MS-MLPA: an attractive alternative laboratory assay for robust, reliable, and semiquantitative detection of MGMT promoter hypermethylation in gliomas.

Expression of the DNA repair protein O6-alkylguanine-DNA-alkyltransferase (AGT), encoded by the O6-methylguanine (O6-mG) -DNA-methyltransferase (MGMT) DNA repair gene, results in resistance to alkylating agents, and hypermethylation of the MGMT promoter is associated with chemosensitivity as it prevents AGT expression. As the interpretation of the results of immunohistochemistry to evaluate AGT expression proved to be difficult, the aim of our present study is to establish a feasible, reliable, and robust method for MGMT promoter hypermethylation testing that can be easily implemented in a diagnostic setting and is applicable to routinely processed tissue. MGMT hypermethylation analysis using methylation-specific (MS-) multiplex ligation-dependent probe amplification (MLPA) was performed on 62 glioma samples of 55 individual tumors (including 12 cell lines) and compared to the more conventionally used, but improved, MS-polymerase chain reaction (PCR). In contrast to MS-PCR, MS-MLPA (i) is not based on bisulfite conversion of unmethylated cytosines (a somewhat troublesome step in MS-PCR), (ii) provided methylation status of all samples, (iii) proved to be semiquantitative, (iv) can be used to evaluate methylation status of multiple sequences (CpG dinucleotides) simultaneously, and (v) allows for a combined copy number detection and methylation specific analysis. The potential therapeutic value of MGMT hypermethylation evaluation using MS-MLPA was shown in a group of 20 glioblastoma patients receiving temozolomide chemotherapy. We conclude that MS-MLPA is a robust and reliable method that can be easily applied to differently processed tissues, including those fixed in formalin and embedded in paraffin. The semiquantitative aspect of MS-MLPA may prove to be of great value, especially in predicting response to alkylating agents, not only for gliomas as evaluated in this study but also for tumors in general.

Uncommon breast tumors in perspective: incidence, treatment and survival in the Netherlands.

The relatively small group of patients with breast tumors other than the ductal, lobular or mixed ducto-lobular types, has reached nonnegligible numbers due to the ongoing increase in the incidence of breast cancer. We investigated stage and grade distribution of uncommon breast tumors using the nation-wide Netherlands Cancer Registry (population 16.5 million) and incidence patterns, treatment and long-term survival (up to 19 years) using the regional Eindhoven Cancer Registry (population 2.4 million). Incidence of all uncommon breast tumors together was 9.2/100,000 person years (age-standardized, ESR). The proportion of stage I tumors was 70% among patients with tubular (n = 3,456) and 40-50% for mucinous (n = 3,482), papillary (n = 1,078), cribriform (n = 503) and neuroendocrine (n = 76) tumors, contrasting to 27, 28 and 36%, respectively among patients with Signet ring cell cancer (n = 75), Paget's disease (n = 818) and the common invasive ductal carcinomas (n = 121,656). A better age-, stage-, and grade-adjusted prognosis was observed for patients with lobular (death risk ratio 0.8, 95%CI: 0.7-0.9), mucinous (0.5, 0.3-0.9), medullary (0.5, 0.3-0.9) and tubular (0.4, 0.2-0.6) carcinoma or phyllodes tumor (0.02, 0.0-0.2), compared with invasive ductal carcinomas. For patients with papillary (0.6, 0.2-1.6) and cribriform (0.1, 0.0-5.1) tumors better prognosis was not statistically significant. In conclusion, histologic type was an essential determinant of survival for about 10% of all newly diagnosed women with invasive breast cancer. Because patients with mucinous, tubular, medullary and phyllodes tumors have such a good prognosis, less aggressive treatment should be considered in some cases whereby specific guidelines are becoming increasingly desirable. Communication to patients with these specific histological types should reflect this.