Myung-Shin Sim

Downregulation of microRNA-29c is associated with hypermethylation of tumor-related genes and disease outcome in cutaneous melanoma

Hypermethylation of the promoter region of tumor-related genes (TRGs) has been shown to silence gene expression during melanoma progression, whereas microRNA-29(miR-29) has been found to downregulate DNA methyltransferases DNMT3A and DNMT3B which were shown as essential to the methylation of TRGs. We hypothesized that the expression level of miR-29 is associated to TRG methylation status and may have prognostic utility in melanoma. AJCC stage I-IV cutaneous melanoma paraffin-embedded archival tissue (PEAT) specimens (n=149) were assessed. Expression of miR-29 isoforms a, b, and c were analyzed by reverse-transcription quantitative real-time polymerase chain reaction(RT-qPCR). Expression of DNMT3A and DNMT3B was assessed by immunohistochemistry(IHC) on defined clinically annotated tissue microarrays (TMA) of AJCC stage III melanoma lymph node metastases. Promoter region CpG island methylation status of RASSF1A, TFPI-2, RAR-β, SOCS, GATA4 and genomic repeat sequence MINT17 and MINT31 were previously evaluated in melanoma tissues. Only miR-29c isoform expression was correlated to advancing AJCC stages in melanoma. miR-29c expression was significantly downregulated in AJCC stage IV melanoma tumors compared to primary melanomas. Hypermethylation status of TRGs and non-coding MINT loci in different stages of melanoma showed an inverse association with miR-29c expression. Overall, an increase in miR-29c expression inversely correlated to both DNMT3A and DNMT3B protein expression in melanomas. Expression of DNMT3B and miR-29c were significantly (p=0.004 and p=0.002, respectively) associated with overall survival(OS) in AJCC stage III melanoma patients by multivariate analysis. The studies demonstrated that both miR-29c and DNMT3B have significant roles in melanoma progression, and may be useful epigenetic biomarkers for disease outcome.

CpG Island Methylator Phenotype Predicts Progression of Malignant Melanoma

Purpose: The CpG island methylator phenotype (CIMP) may be associated with development of malignancy through coordinated inactivation of tumor suppressor and tumor-related genes (TRG) and methylation of multiple noncoding, methylated-in-tumor (MINT) loci. These epigenetic changes create a distinct CIMP pattern that has been linked to recurrence and survival in gastrointestinal cancers. Because epigenetic inactivation of TRGs also has been shown in malignant melanoma, we hypothesized the existence of a clinically significant CIMP in cutaneous melanoma progression. Experimental Design: The methylation status of the CpG island promoter region of TRGs related to melanoma pathophysiology (WIF1, TFPI2, RASSF1A, RARβ2, SOCS1, and GATA4) and a panel of MINT loci (MINT1, MINT2, MINT3, MINT12, MINT17, MINT25, and MINT31) in primary and metastatic tumors of different clinical stages (n = 122) was assessed. Results: Here, we show an increase in hypermethylation of the TRGs WIF1, TFPI2, RASSF1A, and SOCS1 with advancing clinical tumor stage. Furthermore, we find a significant positive association between the methylation status of MINT17, MINT31, and TRGs. The methylation status of MINT31 is associated with disease outcome in stage III melanoma. Conclusions: These findings show the significance of a CIMP pattern that is associated with advancing clinical stage of malignant melanoma. Future prospective large-scale studies may determine if CIMP-positive primary melanomas are at high risk of metastasis or recurrence.

A Collagen-Remodeling Gene Signature Regulated by TGF-β Signaling Is Associated with Metastasis and Poor Survival in Serous Ovarian Cancer

