Angela A. Guzzetta

Novel Methylation Biomarker Panel for the Early Detection of Pancreatic Cancer

Purpose: Pancreatic cancer is the fourth leading cause of cancer deaths and there currently is no reliable modality for the early detection of this disease. Here, we identify cancer-specific promoter DNA methylation of BNC1 and ADAMTS1 as a promising biomarker detection strategy meriting investigation in pancreatic cancer. Experimental Design: We used a genome-wide pharmacologic transcriptome approach to identify novel cancer-specific DNA methylation alterations in pancreatic cancer cell lines. Of eight promising genes, we focused our studies on BNC1 and ADAMTS1 for further downstream analysis, including methylation and expression. We used a nanoparticle-enabled methylation on beads (MOB) technology to detect early-stage pancreatic cancers by analyzing DNA methylation in patient serum. Results: We identified two novel genes, BNC1 (92%) and ADAMTS1 (68%), that showed a high frequency of methylation in pancreatic cancers (n = 143), up to 100% in PanIN-3 and 97% in stage I invasive cancers. Using the nanoparticle-enabled MOB technology, these alterations could be detected in serum samples (n = 42) from patients with pancreatic cancer, with a sensitivity for BNC1 of 79% [95% confidence interval (CI), 66%–91%] and for ADAMTS1 of 48% (95% CI, 33%–63%), whereas specificity was 89% for BNC1 (95% CI, 76%–100%) and 92% for ADAMTS1 (95% CI, 82%–100%). Overall sensitivity using both markers is 81% (95% CI, 69%–93%) and specificity is 85% (95% CI, 71%–99%). Conclusions: Promoter DNA methylation of BNC1 and ADAMTS1 is a potential biomarker to detect early-stage pancreatic cancers. Assaying the promoter methylation status of these genes in circulating DNA from serum is a promising strategy for early detection of pancreatic cancer and has the potential to improve mortality from this disease. Clin Cancer Res; 19(23); 6544–55. ©2013 AACR.

DNA methylation biomarker candidates for early detection of colon cancer.

Promoter CpG island hypermethylation of tumor suppressor genes is a common hallmark of all human cancers. Many researchers have been looking for potential epigenetic therapeutic targets in cancer using gene expression profiling with DNA microarray approaches. Our recent genome-wide platform of CpG island hypermethylation and gene expression in colorectal cancer (CRC) cell lines revealed that FBN2 and TCERG1L gene silencing is associated with DNA hypermethylation of a CpG island in the promoter region. In this study, promoter DNA hypermethylation of FBN2 and TCERG1L in CRC occurs as an early and cancer-specific event in colorectal cancer. Both genes showed high frequency of methylation in colon cancer cell lines (>80% for both of genes), adenomas (77% for FBN2, 90% for TCERG1L, n = 39), and carcinomas (86% for FBN2, 99% for TCERG1L, n = 124). Bisulfite sequencing confirmed cancer-specific methylation of FBN2 and TCERG1L of promoters in colon cancer cell line and cancers but not in normal colon. Methylation of FBN2 and TCERG1L is accompanied by downregulation in cell lines and in primary tumors as described in the Oncomine™ website. Together, our results suggest that gene silencing of FBN2 and TCERG1L is associated with promoter DNA hypermethylation in CRC tumors and may be excellent biomarkers for the early detection of CRC.

CpG Island Methylator Phenotype Positive Tumors in the Absence of MLH1 Methylation Constitute a Distinct Subset of Duodenal Adenocarcinomas and Are Associated with Poor Prognosis

Purpose: Little information is available on genetic and epigenetic changes in duodenal adenocarcinomas. The purpose was to identify possible subsets of duodenal adenocarcinomas based on microsatellite instability (MSI), DNA methylation, mutations in the KRAS and BRAF genes, clinicopathologic features, and prognosis. Experimental Design: Demographics, tumor characteristics, and survival were available for 99 duodenal adenocarcinoma patients. Testing for KRAS and BRAF mutations, MSI, MLH1 methylation, and CpG island methylator phenotype (CIMP) status was conducted. A Cox proportional hazard model was built to predict survival. Results: CIMP+ was detected in 27 of 99 (27.3%) duodenal adenocarcinomas and was associated with MSI (P = 0.011) and MLH1 methylation (P < 0.001), but not with KRAS mutations (P = 0.114), as compared with CIMP− tumors. No BRAF V600E mutation was detected. Among the CIMP+ tumors, 15 (55.6%) were CIMP+/MLH1-unmethylated (MLH1-U). Kaplan–Meier analysis showed that tumors classified by CIMP, CIMP/MLH1 methylation status, or CIMP/MSI status could predict overall survival (OS; P = 0.047, 0.002, and 0.002, respectively), whereas CIMP/MLH1 methylation status could also predict time-to-recurrence (TTR; P = 0.016). In multivariate analysis, CIMP/MLH1 methylation status showed a significant prognostic value in both OS (P < 0.001) and TTR (P = 0.023). Patients with CIMP+/MLH1-U tumors had the worst OS and TTR. Conclusions: Our results showed existence of CIMP in duodenal adenocarcinomas. The combination of CIMP+/MLH1-U seems to be independently associated with poor prognosis in patients with duodenal adenocarcinomas. This study also suggests that BRAF mutations are not involved in duodenal tumorigenesis, MSI, or CIMP development. Clin Cancer Res; 18(17); 4743–52. ©2012 AACR.

