Question

Clinical Background: A 40-year-old male patient complained of a right frontal headache and right neck pain for the duration of 4 weeks. The pain was associated with intermittent blurred vision in the right eye and three episodes of morning emesis without nausea. The patient denied dizziness, fever, diplopia, change in hearing, tinnitus, vertigo, weakness, numbness, difficulty speaking or with language, swallowing, eating, or gait. He reported no recent travel and no ill contacts. A head CT scan showed a large, primarily cystic lesion within the right frontal lobe containing areas of calcification with surrounding vasogenic edema. There was significant mass effect on the right lateral ventricle with a shift of midline structures to the left and evidence of subfalcine herniation. The patient underwent exploratory craniotomy and a frozen section was diagnosed as high-grade glioma. A total resection of the tumor was performed. The final pathological diagnosis was anaplastic oligodendroglioma (Figure a). A hematoxylin and eosin stain of the patient's tumor revealed classical histological features of oligodendroglioma with round nuclei, perinuclear halos, and delicate capillaries. Glial tumors are classified based on their morphological resemblance to normal anatomic counterparts. Anaplastic oligodendrogliomas have aggressive behavior with early recurrence and progression and short overall survival. Loss of chromosome arms 1p and 197 is seen in 85% of oligodendrogliomas and 50-60% of anaplastic oligodendrogliomas. Generally, tumors with 1p/19q co-deletion have a good response to chemotherapy and radiotherapy and oligodendrogliomas with 1p/19 loss have significantly better progression free and overall survival than those without the co-deletion. Therefore, 1p/19q loss represents a reliable marker of biologic behavior and therapeutic response. The 1p/19 co-deletion is also strongly associated with tumor location. Oligodendrogliomas most commonly arise in the frontal lobe, less commonly in the occipital lobe. Almost 90% of oligodendroglial tumors arising in the frontal lobe have 1p/19q co- deletion.

1. Explain the results in the Methylation Specific PCR (MSP) above. Be specific

2. The positive control in the lower panel is much stronger than the patient samples. What control would be better (or in addition to this control) to detect the weak expression in T3 and T4? Be specific

Answer 1

Ans)Methylation analysis is the study of chromosomal patterns of DNA or histone modification by methyl groups. Methylation constitutes an epigenetic mark because its location can affect the regulation of gene expression in a heritable fashion.

- One of the most common methods for determining methylation status on DNA sequences is sodium bisulfite conversion. Due to similarities in base pairing characteristics between methylated and unmethylated cytosines on CpG dinucleotides, standard methods cannot distinguish between different methylation states.

- Vertebrate DNA methylation typically occurs at sites of CpG sequence in the genome when DNA methyltransferase converts cytosine to 5-methylcytosine (Me-CpG). CpG methylation results in reduced transcriptional activity and, thus, is a mechanism for regulating gene expression.

- Production rates of T4 and T3 can be estimated, but the techniques are time-consuming and expensive, and would be indicated only infrequently even if readily available. Similarly, a free T3 has been used on a limited basis, but it too is not generally available.