Purpose: To elucidate molecular pathways contributing to metastatic cancer progression and poor clinical outcome in serous ovarian cancer. Experimental Design: Poor survival signatures from three different serous ovarian cancer datasets were compared and a common set of genes was identified. The predictive value of this gene signature was validated in independent datasets. The expression of the signature genes was evaluated in primary, metastatic, and/or recurrent cancers using quantitative PCR and in situ hybridization. Alterations in gene expression by TGF-β1 and functional consequences of loss of COL11A1 were evaluated using pharmacologic and knockdown approaches, respectively. Results: We identified and validated a 10-gene signature (AEBP1, COL11A1, COL5A1, COL6A2, LOX, POSTN, SNAI2, THBS2, TIMP3, and VCAN) that is associated with poor overall survival (OS) in patients with high-grade serous ovarian cancer. The signature genes encode extracellular matrix proteins involved in collagen remodeling. Expression of the signature genes is regulated by TGF-β1 signaling and is enriched in metastases in comparison with primary ovarian tumors. We demonstrate that levels of COL11A1, one of the signature genes, continuously increase during ovarian cancer disease progression, with the highest expression in recurrent metastases. Knockdown of COL11A1 decreases in vitro cell migration, invasion, and tumor progression in mice. Conclusion: Our findings suggest that collagen-remodeling genes regulated by TGF-β1 signaling promote metastasis and contribute to poor OS in patients with serous ovarian cancer. Our 10-gene signature has both predictive value and biologic relevance and thus may be useful as a therapeutic target. Clin Cancer Res; 20(3); 711–23. ©2013 AACR.

Estrogen receptor and HER2/neu status affect epigenetic differences of tumor-related genes in primary breast tumors

IntroductionEstrogen receptor (ER)-positive breast cancers are considered prognostically more favorable than ER-negative tumors, whereas human epidermal growth factor receptor (HER)2/neu-positive breast cancers are associated with worse prognosis. The objective of the present study was to determine whether ER-positive and ER-negative status relates to epigenetic changes in breast cancer-related genes. To evaluate epigenetic differences in tumor-related genes relating to ER and HER2/neu status of primary tumors, we examined the promoter methylation status of the promoter region CpG islands of eight major breast tumor-related genes (RASSF1A, CCND2, GSPT1, TWIST, APC, NES1, RARβ2, and CDH1).MethodsPaired ER-positive (n = 65) and ER-negative (n = 65) primary breast tumors (n = 130) matched for prognostic factors were assessed. DNA was extracted from paraffin-embedded tumor tissue after microdissection, and methylation-specific PCR and capillary-array electrophoresis analysis were performed.ResultsIn early stages of tumor progression (T1 and N0), RASSF1A and CCND2 were significantly (P < 0.05) more methylated in ER-positive than in ER-negative tumors. GSTP1 hypermethylation was more frequent in the lymph node metastasis positive group than in the negative group. Double negative (ER-negative, HER2/neu-negative) breast cancers had significantly lesser frequencies of RASSF1A, GSTP1, and APC methylation (P < 0.0001, P < 0.0001, and P = 0.0035, respectively). Both ER and HER2/neu status correlated independently with these epigenetic alterations.ConclusionWe demonstrated significant differences in tumor-related gene methylation patterns relevant to ER and HER2/neu status of breast tumors. This may be of significance in the assessment of targeted therapy resistance related to ER and HER2/neu status in breast cancer patients.

Comparison of Patient Characteristics and Outcomes of Contralateral Prophylactic Mastectomy and Unilateral Total Mastectomy in Breast Cancer Patients

BackgroundThere has been an increasing trend toward contralateral prophylactic mastectomy (CPM) in the management of breast cancer (BCa). This study’s objective was to compare clinicopathologic characteristics of BCa patients who elected CPM to those who elected unilateral total mastectomy (UTM) and to determine whether CPM improved survival.MethodsComparison was performed on 355 patients with stage 0–III BCa matched by age and stage who underwent mastectomy from 1995 to 2008: 177 patients had CPM; 178 patients had UTM. Clinicopathological characteristics and survival outcomes were analyzed.ResultsWomen who underwent preoperative MRI were twice as likely to have CPM (40.9 vs. 19.7%, Pxa0<xa00.001). MRI identified additional suspicious foci in 45% CPM and 19% UTM. Patients with history of previous breast biopsies, family history, or BRCA mutation were more likely to choose CPM than UTM (40.1 vs. 24%, Pxa0=xa00.001; 64.3 vs. 41.4%, Pxa0<xa00.001; 20.3 vs. 6.5%, Pxa0=xa00.04, respectively). CPM patients elected nipple preservation (26 vs. 5.2%, Pxa0<xa00.001) and immediate reconstruction more often (92.2 vs. 73.5%, Pxa0<xa00.001); UTM patients were more likely to have attempted breast conservation prior to mastectomy (52.8 vs. 39.5%, Pxa0=xa00.01). CPM identified occult BCa in 11 patients (6.6%), and three UTM patients (1.7%) developed contralateral BCa. With median follow-up of 61xa0months, by univariable/multivariable analyses, CPM did not improve overall, disease-free, or distant metastases-free survival.ConclusionFactors that may influence choice of CPM included preoperative MRI, history of prior breast biopsies, immediate reconstruction, nipple preservation, family history, and BRCA status. Those who chose CPM did not have improved survival.

