Methylated Tumors Research Articles

Abstract 1192: Triple negative breast cancer: BRCAness and concordance of clinical features and treatment response with BRCA1 mutation carriers.

Abstract Introduction Previous work from our group and other groups has shown that triple negative breast cancers (TNBC) frequently show BRCAness, the phenotype that some sporadic tumors share with BRCA-mutated cancers. We propose the use of a BRCA1-like genomic profile and of BRCA1 promoter methylation as indicators of BRCAness. In a randomized trial, tumors with this BRCA1-like profile responded more favorably to intensified alkylating chemotherapy. We now assessed the frequency of BRCAness in a large cohort of TNBCs. In addition, we determined the association with clinicopathological variables and treatment response. Methods A total of 388 TNBCs were included in this study. These were obtained from three different sources: a neoadjuvant chemotherapy trial (n=152), a series of patients from breast cancer families but without germline mutations (n=145), and a series of patients treated with adjuvant chemotherapy (n=91). Two assays for BRCAness were employed: array Comparative Genomic Hybridization (aCGH) to asses a BRCA1-like profile and methylation specific Multiplex Ligation-dependent Probe Amplification (MLPA) to determine BRCA1 promoter methylation. BRCA1 germline mutation status was known for half of the patients. Clinicopathological characteristics were available for most patients, and chemotherapy response data was available for the neoadjuvant series. Results In the three series, 66% to 69% of the tumors had an aCGH BRCA1-like profile and 27% to 37% showed BRCA1 promoter methylation. 87% of the tumors with promoter methylation showed a BRCA1-like aCGH profile. A germline mutation in BRCA1 and somatic BRCA1 promoter methylation did not occut together (n=155; p = 10-6). Patients with aCGH BRCA1-like or BRCA1 methylated tumors were younger than patients with non-BRCA1-like or unmethylated tumors (p=0.02 and p<0.01, respectively). BRCA1 mutated tumors and aCGH BRCA1-like tumors were more often of histological grade 3 (97% vs. 74% (p<0.01) for BRCA1-mutated vs. BRCA1 wildtype and 86% vs 67% (p<0.01) for aCGH BRCA1-like vs. non-BRCA1-like tumors). BRCA1-mutated tumors had a higher response rate to neoadjuvant chemotherapy than BRCA1 wildtype tumors (71% vs. 37% (p=0.04) pathological complete remission rate). For aCGH BRCA1-like and BRCA1 methylated tumors, a non-significant trend for a better chemtherapy response was observed. Conclusion The majority of the TNBCs in this study show signs of BRCAness, and these tumors share clinicopathological characteristics with BRCA1 mutated tumors. Somatic BRCA1 promoter methylation and BRCA1 germline mutations are mutually exclusive events. A better characterization of TNBC and the presence of BRCAness will not only have consequences for treatment, but also for hereditary breast cancer screening. Citation Format: Esther H. Lips, Lennart Mulder, Lizet E. van der Kolk, Anne M.M. Oonk, Alex L.T. Imholz, Frans B.L. Hogervorst, Jelle Wesseling, Sjoerd Rodenhuis, Petra M. Nederlof. Triple negative breast cancer: BRCAness and concordance of clinical features and treatment response with BRCA1 mutation carriers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1192. doi:10.1158/1538-7445.AM2013-1192

Abstract 666: Validation of nucleolar protein 4 as a novel methylated tumor suppressor gene in head and neck cancer.

Abstract Purpose: Methylation of CpG islands in the promoter region of genes acts as a significant mechanism of epigenetic gene silencing in head and neck cancer. In this study, we assessed the utility of epigenetic alterations of a panel of twelve genes (NOL4, IRX1, SLC5A8, LRRC3B, FUSSEL18, EBF3, GBX2, HMX2, SEPT9, ALX3, SOCS3, LHX6) previously reported. Experimental Design: In this study we investigated methylation of the 12 gene promoters via a candidate gene approach. After the initial screening of methylated CpG islands on the promoter regions by bisulfite sequencing using salivary rinse samples, only two genes had methylated CpG dinucleotides on their promoter regions in tumor samples and absence of methylated CpGs were found in normal salivary rinse samples after bisulfite modification and bisulfite sequencing. We then performed quantitative methylation specific PCR (Q-MSP) on 16 salivary rinse and 14 normal mucosal samples from healthy people and 33 HNSCC tumor samples for two genes selected. Results: After validation with QMSP, one gene, nucleolar protein 4 (NOL4), was highly methylated (91%) in tumor samples and unmethylated in normal salivary rinse samples and minimally methylated in normal mucosal samples demonstrating cancer specific methylation in HNSCC tissues. Although iroquois homeobox 1 (IRX1) gene was observed as methylated in normal mucosal and salivary rinse samples, indeed, the methylation values of these normal samples were very low (less than 10%). Conclusions: We identified NOL4, as a highly specific promoter methylated gene was associated with HNSCC. IRX1 which may have a potential as a biomarker for HNSCC and should also be assessed in a larger cohort. Citation Format: Semra Demokan, Alice Y. Chuang, Kavita M. Pattani, David Sidransky, Wayne Koch, Joseph A. Califano. Validation of nucleolar protein 4 as a novel methylated tumor suppressor gene in head and neck cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 666. doi:10.1158/1538-7445.AM2013-666

