter methylation may serve as a "first hit," much like an inherited germ line mutation, and promote tumor progression down a restricted set of molecular pathways.

Molecular-cytogenetic analysis of HER-2/neu gene in BRCA1-associated breast cancers.

The BRCA1 tumor suppressor gene and the HER-2/neu oncogene are located in close proximity on the long arm of chromosome 17 (17q11-21). Absence of BRCA1 or functional overexpression of the HER-2/neu gene presumably contributes to the somatic phenotype of breast cancer in premenopausal women, characterized by unfavorable prognostic features such as high tumor grade, hormone receptor negativity, and high proliferation rate. To examine whether amplification of HER-2/neu contributes to the aggressive biology of BRCA1-associated tumors, we have performed fluorescence in situ hybridization on formalin-fixed paraffin-embedded breast tumor tissue sections from 53 BRCA1 mutation carriers and 41 randomly selected, age-matched sporadic breast cancer cases. Although BRCA1-associated and sporadic tumors were equally likely (19% versus 22%) to exhibit HER-2/neu amplification [defined as a ratio of HER-2/neu copies to chromosome 17 centromere (CEP17) copies > or = 2], 6 (15%) of the sporadic tumors were highly amplified (defined as a ratio greater-than-or-equal 5) versus none of the BRCA1-associated tumors (P = 0.048). HER-2 protein overexpression as measured by immunohistochemical analysis was not observed among the BRCA1-associated cases (P = 0.042). Four of 21 (19%) sporadic tumors exhibited strong membranous staining of HER-2 (intensity level of 3+) as compared with 0 of 39 BRCA1-associated tumors. Our data suggest that a germ-line mutation in the BRCA1 tumor suppressor gene is associated with a significantly lower level of HER-2/neu amplification. Thus, it is possible that BRCA1-associated and HER-2/neu-highly amplified tumors progress through distinct molecular pathways, and the aggressive pathological features of BRCA1-associated tumors appear unrelated to amplification of the adjacent HER-2/neu oncogene.

Association of Metformin Use with Outcomes in Advanced Endometrial Cancer Treated with Chemotherapy.

There is increasing evidence that metformin, a commonly used treatment for diabetes, might have the potential to be repurposed as an economical and safe cancer therapeutic. The aim of this study was to determine whether stage III-IV or recurrent endometrial cancer patients who are using metformin during treatment with chemotherapy have improved survival. To test this we analyzed a retrospective cohort of subjects at two independent institutions who received chemotherapy for stage III-IV or recurrent endometrial cancer from 1992 to 2011. Diagnosis of diabetes, metformin use, demographics, endometrial cancer clinico-pathologic parameters, and survival duration were abstracted. The primary outcome was overall survival. The final cohort included 349 patients, 31 (8.9%) had diabetes and used metformin, 28 (8.0%) had diabetes but did not use metformin, and 291 (83.4%) did not have diabetes. The results demonstrate that the median overall survival was 45.6 months for patients with diabetes who used metformin compared to 12.5 months for patients with diabetes who did not use metformin and 28.5 months for patients without diabetes (log-rank test comparing the three groups P = 0.006). In a model adjusted for confounders, the difference in survival between the three groups remained statistically significant (P = 0.023). The improvement in survival among metformin users was not explained by better baseline health status or more aggressive use of chemotherapy. Overall, the findings in this retrospective cohort of endometrial cancer patients with stage III-IV or recurrent disease treated with chemotherapy indicate that patients with diabetes who were concurrently treated with metformin survived longer than patients with diabetes who did not use metformin.

MYC is amplified in BRCA1-associated breast cancers.

PURPOSE: Germ-line mutations in the BRCA1 tumor suppressor gene predispose to early onset breast cancers with a distinct phenotype characterized by high tumor grade, aneuploidy, high proliferation rate, and estrogen receptor-negativity. The molecular mechanisms and cooperative oncogenes contributing to multistep tumor progression in cells lacking BRCA1 are not well defined. To examine whether C-MYC (MYC), a transforming oncogene associated with genetic instability, contributes to multistep tumor progression in BRCA1-associated breast cancer, we have analyzed tumors from women with hereditary BRCA1-mutated and sporadic breast cancers. EXPERIMENTAL DESIGN: We performed fluorescence in situ hybridization using a MYC:CEP8 assay on formalin-fixed paraffin-embedded tumor tissues from 40 women with known deleterious germ-line BRCA1 mutations and 62 sporadic cases, including 20 cases with hypermethylation of the BRCA1 gene promoter. RESULTS: We observed a MYC:CEP8 amplification ratio >/=2 in 21 of 40 (53%) BRCA1-mutated tumors compared with 14 of 62 (23%) sporadic tumors (P = 0.003). Of the 14 sporadic cases with MYC amplification, 8 (57%) were BRCA1-methylated. In total, MYC amplification was found in a significantly higher proportion of tumors with BRCA1 dysfunction (29 of 60, 48% versus 6 of 42, 14%; P = 0.0003). In a multivariable regression model controlling for age, tumor size, and estrogen receptor status, BRCA1-mutated tumors demonstrated significantly greater mean MYC:CEP8 ratio than sporadic tumors (P = 0.02). CONCLUSIONS: Our data indicate that MYC oncogene amplification contributes to tumor progression in BRCA1-associated breast cancers. Thus, we conclude that the aggressive histopathological features of BRCA1-associated tumors are in part due to dysregulated MYC activity.