Age-Related Lymphatic Dysfunction in Melanoma Patients

BackgroundAge-related outcomes have become increasingly common in evaluating patients with melanoma. For instance, as age increases, sentinel node (SN) nonidentification increases and SN positivity decreases. Furthermore, advanced age is a risk factor for in-transit disease. We hypothesized that increasing age is accompanied by alterations in lymphatic function, possibly explaining these findings.MethodsOur center’s melanoma database was queried to identify patients who underwent successful sentinel node biopsy after lymphoscintigraphy. Records of those treated between 2000 and 2005 were reviewed for age, sex, drainage basin, intraoperative radioactivity, and SN pathology.ResultsThe 858 patients had a mean age of 55xa0years; 59% were men. Mean radioactivity in the hottest SN was 5232 counts per second; 179 patients (21%) had SN metastases. SN count rates were significantly and inversely related to age (P < .001 by Pearson correlation, analysis of variance, and χ2 test). Mean counts per second were 6105, 5883, and 2720 for axillary, inguinal, and cervical basins, respectively (P < .01), and count rates in these basins were consistently lower with increasing age (neck and axilla, P < .001; groin, Pxa0=xa0.060; Pearson correlation). Multivariate analysis confirmed an independent inverse association between age and count rates (P < .001), overall and within each primary tumor site.ConclusionsLymphatic function, as assessed by radiocolloid transit to and uptake within the SN, declines with age. Altered lymphatic function in older patients may modify metastatic patterns; knowledge of this may help clarify findings of reduced nodal positivity and increased in-transit disease in this population.

AIM1 and LINE-1 Epigenetic Aberrations in Tumor and Serum Relate to Melanoma Progression and Disease Outcome

Aberrations in the methylation status of non-coding genomic repeat DNA sequences and specific gene promoter region are important epigenetic events in melanoma progression. Promoter methylation status in LINE-1 and Absent in melanoma-1(AIM1;6q21) associated with melanoma progression and disease outcome was assessed. LINE-1 and AIM1 methylation status was assessed in paraffin-embedded archival tissues(PEAT)(n=133) and melanoma patients’ serum(n=56). LINE-1 U-Index(hypomethylation) and AIM1 were analyzed in microdissected melanoma PEAT sections. The LINE-1 U-Index of melanoma(n=100) was significantly higher than that of normal skin(n=14) and nevi(n=12)(P=0.0004). LINE-1 U-Index level was elevated with increasing AJCC stage(P<0.0001). AIM1 promoter hypermethylation was found in higher frequency(P=0.005) in metastatic melanoma(65%) than in primary melanomas(38%). When analyzed, high LINE-1 U-Index and/or AIM1 methylation in melanomas were associated with disease-free survival(DFS) and overall survival(OS) in Stage I/II patients (P=0.017, 0.027; respectively). In multivariate analysis, melanoma AIM1 methylation status was a significant prognostic factor of OS(P=0.032). Furthermore, serum unmethylated LINE-1 was at higher levels in both stage III(n=20) and stage IV(n=36) patients compared to healthy donors(n=14)(P=0.022). Circulating methylated AIM1 was detected in patients’ serum and was predictive of OS in Stage IV patients (P=0.009). LINE-1 hypomethylation and AIM1 hypermethylation have prognostic utility in both melanoma patients’ tumors and serum.