Treatment in Elderly Glioblastoma Patients: General Reduction of Standard Therapy Should Be Avoided. Prognostic Value of MGMT Is Questionable

Purpose: Aim of this single center study was to determine whether elderly patients benefit from individualized treatment not excluding full standard therapy. Additionally predictive and prognostic factors influencing outcome in this patient’s population were evaluated. Material and Methods: Between 1997 and 2010, 119 patients equal or older than 60 years were enrolled in this retrospective review. All patients had neuropathology confirmed diagnosis of glioblastoma. Treatment outcome concerning progression free survival was measured by MRI. For evaluation of O6-Methylguanin-DNA-methyltransferase (MGMT) Methylation-specific PCR was used. The log rank test and the Cox proportional hazards model were used to analyze the data. Factors considered in univariate and multivariate analyses included age, gender, Karnofsky performance scale (KPS), extent of resection, treatment with radioand chemotherapy and MGMT status. Survival probabilities were estimated by means of the Kaplan Meier method. Results: Multivariate analysis demonstrated age, KPS and treatment more than surgery as prognostic factors for survival and sex, KPS, type of surgery and standard treatment as independent factors for PFS. MGMT status revealed no difference in survival between the methylated and unmethylated tumours in the whole study population (12.7 and 12.0, respectively). Surgery had an impact on survival with a significant advantage for complete resection. Conclusion: Extent of resection is essential even in elderly patients. Full standard treatment should be offered to elderly GBM patients with good clinical performance, there is no reason to withhold radioor chemotherapy from these patients. MGMT promotor methylation of the tumour is not relevant for treatment decision.

5-Aza-2’-deoxycytidine may influence the proliferation and apoptosis of cervical cancer cells via demethylation in a dose- and time-dependent manner

The methylation of tumor suppressor genes has been shown to be involved in many human cancers. 5-Aza-2'-deoxycytidine (5-Aza-CdR) can reactivate the expression of methylated tumor suppressor genes. In our study, 2 human cervical cancer cell lines, HeLa and SiHa, were treated with different concentrations (20, 10, 5, and 2.5 μM) of 5-Aza-CdR for 24, 48, and 72 h. After incubation, cells were analyzed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay and flow cytometry. The expression of RASSF1A and APAF-1 was detected by RT-PCR. 5-Aza-CdR inhibited the growth of HeLa and SiHa cells at different concentrations. The strongest inhibition and apoptosis rates were obtained after incubation for 72 h (5.63 ± 1.38 and 8.24 ± 2.40%, respectively). No significant difference in the expression of RASSF1A was found upon drug treatment, while APAF-1 expression increased in HeLa cells after treatment (0.790 ± 0.056%). Our results suggest that the tumor-suppressive effect of 5-Aza-CdR may result from the reactivation of silenced APAF-1 through demethylation.

MGMT promoter methylation status and MGMT and CD133 immunohistochemical expression as prognostic markers in glioblastoma patients treated with temozolomide plus radiotherapy.

BackgroundThe CD133 antigen is a marker of radio- and chemo-resistant stem cell populations in glioblastoma (GBM). The O6-methylguanine DNA methyltransferase (MGMT) enzyme is related with temozolomide (TMZ) resistance. Our propose is to analyze the prognostic significance of the CD133 antigen and promoter methylation and protein expression of MGMT in a homogenous group of GBM patients uniformly treated with radiotherapy and TMZ. The possible connection between these GBM markers was also investigated.MethodsSeventy-eight patients with GBM treated with radiotherapy combined with concomitant and adjuvant TMZ were analyzed for MGMT and CD133. MGMT gene promoter methylation was determined by methylation-specific polymerase chain reaction after bisulfite treatment. MGMT and CD133 expression was assessed immunohistochemically using an automatic quantification system. Overall and progression-free survival was calculated according to the Kaplan–Meier method.ResultsThe MGMT gene promoter was found to be methylated in 34 patients (44.7%) and unmethylated in 42 patients (55.3%). A significant correlation was observed between MGMT promoter methylation and patients’ survival. Among the unmethylated tumors, 52.4% showed low expression of MGMT and 47.6% showed high-expression. Among methylated tumors, 58.8% showed low-expression of MGMT and 41.2% showed high-expression. No correlation was found between MGMT promoter methylation and MGMT expression, or MGMT expression and survival. In contrast with recent results, CD133 expression was not a predictive marker in GBM patients. Analyses of possible correlation between CD133 expression and MGMT protein expression or MGMT promoter methylation were negative.ConclusionsOur results support the hypothesis that MGMT promoter methylation status but not MGMT expression may be a predictive biomarker in the treatment of patients with GBM. In addition, CD133 should not be used for prognostic evaluation of these patients. Future studies will be necessary to determine its clinical utility.