Epithelioid sarcoma: one institution’s experience with a rare sarcoma

BACKGROUND Epithelioid sarcomas (ES) are extremely rare soft tissue sarcomas. As such, their clinical behavior and response to treatment are poorly described in the literature. METHODS We queried the centralized cancer registry and pathology archives at the Johns Hopkins Medical Institution and identified 22 patients with a diagnosis of ES. We excluded two patients because of inadequate data. A pathologist reviewed patient charts and reexamined available histological slides. This study was performed with institutional review board approval. RESULTS The median age at diagnosis was 27.8 y; most patients (75%) were male. Regional lymph node metastases were present in 10% of patients at presentation. The majority of tumors (57.9%) recurred and 35% recurred more than once, although the number of recurrences did not affect survival (P = 0.48). Patients did not experience a decrease in time to recurrence with increasing number of resections. The median time between resection and recurrence was 1.23 y and the maximum was 18.8 y. Median overall survival was 56.2 mo and 5-y survival was 92%. CONCLUSIONS Our study reveals that ES is an extremely rare tumor with a protracted and recurrent course, but overall survival may be more favorable than in the past. Patients benefit from aggressive and repeated resection. Epithelioid sarcoma is unique because it metastasizes to regional nodal basins. Extended surveillance is indicated, because recurrences can appear after decades of quiescence.

Inhibiting DNA methylation activates cancer testis antigens and expression of the antigen processing and presentation machinery in colon and ovarian cancer cells

Innovative therapies for solid tumors are urgently needed. Recently, therapies that harness the host immune system to fight cancer cells have successfully treated a subset of patients with solid tumors. These responses have been strong and durable but observed in subsets of patients. Work from our group and others has shown that epigenetic therapy, specifically inhibiting the silencing DNA methylation mark, activates immune signaling in tumor cells and can sensitize to immune therapy in murine models. Here we show that colon and ovarian cancer cell lines exhibit lower expression of transcripts involved in antigen processing and presentation to immune cells compared to normal tissues. In addition, treatment with clinically relevant low doses of DNMT inhibitors (that remove DNA methylation) increases expression of both antigen processing and presentation and Cancer Testis Antigens in these cell lines. We confirm that treatment with DNMT inhibitors upregulates expression of the antigen processing and presentation molecules B2M, CALR, CD58, PSMB8, PSMB9 at the RNA and protein level in a wider range of colon and ovarian cancer cell lines and treatment time points than had been described previously. In addition, we show that DNMTi treatment upregulates many Cancer Testis Antigens common to both colon and ovarian cancer. This increase of both antigens and antigen presentation by epigenetic therapy may be one mechanism to sensitize patients to immune therapies.

Long-term outcomes in treatment of retroperitoneal sarcomas: A 15 year single-institution evaluation of prognostic features.

Retroperitoneal sarcomas are connective tissue tumors arising in the retroperitoneum. Surgical resection is the mainstay of treatment. Debate has arisen over extent of resection, changes in histological classification/grading, and interest in incorporating radiotherapy. Therefore, we reviewed our institutions experience to evaluate prognostic factors.