Afatinib efficacy against squamous cell carcinoma of the head and neck cell lines in vitro and in vivo.

Epidermal growth factor receptor (EGFR) inhibitors have demonstrated efficacy in squamous cell carcinoma of the head and neck (SCCHN). In addition to EGFR, other ErbB family members are expressed and activated in SCCHN. Afatinib is an ErbB family blocker that has been approved for treating patients with EGFR-mutated nonsmall cell lung cancer. We sought to determine the efficacy of afatinib in preclinical models and compare this to other EGFR-targeted agents. Afatinib efficacy was characterized in a panel of ten SCCHN cell lines and found to be most effective against cell lines amplified for EGFR. Afatinib had lower IC(50) values than did gefitinib against the same panel. Two EGFR-amplified cell lines that are resistant to gefitinib are sensitive to afatinib. Cetuximab was not found to have a synergistic effect with afatinib either in vitro or in vivo. Both afatinib and cetuximab were effective in tumor xenograft model. Afatinib is an effective agent in SCCHN especially in models with EGFR amplification.

Phase II study of gefitinib adaptive dose escalation to skin toxicity in recurrent or metastatic squamous cell carcinoma of the head and neck.

BACKGROUND: Gefitinib has activity in patients with advanced squamous cell carcinoma of the head and neck (SCCHN) and skin toxicity has been postulated to be a predictor of response and improved outcome. METHODS: This open-label, multi-institution, phase II study evaluated the activity of gefitinib at individually escalated doses up to 750 mg to achieve the skin toxicity grade ≥2. RESULTS: Forty four patients were enrolled. Only twenty-three (52%) experienced skin rash grade ≥2. Of 44 patients, partial responses were noted in 3 (7%), stable disease in 8 (18%) and progressive disease in 33 patients. Median progression-free survival was 1.9 months (95% CI 1.6-2.2) and median overall survival was 5.1 months (95% CI 2.4-7.8). Grade of skin rash was not associated with response rate (p=0.169) nor tumor control rate (p=0.284); however, higher gefitinib trough levels were associated with disease control. Of the 11 tissue samples analyzed for EGFR gene copy by FISH, 7 were EGFR FISH positive, but this was not associated with improved tumor control or survival. CONCLUSIONS: Gefitinib has clinical activity as monotherapy in SCCHN. Dose escalation of gefitinib is feasible and may increase skin toxicity, but our data do not support increased activity.

RON (MST1R) is a novel prognostic marker and therapeutic target for gastroesophageal adenocarcinoma.

RON (MST1R) is one of two members of the MET receptor tyrosine kinase family, along with parent receptor MET. RON has a putative role in several cancers, but its expression and function is poorly characterized in gastroesophageal adenocarcinoma. A recognized functional role of MET tyrosine kinase in gastroesophageal cancer has led to early phase clinical trials using MET inhibitors, with unimpressive results. Therefore, the role of RON in gastroesophageal cancer, as well as its role in cooperative signaling with MET and as a mechanism of resistance to MET inhibition, was studied in gastroesophageal tissues and cell lines. By IHC, RON was highly over-expressed in 74% of gastroesophageal samples (n=94), and over-expression was prognostic of poor survival (p=0.008); RON and MET co-expression occurred in 43% of samples and was prognostic of worst survival (p=0.03). High MST1R gene copy number by quantitative polymerase chain reaction, and confirmed by fluorescence in situ hybridization and/or array comparative genomic hybridization, was seen in 35.5% (16/45) of cases. High MST1R gene copy number correlated with poor survival (p=0.01), and was associated with high MET and ERBB2 gene copy number. A novel somatic MST1R juxtamembrane mutation R1018G was found in 11% of samples. RON signaling was functional in cell lines, activating downstream effector STAT3, and resulted in increased viability over controls. RON and MET co-stimulation assays led to enhanced malignant phenotypes over stimulation of either receptor alone. Growth inhibition as evidenced by viability and apoptosis assays was optimal using novel blocking monoclonal antibodies to both RON and MET, versus either alone. SU11274, a classic MET small molecule tyrosine kinase inhibitor, blocked signaling of both receptors, and proved synergistic when combined with STAT3 inhibition (combination index < 1). These preclinical studies define RON as an important novel prognostic marker and therapeutic target for gastroesophageal cancer warranting further investigation.