Methylation of the WNT5A gene is frequently detected in early gastric carcinoma.

Recently, it has been reported that WNT5A methylation was frequently detected in colorectal cancers. However, the relationship between the WNT5A methylation and the characteristics of gastric cancer remains unknown. Methylation status of the WNT5A gene was examined in primary carcinomas and the corresponding normal tissues derived from 38 patients with gastric cancer using quantitative methylation-specific PCR (qMSP) and the correlation between the methylation status and the clinicopathological findings was evaluated. Aberrant methylation of the WNT5A gene was detected in 7 out of the 38 (18%) primary gastric carcinomas, suggesting that the methylation of WNT5A is observed in gastric carcinomas as well as colorectal ones. The clinicopathological data were correlated with the methylation results. A significant difference was observed in the extent of tumor (p=0.0226). Moreover, a trend was shown towards early TNM stages in methylated tumors (p=0.209). WNT5A was more frequently methylated in early gastric carcinomas.

Methylation of MGMT in paired primary and recurrent GBMs.

65 Background: Epigenetic sliencing of the MGMT gene promoter in primary glioblastomas of patients subsequently treated with TMZ is associated with prolonged survival. Further, several studies have observed a change in MGMT silencing in paired primary and recurrent glioblastoma. However, the relationship between this “MGMT switch” and patients outcome is largely unknown. Methods: The study involved primary and recurrent tumor tissue samples from 53 glioblastoma patients diagnosed and treated within the First Hospital of Jilin University from January 2003 to November 2010. After surgical treatment, all patients were subjected to radiotherapy with concomitant administration of TMZ. Patients that experienced recurrent tumors received TMZ. 53 patients underwent 58 further operations after recurrence (5 pats received a third surgery). MGMT promoter methylation levels were determined using qMSP. The relationship between “MGMT switch” and clinical outcome was investigated. Results: 53 (M/F=33/20; median age: 49.2±3.6, 19.5-72.3 ys) underwent a first operation for GBM. qMSP analysis revealed MGMT promoter methylation in 19 pats (35.8%, A, OS 31.5 ms); no methylation in 34 pats (64.2%, B, OS 7.9 ms). In the recurrent tumors, MGMT promoter methylation was detected in 25 pats (47.2%); and no methylation in 28 pats (52.8%). Comparison of individual pairs of primary and recurrent GBMs revealed a changed methylation status in 10 (18.9%), including 8 changed from unmethylated to methylated tumors (15.1%, C, OS 29.4 ms), 2 changed from methylated to unmethylated tumors (3.8%, D, OS 10.5 ms). Median overall survival (OS) was 12.1 months. MGMT promoter methylation was significantly associated with a favorable clinical outcome (A vs B, p=0.0027). The outcome of patients were not significant different between group A and group C (p>0.01). Conclusions: The methylation status of the MGMT promoter was altered in 10 (18.9%) of 53 recurrent GBM after chemoradiotherapy. 8 patients the promoter changed from unmethylated to methylated and these patients had a median overall survival similar to the better prognosis of patients who had a methylated promoter in their primary tumor. 2 pats where a change from methylated to unmethylated was observed had a poorer outcome.

Probabilistic Radiographic Atlas of Glioblastoma Phenotypes

Tumor location is a significant prognostic factor in glioblastoma, which may reflect the genetic profile of tumor precursor cells. The purpose of the current study was to construct and analyze probabilistic radiographic atlases reflecting preoperative tumor locations and corresponding demographic, "-omic," and interventional phenotypes to provide insight into potential niche locations of glioblastoma cells of origin. Preoperative anatomic MR images in 507 patients with de novo glioblastoma were analyzed. Images were registered to stereotactic space, tumors were segmented, and the stereospecific frequency of tumor occurrence was analyzed statistically by age, extent of resection, MGMT methylation, IDH1 mutation, gene expression subclassification, PTEN loss, PTEN deficiency, EGFR amplification, EGFR variant 3 expression, progression-free survival from the start of radiochemotherapy, and overall survival from initial diagnosis. Most glioblastomas grow into the periventricular white matter regions adjacent to the subventricular zone. MGMT promoter methylated tumors occur more frequently in the left temporal lobe, in young patients with glioblastoma, in IDH1 mutant tumors, in tumors having the proneural gene expression subtype, and in tumors lacking loss of PTEN occurring most frequently in the frontal lobe. MGMT methylated tumors with the IDH1 mutation tended to occur in the left frontal lobe. EGFR amplified and EGFR variant 3-expressing tumors occurred most frequently in the left temporal lobe. A similar region in the left temporal lobe was associated with favorable response to radiochemotherapy and increased survival. Radiographic atlases for specific phenotypes provide insight into overlap between prognostic variables and may help to identify niche locations for cancer cells of origin.

