RNA from cytology samples to diagnose disease
View Patent ↗The invention relates to methods and kits for detecting the likelihood that a subject has cancer, e.g., squamous cell carcinoma, by assaying the expression levels of tumor associated genes. More specifically, the expression levels of nucleic acids or proteins can be assayed in the tumor associated genes, e.g., beta-2 microgobulin (B2M) and cytochrome p450 1B1 (CYP1B1). The expression levels compared to standards can be indicative of the likelihood a subject has squamous cell carcinoma. For example, over-expression of B2M and under-expression of CYP1B1 can be indicative of the likelihood a subject has squamous cell carcinoma. Also, over-expression of B2M and over-expression of CYP1B1 can be indicative of the likelihood a subject has a precancerous squamous cell disorder. The expression levels of B2M and CYP1B1 can also be repeatedly assayed to monitor the progression of a squamous cell neoplasia.
1. A method for detecting the likelihood that a subject has oral squamous cell carcinoma, comprising:
obtaining a brush cytology sample from a subject,
extracting nucleic acids from cells in the sample,
assaying the nucleic acids for expression levels of non-degraded genes coding for production of beta-2 microgobulin (B2M) and cytochrome p450 1B1 (CYP1B1);
comparing B2M gene to a standard and CYP1B1 gene to a second standard; and
identifying over-expression of the B2M gene compared to the standard, together with under-expression of the CYP1B1 gene compared to the second standard as indicative of a likelihood that the subject has oral squamous cell carcinoma.
2. The method of claim 1 , wherein the brush cytology sample comprises oral squamous cells.
3. The method of claim 1 , wherein the step of obtaining the sample further comprises at least 20 brush strokes.
4. The method of claim 1 , wherein the step of obtaining the sample further comprises 2 initial brush strokes to prime the surface, followed by at least 20 brush strokes to obtain the sample.
5. The method of claim 1 , wherein the step of assaying the nucleic acids further comprises amplifying and quantifying expression of the B2M gene and the CYP1B1 gene by real time polymerase chain reaction using primers complementary to an mRNA sequence of at least 15 bases found near the 5′ ends of the B2M and CYP1B1 genes.
6. A method for detecting the likelihood that a subject has oral squamous cell carcinoma, comprising detecting beta-2 microgobulin (B2M) and cytochrome p450 1B1 (CYP1B1) protein or nucleic acid expression levels in a brush cytology sample from the subject, and identifying over-expression of the B2M gene compared to a standard together with under-expression of the CYP1B1 gene compared to a second standard as indicative of a likelihood that the subject has oral squamous cell carcinoma.
7. A method for monitoring oral squamous cell neoplasia in a human subject over time, comprising:
obtaining a brush cytology sample from a subject at a first time,
extracting nucleic acids from cells in the sample,
assaying said nucleic acids for the expression level of genes coding for the production of beta-2 microgobulin (B2M) and cytochrome p450 1B1 (CYP1B1), and
repeating the steps of obtaining a sample, extracting nucleic acids and assaying for expression levels of B2M and CYP1B1 at a later time,
wherein increased expression of the B2M gene at a later time or decreased expression of the CYP1B1 gene at a later time is indicative of progression of neoplasia.