Preoperative Breast MRI in the Surgical Treatment of Ductal Carcinoma In Situ

Abstract:u2002 Accurate determination of the size or extent of ductal carcinoma in situ (DCIS) by imaging is uncertain, and incomplete resection of tumor results in involved margins in up to 81% of cases. This study examined the accuracy of magnetic resonance imaging (MRI) for assessment of DCIS size, and evaluated the effect of preoperative breast MRI on achievement of tumor‐free surgical margins after breast‐conserving surgery (BCS). One‐hundred and fifty‐eight female patients with DCIS were identified from a prospective database: 60 patients (62 cases) had preoperative breast MRI, and 98 patients did not have MRI. The accuracy of tumor size assessed by MRI was determined by comparison with histopathologic size. All patients underwent BCS initially. The rate of involved margins after resection was compared in MRI and no‐MRI groups. The overall correlation between MRI size and histopathologic size was high (pu2003<u20030.0001). MRI assessment of size was significantly more accurate when DCIS was high grade (pu2003<u20030.0001) or intermediate grade (pu2003=u20030.005) versus low grade (pu2003=u20030.187). The rate of tumor‐involved margins was not significantly different in MRI and no‐MRI groups (30.7% and 24.7%, respectively; pu2003=u20030.414). The rate of mastectomy was significantly higher in the MRI group than the no‐MRI group (17.7% versus 4.1%; pu2003=u20030.004). These findings indicate that MRI can detect DCIS, especially when lesions are high or intermediate grade, but that MRI does not accurately predict the size of DCIS. In this study, MRI did not improve the surgeon’s ability to achieve clear margins following BCS.

Small Bowel Adenocarcinoma: Understaged and Undertreated?

BackgroundPrimary small bowel adenocarcinoma (SBA) is a rare, chemoresistant tumor with an aggressive clinical nature. Surgery is the mainstay of therapy, but the extent of lymph node (LN) recovery necessary for optimal care of jejunoileal SBA is unknown.Materials and MethodsThe SEER database was queried to identify patients whose primary jejunoileal SBA was diagnosed between 1995 and 2005. Patients were grouped by AJCC stage and number of LNs recovered from the surgical specimen.ResultsOf 1444 patients with primary SBA, 93 (6.4%), 529 (36.6%), 356 (24.7%), and 466 (32.3%) were initially diagnosed with stage I, II, III, and IV disease, respectively. Five-year overall survival (OS) rate was 59.8%, 39.5%, 27.0%, and 3.2% for patients with stage I, II, III, and IV SBA, respectively. Whenxa0≥10 nodes were recovered, OS rate increased nonsignificantly in stage I (73.2% vs. 55.6%) and significantly in stage II (61.8% vs. 32.9%, Pxa0<xa0.001) but was unchanged in stage III (27.4% vs. 27.3%, Pxa0=xa0.13). Recovery ofxa0≥10 nodes occurred in 26.9%, 23.6%, and 42.1% of patients with stage I, II, and III SBA, respectively. Multivariate analysis identified age, AJCC stage, site of primary tumor, recovery ofxa0≥10 LNs, and number of positive nodes as significant for OS.ConclusionsWe have found SBA staging is largely inadequate. Our results suggest recovery ofxa0≥10 LNs ensures accurate staging. Improvement in stage II SBA OS after adequate LN may reflect a high degree of understaging in this dataset rather than a therapeutic effect of LAD.

Redirected lysis of human melanoma cells by a MCSP/CD3-bispecific BiTE antibody that engages patient-derived T cells.

Melanoma-associated chondroitin sulfate proteoglycan (MCSP; also called HMW-MAA, CSPG4, NG2, MSK16, MCSPG, MEL-CSPG, or gp240) is a well characterized melanoma cell-surface antigen. In this study, a new bispecific T-cell engaging (BiTE) antibody that binds to MCSP and human CD3 (MCSP-BiTE) was tested for its cytotoxic activity against human melanoma cell lines. When unstimulated peripheral mononuclear blood cells (PBMCs) derived from healthy donors were cocultured with melanoma cells at effector:target ratios of 1:1, 1:5, or 1:10, and treated with MCSP-BiTE antibody at doses of 10, 100, or 1000 ng/mL, all MCSP-expressing melanoma cell lines (n=23) were lysed in a dose-dependent and effector:target ratio-dependent manner, whereas there was no cytotoxic activity against MCSP-negative melanoma cell lines (n=2). To investigate whether T cells from melanoma patients could act as effector cells, we cocultured unstimulated PBMCs with allogeneic melanoma cells from 13 patients (4 stage I/II, 3 stage III, and 6 stage IV) or with autologous melanoma cells from 2 patients (stage IV). Although cytotoxic activity varied, all 15 PBMC samples mediated significant redirected lysis by the BiTE antibody. When PBMC or CD8+ T cells were prestimulated by anti-CD3 antibody OKT-3 and interleukin-2, the MCSP-BiTE concentrations needed for melanoma cell lysis decreased up to 1000-fold. As MCSP is expressed on most human melanomas, immunotherapy with MCSP/CD3-bispecific antibodies merits clinical investigation.