Immune genes are associated with human glioblastoma pathology and patient survival

BackgroundGlioblastoma (GBM) is the most common and lethal primary brain tumor in adults. Several recent transcriptomic studies in GBM have identified different signatures involving immune genes associated with GBM pathology, overall survival (OS) or response to treatment.MethodsIn order to clarify the immune signatures found in GBM, we performed a co-expression network analysis that grouped 791 immune-associated genes (IA genes) in large clusters using a combined dataset of 161 GBM specimens from published databases. We next studied IA genes associated with patient survival using 3 different statistical methods. We then developed a 6-IA gene risk predictor which stratified patients into two groups with statistically significantly different survivals. We validated this risk predictor on two other Affymetrix data series, on a local Agilent data series, and using RT-Q-PCR on a local series of GBM patients treated by standard chemo-radiation therapy.ResultsThe co-expression network analysis of the immune genes disclosed 6 powerful modules identifying innate immune system and natural killer cells, myeloid cells and cytokine signatures. Two of these modules were significantly enriched in genes associated with OS. We also found 108 IA genes linked to the immune system significantly associated with OS in GBM patients. The 6-IA gene risk predictor successfully distinguished two groups of GBM patients with significantly different survival (OS low risk: 22.3 months versus high risk: 7.3 months; p < 0.001). Patients with significantly different OS could even be identified among those with known good prognosis (methylated MGMT promoter-bearing tumor) using Agilent (OS 25 versus 8.1 months; p < 0.01) and RT-PCR (OS 21.8 versus 13.9 months; p < 0.05) technologies. Interestingly, the 6-IA gene risk could also distinguish proneural GBM subtypes.ConclusionsThis study demonstrates the immune signatures found in previous GBM genomic analyses and suggests the involvement of immune cells in GBM biology. The robust 6-IA gene risk predictor should be helpful in establishing prognosis in GBM patients, in particular in those with a proneural GBM subtype, and even in the well-known good prognosis group of patients with methylated MGMT promoter-bearing tumors.

543P - P16(INK4A) Gene Hypermethylation and Kras Mutation are Independent Predictors of FolFiri and Cetuximab Chemotherapy in Patients with Metastatic Colorectal Cancer (MCRC)

ABSTRACT Background P16 (INK4a) inhibits cyclin dependent kinases (CDKs) and plays various roles in cancer and senescence. P16 aberrancy is frequently detected in various cancers and may contribute to multidrug resistance. Hypermethylation of CpG island of p16 occurs in a significant proportion in colorectal cancer (CRC) and also is one of the CpG island methylator phenotype (CIMP) markers. Patients and method We analyzed tumor samples from 49 patients with metastatic colorectal cancer (mCRC) who were treated with 5-fluorouracil, leucovorin, irinotecan (FOLFIRI) and cetuximab (1st line 22 pts, 2nd line 27 pts). Pyrosequencing was used to identify KRAS mutation and hypermethylation of 6 CpG island loci (p16, p14, MINT1, MINT2, MINT31, hMLH1) in DNA extracted from formalin-fixed paraffin-embedded specimens. To analyze the relation between methylation status of CIMP markers including p16 and clinical outcome, logistic regression and Cox regression were performed. Result Hypermethylation of p16 gene was detected in 14 of 49 patients (28.6%) and was significantly associated with KRAS mutation (Fisher's exact, P = 0.01) and CIMP+ (Fisher's exact, P = 0.002). Patients with p16 unmethylated tumors had significant longer time to progression (TTP, median 9.0 vs 3.5 months; log-rank, P = 0.001) and overall survival (OS, median 44.9 vs 16.4 months; log-rank, P = 0.008) than those with p16 methylated tumors. KRAS mutation was also associated with shortened TTP (median 8.9 vs 4.4 months; HR = 3.1, P = 0.008) and OS (median 44.9 vs 16.1 months; HR = 5.2, P Conclusion Promoter CpG island hypermethylation of p16 was predictive of clinical outcome in mCRC patients treated with cetuximab and FOLFIRI, irrespective of KRAS mutation. Disclosure All authors have declared no conflicts of interest.