Epidermal growth factor receptor in triple-negative and basal-like breast cancer: promising clinical target or only a marker?

Triple-negative breast cancers represent a subset of breast cancers with a particularly aggressive phenotype and poor clinical outcomes. Recent molecular profiling of these tumors has revealed a high frequency of epidermal growth factor receptor (EGFR) dysregulation, among other abnormalities. EGFR status correlates negatively with survival in patients with triple-negative breast cancers, and thus focus has turned on this receptor as a potential clinical target. Two classes of EGFR inhibitors are currently in clinical use: the monoclonal antibodies and the small molecule tyrosine kinase inhibitors. Trials of these drugs in breast cancer, however, have been largely disappointing. It remains to be seen whether advances in our understanding of the mechanisms of EGFR dysregulation and effects of multiple compensatory pathways in breast cancer, coupled with improved targeting to appropriate patient populations, will yield meaningful improvements in clinical outcomes.

Epidermal growth factor receptor inhibitor gefitinib added to chemoradiotherapy in locally advanced head and neck cancer.

PURPOSE: Assess efficacy and toxicity of gefitinib, an epidermal growth factor receptor (EGFR) inhibitor, added to, and in maintenance after, concurrent chemoradiotherapy (CCRT) in locally advanced head and neck cancer (LA-HNC) and correlate outcomes with EGFR gene copy number alterations. PATIENTS AND METHODS: Patients with stage III to IV LA-HNC received two cycles of carboplatin/paclitaxel induction chemotherapy (IC) followed by split-course CCRT with fluorouracil, hydroxyurea, twice daily radiotherapy (FHX), and gefitinib (250 mg daily) followed by continued gefitinib for 2 years total. The primary end point was complete response (CR) rate after CCRT. EGFR gene copy number was assessed by fluorescent in situ hybridization. RESULTS: Sixty-nine patients (66 with stage IV disease, 37 with oropharynx primary tumors, and 67 with performance status 0 to 1) were enrolled with a median age of 55 years. Predominant grade 3 or 4 toxicities during IC and CCRT were neutropenia (n = 20) and in-field mucositis (n = 59) and dermatitis (n = 23), respectively. CR rate after CCRT was 90%. After median follow-up of 3.5 years, 4-year overall, progression-free, and disease-specific survival rates were 74%, 72%, and 89%, respectively. To date, one patient has developed a second primary tumor in the aerodigestive tract. In 31 patients with available tissue, high EGFR gene copy number was associated with worse overall survival (P = .02). CONCLUSION: Gefitinib can be administered with FHX and as maintenance therapy for at least 2 years, demonstrating CR and survival rates that compare favorably with prior experience. High EGFR gene copy number may be associated with poor outcome in patients with LA-HNC treated with this regimen.

Estrogen receptor alpha, BRCA1, and FANCF promoter methylation occur in distinct subsets of sporadic breast cancers.

Estrogen receptor alpha (ER) and its ligand estrogen play vital roles in the development, progression and treatment of breast cancer. An increasing number of studies have also provided evidence linking disruption of the Fanconi anemia/BRCA cascade to breast cancer. Our objectives were to examine the methylation status and expression profiles of ER, correlate the findings with BRCA1 and FANCF methylation and map the critical CpGs for ER expression. We found that the CpG islands in the 5' region of the ER gene are methylated in 59 of 120 (49.2%) primary breast cancers, including 45 of 59 ER-negative tumors (76.3%, P < 0.00001). In addition, we observed a strong correlation between ER promoter and BRCA1 promoter methylation (odds ratio 3.12, 95% confidence interval 1.10-9.68, P = 0.02). In contrast, FANCF methylation was rare in breast tumors: one of 120 (0.8%). ER methylation was associated with high tumor grade (60.4% methylated vs. 39.6% unmethylated in grade 3 tumors, P = 0.04) and tumor subtype (P = 0.03). Though small in number, all tumors of the medullary subtype were ER methylated. In contrast, the lobular subtype had the least methylation (23.1% methylated vs. 76.9% unmethylated). After treatment of MDA-MB-231 cells with 5-aza-cytidine (5-aza-dC) and trichostatin, which resulted in re-expression of ER mRNA, we localized dramatic demethylation effects to CpG islands in positions +68, +165, +192, +195, +337, +341 and +405 from transcription start site of the ER promoter. These data suggest that unlike FANCF, both ER and BRCA1 are specifically targeted for methylation in sporadic breast cancers, a phenomenon that should be explored for development of novel diagnostic and therapeutic approaches.