KRAS G>A Mutation Favors Poor Tumor Differentiation but May Not be Associated with Prognosis in Patients with Curatively Resected Duodenal Adenocarcinoma

KRAS mutations have been found in duodenal adenocarcinomas and may have prognostic significance. The purpose of this study was to classify clinicopathological characteristics, microsatellite instability and KRAS mutations and identify possible prognostic role of KRAS mutations in duodenal adenocarcinomas. Demographics, tumor characteristics and survival were recorded for 78 patients with duodenal adenocarcinomas (Stages I–III). KRAS mutations were detected in 27 (34.6%) cases, of which the majority (74.1%) were G>A transitions. Multivariate logistic regression analysis showed that KRAS G>A mutation was significantly associated with late stage (p = 0.025) and poor tumor differentiation (p = 0.035), when compared with wild‐type and other than G>A mutations. KRAS G>A mutation carriers were at increased risk for distant relapse (p = 0.022) and had significantly shorter overall survival (OS; log‐rank p = 0.045) and a trend toward shorter relapse‐free survival (RFS; log‐rank p = 0.062) when compared with those who did not carry the KRAS G>A mutation. In multivariate analyses, there was a significant correlation between ≥ 3 positive lymph nodes and poor OS (p < 0.001) and RFS (p = 0.001) and KRAS G>A mutation carriers demonstrated no effect on clinical outcome. In conclusion, KRAS G>A mutation correlates significantly with late stage and poor tumor differentiation in duodenal adenocarcinoma. Among patients who undergo a curative resection of duodenal adenocarcinoma, KRAS G>A mutation carriers will more likely experience distant relapse but may not exhibit a poor prognosis. The number of positive lymph nodes should be incorporated in future staging systems.

Long-term outcomes in treatment of retroperitoneal sarcomas

Retroperitoneal sarcomas are connective tissue tumors arising in the retroperitoneum. Surgical resection is the mainstay of treatment. Debate has arisen over extent of resection, changes in histological classification/grading, and interest in incorporating radiotherapy. Therefore, we reviewed our institutions experience to evaluate prognostic factors.

IGFBP-3 Gene Methylation in Primary Tumor Predicts Recurrence of Stage II Colorectal Cancers

Objectives:To evaluate the influence of IGFBP-3 methylation on recurrence in patients with stage II colorectal cancer (CRC) from 2 independent cohorts. Background:The relationship between IGFBP-3 methylation in primary tumors (PTs) or lymph nodes (LNs) and risk of recurrence in patients with stage II CRC treated with surgery alone is unknown. Methods:IGFBP-3 methylation of DNA from 115 PTs and 1641 LNs in patients with stage II CRC from 2 independent cohorts was analyzed. Forty patients developed recurrence, whereas 75 matched patients remained recurrence free for more than 2 years after surgery. Cox proportional hazard models were used to calculate hazard ratios (HRs) of recurrence, adjusted for patient and tumor characteristics. Results:Methylation of IGFBP-3 in PTs was identified to be significantly associated with risk of recurrence in the training set. The signature was tested in a validation set and classified 40.7% of patients as high risk. Five-year recurrence-free survival rates were 76.4% and 58.3% for low- and high-risk patients, respectively, with an HR of 2.21 (95% confidence interval, 1.04–4.68; P = 0.039). In multivariate analysis, the signature remained the most significant prognostic factor, with an HR of 2.40 (95% confidence interval, 1.10–5.25; P = 0.029). A combined analysis of 1641 LNs from the 2 sets identified IGFBP-3 methylation in LNs was not associated with risk of recurrence. Conclusions:Detection of IGFBP-3 methylation in PTs, but not in LNs, provides a powerful tool for the identification of patients with stage II CRC at high risk of recurrence.

Hypomethylating agents synergize with irinotecan to improve response to chemotherapy in colorectal cancer cells

Colorectal cancer (CRC) is the second leading cause of cancer death in the United States. In the metastatic setting, the majority of patients respond to initial therapies but eventually develop resistance and progress. In this study, we test the hypothesis that priming with epigenetic therapy sensitizes CRC cell lines, which were previously resistant to subsequent chemotherapeutic agents. When multiple CRC cell lines are first exposed to 500 nM of the DNA demethylating agent, 5-aza-cytidine (AZA) in-vitro, and the cells then established as in-vivo xenografts in untreated NOD-SCID mice; there is an enhanced response to cytotoxic chemotherapy with agents commonly used in CRC treatment. For irinotecan (IRI), growth diminished by 16–62 fold as assessed, by both proliferation (IC50) and anchorage independent cell growth soft agar assays. Treatment of resistant HCT116 cell line along with in-vivo, for CRC line xenografts, AZA plus IRI again exhibits this synergistic response with significant improvement in survival and tumor regression in the mice. Genome-wide expression correlates changes in pathways for cell adhesion and DNA repair with the above responses. A Phase 1/2 clinical trial testing this concept is already underway testing the clinical efficacy of this concept in IRI resistant, metastatic CRC (NCT01896856).