The Retinoblastoma Gene Undergoes Rearrangements in BRCA1-Deficient Basal-like Breast Cancer

Breast tumors from BRCA1 germ line mutation carriers typically exhibit features of the basal-like molecular subtype. However, the specific genes recurrently mutated as a consequence of BRCA1 dysfunction have not been fully elucidated. In this study, we used gene expression profiling to molecularly subtype 577 breast tumors, including 73 breast tumors from BRCA1/2 mutation carriers. Focusing on the RB1 locus, we analyzed 33 BRCA1-mutated, 36 BRCA2-mutated, and 48 non-BRCA1/2-mutated breast tumors using a custom-designed high-density oligomicroarray covering the RB1 gene. We found a strong association between the basal-like subtype and BRCA1-mutated breast tumors and the luminal B subtype and BRCA2-mutated breast tumors. RB1 was identified as a major target for genomic disruption in tumors arising in BRCA1 mutation carriers and in sporadic tumors with BRCA1 promoter methylation but rarely in other breast cancers. Homozygous deletions, intragenic breaks, or microdeletions were found in 33% of BRCA1-mutant tumors, 36% of BRCA1 promoter-methylated basal-like tumors, 13% of non-BRCA1-deficient basal-like tumors, and 3% of BRCA2-mutated tumors. In conclusion, RB1 was frequently inactivated by gross gene disruption in BRCA1 hereditary breast cancer and BRCA1-methylated sporadic basal-like breast cancer but rarely in BRCA2 hereditary breast cancer and non-BRCA1-deficient sporadic breast cancers. Together, our findings show the existence of genetic heterogeneity within the basal-like breast cancer subtype that is based upon BRCA1 status.

Promoter methylation of MGMT in paired primary and recurrent glioblastomas.

10543 Background: Epigenetic sliencing of MGMT gene promoter in primary glioblastomas (GBM) of pts subsequently treated with temozolomide (TMZ) is associated with prolonged survival. Further, several studies have observed a change in MGMT silencing in paired primary and recurrent GBM. However, the relationship between this “MGMT switch” and patients outcome is largely unknown. Methods: The study involved primary and recurrent tumor tissue samples from 53 GBM pts diagnosed and treated within the First Hospital of Jilin University from Jan 2003 to Nov 2010. After surgical treatment, all pts were subjected to radiotherapy with concomitant administration of TMZ (75 mg/m2/d), followed by 5 cycles of adjuvant TMZ given at 4-weeks intervals. Pts that experienced recurrent tumors received TMZ at a dose of 200 mg/m2/5 days for 4 weeks. 53 pts underwent 58 further operations after recurrence (5 pts received a third surgery). MGMT promoter methylation levels were determined using qMSP. The relationship between “MGMT switch” and clinical outcome was investigated. Results: Fifty three (M/F=33/20; median age: 49.2±3.6, range: 19.5-72.3 ys) underwent a first operation for GBM. qMSP analysis revealed MGMT promoter methylation in 19 pts (35.8%, group A, OS 31.5 ms); no methylation in 34 pts (64.2%, group B, OS 7.9 ms). In the recurrent tumors, MGMT promoter methylation was detected in 25 pts (47.2%); and no methylation in 28 pts (52.8%). Comparison of individual pairs of primary and recurrent GBMs revealed a changed methylation status in 10 (18.9%), including eight changed from unmethylated to methylated tumors (15.1%, group C, OS 29.4 ms), two changed from methylated to unmethylated tumors (3.8%, group D, OS 10.5 ms). Median overall survival was 12.1 ms. MGMT promoter methylation was significantly associated with a favorable clinical outcome (group A vs group B, p=0.0027). The outcome of pts were not significant different between A and C (p&gt;0.01). Conclusions: The methylation status of the MGMT promoter was altered in 10 (18.9%) of 53 recurrent GBM after chemoradiotherapy. Eight pts changed from unmethylated to methylated who have a median overall survival similar to methylated pts. The pts changed from methylated to unmethylated who have poor outcome.

Abstract 4549: SNP array-based molecular analysis predicts response to neoadjuvant chemotherapy in triple negative and HER2-positive breast cancers

Abstract Patients with breast cancer (BC) who achieved pathological complete response (pCR) after neoadjuvant chemotherapy are expected to enjoy a long-term remission. However, the pCR rates ranged between 8% and 80% among the four types of BCs classified by immunohistochemistry, and it is important to predict the response to the chemotherapy to avoid unnecessary burden. We examined SNP array patterns in 144 untreated BC specimens; Luminal A (LA) 77 tumors, Luminal B (LB) 29, HER2 18, and triple-negative (TN) 20. The efficacy of the chemotherapy was pathologically assessed in 109 BCs after surgical resection. Anthracycline, cyclophosphamide, and taxane were given for LA and TN type patients and the 3 drugs plus trastuzumab and 5FU for LB and HER2 type patients. pCR was attained in 8%, 36%, 80%, and 27% of LA, LB, HER2, and TN type patients, respectively. Total numbers of genomic alteration (TGA) and uniparental disomy (UPD) per tumor were found in 15.2 and 2.3, 19.4 and 2.9, 20.2 and 3.3, and 30.5 and 7.6 in LA, LB, HER2, and TN type tumors, respectively. The frequency of TGA and UPD was higher in TNBCs than in non-TNBCs (P&amp;lt;0.01). Promoter methylation of BRCA1 examined with MethyLight was found in 45% of TNBCs and 7% of non-TNBCs (P&amp;lt;0.01). The Spearman correlation coefficient analysis showed that the methylation levels of the BRCA1 promoter correlated with the numbers of TGA and UPD in 134 BCs examined (0.325, P&amp;lt;0.01 and 0.362, P&amp;lt;0.01). Among TNBCs, pCR was more frequent in patients with the methylated tumor than in those with the unmethylated tumor (P=0.04). In contrast, no such association was found in LA or LB type patients. These findings suggest that a subgroup of TNBCs characterized by BRCA1 methylation and frequent genomic changes are likely to be induced into pCR, and that activation of the hormone receptor (HR) signaling pathway may confer the chemotherapy-resistance on HR-positive tumors with such molecular changes. Among 38 patients with HER2-positive BCs, 30 had HER2 amplification and 8 had a single copy of HER2. pCR was achieved in 63% of the 30 amplified tumors and only 13% of the 8 non-amplified tumors (P=0.01). The single copy number of HER2 was more frequent in LB type tumors than in HER2 type tumors (P=0.01). A HER2 splicing variant (HER2 del16) was reported to be a poor prognostic factor, and we quantified the levels of normal and del16 HER2 transcripts to identify whether the amount of del16 HER2 is associated with a single copy of HER2 or attainability of pCR. The amount of del16 HER2 was not associated with the HER2 copy number or the percentage of pCR. Loss or UPD of PTEN was found in 6 of 38 BCs. pCR was achieved in 9 of 18 tumors with normal PTEN, but in none of 6 tumors with abnormal PTEN. Because only 1 of 11 tumors with the single copy of HER2 and/or abnormal PTEN achieved pCR after receiving trastuzumab containing regimens, tyrosine kinase inhibitors should be given to the patients with such genetic changes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4549. doi:1538-7445.AM2012-4549

Highlights of This Issue

Successful development of targeted therapeutics now and in the future will hinge on defining patient subsets that are most likely to benefit from new drug candidates. Walter and colleagues combined microfluidics-based gene expression analysis and genome-wide methylation profiling to determine if DNA methylation patterns could classify clinically relevant phenotypic subsets of non–small cell lung cancer (NSCLC). DNA methylation patterns divided NSCLC cell lines and tumors into epithelial-like and mesenchymal-like subtypes. These findings show that DNA methylation profiles can serve as surrogates for gene expression profiles and provide proof-of-principle that DNA methylation differences can be used as a platform for predictive biomarker discovery and development.Overexpression of Auroras is implicated in oncogenic transformation associated with chromosomal instability in human malignancy. In mantle cell lymphoma, Aurora A and B are prognostic markers and therapeutic targets. To investigate Auroras as therapeutic targets, Mahadevan and colleagues evaluated the synergistic combination of MLN8237 with vincristine (MV) in a mouse xenograft model of mantle cell lymphoma and observed that the combination led to tumor regression. However, the tumors returned 2 weeks after treatment. Addition of rituximab to MV resulted in tumor regression and cure, with no relapses 120 days after treatment. Hence, rituximab has synthetic lethality with the MV regimen. A phase I/II study is evaluating MV combined with rituximab in relapsed refractory B-cell lymphoma.The dual-targeting strategy involving phosphoinositide 3-kinase/AKT and RAS/MEK pathways in cancer treatment is particularly intriguing because there is evidence that both pathways are extensively interconnected. Shimizu and colleagues found, through an analysis from a phase I database, that dual inhibition of both pathways may show favorable efficacy compared with single inhibition of either pathway alone, at the expense of greater toxicity in a clinical setting. They also report potentially promising clinical benefits in patients who were treated with combination drug regimens simultaneously targeted to both pathways. These data could provide a platform for further in-depth investigation of molecular profiling and matching of patients with combinations of these targeted drugs for personalized treatment.Diagnostic evaluation of advanced non–small cell lung cancer (NSCLC) could be aided considerably by robust circulating biomarkers. Punnoose and colleagues showed that evaluation of circulating tumor cells (CTC) and circulating tumor DNA can provide information about the molecular characteristics of a patient's tumor from a noninvasive blood draw. They found that mutational analysis of cell-free DNA from blood was more sensitive than analyses of captured CTCs for determining the epidermal growth factor receptor genotype. Moreover, decreases in CTC numbers during treatment of advanced NSCLC patients with targeted therapies were associated with antitumor activity based on imaging endpoints and progression-free survival.

Methylation of the RASSF1A promoter is predictive of poor outcome among patients with Wilms tumor

Wilms tumor (WT) has a survival rate of 90% following multimodality therapy. Nevertheless, there are some groups of patients with event-free survival rates less than 75%. In addition to clinical prognostic factors, loss of heterozygosity at 1p and/or 16q has been used to determine treatment intensity. However, the incidence of this abnormality is low, and new biomarkers are still needed. We analyzed methylation status of three tumor suppressor genes; Ras-association domain family 1 protein, isoform A (RASSF1A), DCR2, and CASP8, in 84 WTs using conventional methylation-specific PCR (cMSP), and the results were correlated with outcome. Furthermore, we analyzed the methylation status of RASSF1A by quantitative MSP (qMSP) in 171 WTs, and evaluated clinical and genetic differences between the methylated and unmethylated tumors. RASSF1A was the most frequently methylated gene identified by cMSP, and associated with a poor outcome. Patients with a RASSF1A-methylated tumor had shorter overall and event-free survival periods (P = 0.043 and 0.018, respectively), when a cut-off value of 7% by qMSP was used. The methylation was more frequent in tumors of older children than younger children (P < 0.001), and in advanced-stage tumors than early stage tumors (P = 0.001). However, multivariate analysis could not confirm the prognostic significance of RASSF1A methylation, possibly because of a small number of advanced stage tumors examined. RASSF1A methylation was correlated with LOH at 1p and/or 16q (P = 0.017), but not with WT1 abnormality, suggesting the methylation and LOH to involve the same tumorigenic pathway. The methylation status of RASSF1A might be a novel biomarker to predict outcome of WT patients.

CpG-island methylation of GATA-family members GATA3 and GATA5 in renal cell carcinoma and association with clinicopathological parameters and progression-free survival.

370 Background: Transcriptional inactivation and CGI methylation of GATA3 and −5 has been reported to be involved in mammary carcinoma, pancreatic cancer, colorectal and gastric carcinogenesis. A recent study demonstrated that a loss of GATA-3 expression due to partially methylation silencing in several renal cell carcinoma (RCC) patients. We quantitatively investigated GATA3 and −5 CGI methylation in RCC and analyzed its association with clinical characteristics as well as progression free survival of patients. Methods: Methylation data were obtained from a quantitative methylation-specific polymerase chain reaction assay (QMSP) for both genes. We investigated 108 RCC and 77 paired tissue samples as well as six RCC cell lines. Statistical analyses were carried out using the paired t-test for matched tumor (TU) and adjacent normal (adN) samples, logistic regression for comparisons of independent samples and cox regression for survival analysis. Results: In paired samples we found a significant higher methylation in TU compared to adN for GATA3 (P=0.007) and for GATA5 (P=3.6*10−9) for all RCCs. GATA5 showed also strong correlations between methylation and status of metastasis (P=0.05) and advanced (pT≥3 and/or N+, M+) tumor samples compared to localized (pT≤2, N0, M0) tumors (P=4.7*10−9). A decreased progression free survival in cox proportional hazard model analysis could be demonstrated for patients with a high GATA5 methylation (P=0.0006, HR=6.5) and a trend could also be seen for GATA3 methylated patients (P=0.06). Conclusions: GATA3 and −5 were identified to demonstrate tumor-specific CGI hypermethylation in renal cell cancer patients. The association of GATA5 CGI methylation with metastasis, advanced disease and progression free survival of patients indicates that epigenetic alterations of both genes are involved in renal cell carcinogenesis. GATA5 methylation could serve as a biomarker for tumor progression. Further prospective and functional investigations are necessary to clarify whether CGI methylation of GATA family members can provide independent information for future clinical management of patients with RCC.

Relationship between Tumor Enhancement, Edema,IDH1Mutational Status,MGMTPromoter Methylation, and Survival in Glioblastoma

Both IDH1 mutation and MGMT promoter methylation are associated with longer survival. We investigated the ability of imaging correlates to serve as noninvasive biomarkers for these molecularly defined GBM subtypes. MR imaging from 202 patients with GBM was retrospectively assessed for nonenhancing tumor and edema among other imaging features. IDH1 mutational and MGMT promoter methylation status were determined by DNA sequencing and methylation-specific PCR, respectively. Overall survival was determined by using a multivariate Cox model and the Kaplan-Meier method with a log rank test. A logistic regression model followed by ROC analysis was used to classify the IDH1 mutation and methylation status by using imaging features. MGMT promoter methylation and IDH1 mutation were associated with longer median survival. Edema levels stratified survival for methylated but not unmethylated tumors. Median survival for methylated tumors with little/no edema was 2476 days (95% CI, 795), compared with 586 days (95% CI, 507-654) for unmethylated tumors or tumors with edema. All IDH1 mutant tumors were nCET positive, and most (11/14, 79%) were located in the frontal lobe. Imaging features including larger tumor size and nCET could be used to determine IDH1 mutational status with 97.5% accuracy, but poorly predicted MGMT promoter methylation. Imaging features are potentially predictive of IDH1 mutational status but were poorly correlated with MGMT promoter methylation. Edema stratifies survival in MGMT promoter methylated but not in unmethylated tumors; patients with methylated tumors with little or no edema have particularly long survival.

Silencing of tumor suppressor genes RASSF1A, SLIT2, and WIF1 by promoter hypermethylation in hereditary breast cancer

Promoter hypermethylation is gaining strength as one of the main mechanisms through which tumor suppressor genes are silenced during tumor progression. Three tumor suppressor genes are frequently found methylated in their promoter, in concordance with absence of expression, RASSF1A, SLIT2, and WIF1. In addition, a previous array-CGH analysis from our group showed that these genes are found in deleted genomic regions observed in hereditary breast cancer tumors. In the present work we analyzed the methylation status of these three tumor suppressor gene promoters in 47 hereditary breast cancer tumors. Promoter methylation status analysis of hereditary breast tumors revealed high methylation frequencies for the three genes (67% RASSF1A, 80% SLIT2, and 72% WIF1). Additionally, the presence of methylated PCR products was associated with absence of protein expression for the three genes and statistically significant for RASSF1A and WIF1. Interestingly, methylation of all the three genes was found in 4 out of 6 grade I invasive ductal carcinoma tumors. Association between RASSF1A methylation and DCIS tumors was found. These results suggest that silencing of these tumor suppressor genes is an early event in hereditary breast cancer, and could be a marker for pre-malignant phenotypes.

DNA methylation silences miR-132 in prostate cancer

Silencing of microRNAs (miRNAs) by promoter CpG island methylation may be an important mechanism in prostate carcinogenesis. To screen for epigenetically silenced miRNAs in prostate cancer (PCa), we treated prostate normal epithelial and carcinoma cells with 5-aza-2'-deoxycytidine (AZA) and subsequently examined expression changes of 650 miRNAs by megaplex stemloop reverse transcription-quantitative PCR. After applying a selection strategy, we analyzed the methylation status of CpG islands upstream to a subset of miRNAs by methylation-specific PCR. The CpG islands of miR-18b, miR-132, miR-34b/c, miR-148a, miR-450a and miR-542-3p showed methylation patterns congruent with their expression modulations in response to AZA. Methylation analysis of these CpG islands in a panel of 50 human prostate carcinoma specimens and 24 normal controls revealed miR-132 to be methylated in 42% of human cancer cases in a manner positively correlated to total Gleason score and tumor stage. Expression analysis of miR-132 in our tissue panel confirmed its downregulation in methylated tumors. Re-expression of miR-132 in PC3 cells induced cell detachment followed by cell death (anoikis). Two pro-survival proteins-heparin-binding epidermal growth factor and TALIN2-were confirmed as direct targets of miR-132. The results of this study point to miR-132 as a methylation-silenced miRNA with an antimetastatic role in PCa controlling cellular adhesion.

Prospective cohort study of radiotherapy with concomitant and adjuvant temozolomide chemotherapy for glioblastoma patients with no or minimal residual enhancing tumor load after surgery

Survival of glioblastoma patients has been linked to the completeness of surgical resection. Available data, however, were generated with adjuvant radiotherapy. Data confirming that extensive cytoreduction remains beneficial to patients treated with the current standard, concomitant temozolomide radiochemotherapy, are limited. We therefore analyzed the efficacy of radiochemotherapy for patients with little or no residual tumor after surgery. In this prospective, non-interventional multicenter cohort study, entry criteria were histological diagnosis of glioblastoma, small enhancing or no residual tumor on post-operative MRI, and intended temozolomide radiochemotherapy. The primary study objective was progression-free survival; secondary study objectives were survival and toxicity. Furthermore, the prognostic value of O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation was investigated in a subgroup of patients. One-hundred and eighty patients were enrolled. Fourteen were excluded by patient request or failure to initiate radiochemotherapy. Twenty-three patients had non-evaluable post-operative imaging. Thus, 143 patients qualified for analysis, with 107 patients having residual tumor diameters ≤1.5 cm. Median follow-up was 24.0 months. Median survival or patients without residual enhancing tumor exceeded the follow-up period. Median survival was 16.9 months for 32 patients with residual tumor diameters >0 to ≤1.5 cm (95% CI: 13.3–20.5, p = 0.039), and 13.9 months (10.3–17.5, overall p < 0.001) for 36 patients with residual tumor diameters >1.5 cm. Patient age at diagnosis and extent of resection were independently associated with survival. Patients with MGMT promoter methylated tumors and complete resection made the best prognosis. Completeness of resection acts synergistically with concomitant and adjuvant radiochemotherapy, especially in patients with MGMT promoter methylation.Electronic supplementary materialThe online version of this article (doi:10.1007/s11060-012-0798-3) contains supplementary material, which is available to authorized users.