Evaluation of Novel Melanoma Biomarkers in Human Patients Michelle Min, Byungwoo Ryu, Rhoda Alani Department of Dermatology, Boston University School of Medicine Boston, MA 02118 Introduction Methods: Quantitative Real-time PCR Results Cont. Melanoma and its Significance Tissue mRNA cDNA Quantifying HELLS, NCAPH, SPINT2 Definition: A form of cancer that begins in melanocytes (cells that 1. Scrape human tissue from glass slides: 12 each for nevi (N), primary 1. Set up qPCR reaction (cDNA, primers, probe, Taqman PCR master mix solution) 5A) HELLS 5B) SPINT2 make the pigment melanin). It most commonly begins in moles but can melanoma (PM), metastatic melanoma (MM)). in 3 repeats of each reaction. 2. Add equal amounts of Luciferase to each sample in order to ensure that all P=0.03 P=0.04 also begin in other pigmented tissues, such as the eye or intestines. • Positive control: cDNA from melanoma cell line; Negative control: water samples and reactions are treated identically. Separately quantify HELLS, NCAPH, SPINT2, Actin, and Melan-A. • 3. Extract and purify mRNA: proteinase K digestion, DNase treatment o Actin and Melan-A eliminate differences in gene expression of our Melanoma accounts for less than 5% of skin cancer cases yet causes 4. Perform reverse transcription to convert mRNA into cDNA. proteins of interest (HELLS, NCAPH, and SPINT2) due to sample size. the majority of skin cancer-related deaths. Frighteningly, (Tissue samples were not provided in equal volumes.) Actin is present in 1.0 incidences of melanoma have been on a steep rise. In fact, in 2012, Optimizing qPCR Conditions/ Standard Curve all eukaryotic cells. Melan-A is a surface protein on melanocytes. P=0.05 P=0.03 Each qPCR reaction quantifies Luciferase expression, which should be equal 76,250 new cases were reported in the US. It is estimated that 1 in 1. Isolate and purify cDNA of HELLS, NCAPH, SPINT2, Actin, and Melan-A, • -9 -12 -15 in each reaction; variations are due to human error and are normalized. 50 people will be diagnosed with melanoma in their lifetimes. diluting them to 3 concentrations: 10 M, 10 M, and 10 M. 2. Obtain standard curves through qPCR with the purified cDNA of known .50 concentrations, hence confirming our designed primers’ and probes’ accuracy Data Analysis and allowing us to calculate copy numbers from unknown cDNA (N, PM, MM). 1. Obtain “cT ratio” = Individual Luciferase cT / Average Luciferase cT 3. Determine primer and probe concentrations that result in lowest cT values; 2. Normalize HELLS or SPINT2 cT values = HELLS or SPINT2 cT / “cT ratio.” lower cT values correlate with higher expression levels, thus optimal conditions. 3. Obtain HELLS or SPINT2 “Log of Copy Number”: Substitute the above number into .00 4. Identify optimal qPCR temperature and cycling: the linear equation calculated from standard curves. (See data below). 95°C 5 min; 60x: 95°C 30 sec,55°C 30 sec, 62°C 30 sec. 4. Normalize HELLS or SPINT2 copy numbers relative to Actin or Melan-A’s. Ratio (SPINT2 vs. Melan-A copynumbers) Ratio (SPINT2 Melan-A vs. copynumbers) Ratio (HELLS Melan-A vs. Nevi PM MM Nevi PM MM Figure 5. After performing statistical analysis (student’s t-test), we concluded that HELLS expression was significantly higher Results: HELLS and SPINT2 Analysis (5A) while SPINT2 expression was significantly lower (5B) in metastatic melanoma (MM) in comparison to nevi and primary Figure 1. Less than 10% of patients with stage IV melanoma Similar patterns of expression were observed for both HELLS and SPINT2 melanoma (PM) samples (p≤0.05). survive 15 tears after diagnosis, despite treatment. Survival Standard Curves when data was normalized to either Actin or Melan-A. However, normalization against the expression levels of Melan-A seemed to be a curves in localized melanoma (stages I and II), regional 9.00E+00 metastases (stage III), and distant metastases (stage IV) differ better method of analysis since Melan-A is a surface antigen found drastically. Therefore, early detection and treatment is critical. 8.00E+00 specifically on melanocytes while Actin is found in all eukaryotic cells. NCAPH 7.00E+00 y = -0.2807x + 12.753 The Search for New Melanoma Biomarkers R² = 1.0000 HELLS Relative to Melan-A 4A) In cancer, global changes in gene expression can occur in activators of 2.5 6.00E+00 A - cell cycle progression and tumor suppressor genes. 2 5.00E+00 Melan Conclusions 1.5 •HELLS and NCAPH (helicase, lymphoid-specific and non-SMC vs. 4.00E+00 SPINT2 Our assay proved sensitive for the detection of metastatic melanoma: condensin I complex, subunit H): activators of cell cycle y = -0.3607x + 12.565 1 Log (Copy#) •HELLS: an activator of cell cycle progression, detected at higher progression, shown to be upregulated in aggressive metastatic R² = 0.9839 3.00E+00 0.5 Copy Copy Numbers) expression levels in metastatic human tissue by our qPCR assay tumor cell lines 0 •SPINT2: a tumor suppressor gene, detected at lower levels in •SPINT2 (serine peptidase inhibitor, Kunitz type 2): implicated 2.00E+00 HELLS Ratio (HELLS metastatic human tissue by qPCR– sometimes undetected y = -0.2789x + 12.332 Nevi PM MM as a tumor suppressor gene; expression induces cell apoptosis, 1.00E+00 R² = 0.9991 so is assumed to be downregulated in metastatic cancer Clinical implications: As current standard of care uses costly testing 4B) SPINT2 Relative to Melan-A 0.00E+00 0.0014 via PET-CT scanning to evaluate disease recurrence/tumor burden in A 10 15 20 25 30 35 40 - 0.0012 patients with melanoma, development of a simple test for melanoma C Mean T 0.001 Melan will allow for rapid and inexpensive methods of screening. vs. vs. 0.0008 Future studies: We hope to show that levels of our biomarker panel Figure 3. Linear equations were calculated from Standard Curves 0.0006 correlate with disease burden and tumor response to therapy, for HELLS, NCAPH, and SPINT2 2 0.0004 with R values nearly 1.0 in value, Copy Numbers) particularly in stage IV disease. Ultimately detecting these biomarkers suggesting our qPCR assay can reliably quantify cDNA concentrations 0.0002 in serum, rather than tissue, would be an even more non-invasive, rapid -15 Ratio (SPINT2 as low as 10 M for our proteins of interest. 0 screen available for patients. Nevi PM MM Additional standard curves were obtained for Actin and Melan A, also 2 implying a strong assay, with linear equation R values approximately 1.0: Figure 2. HELLS is found on chromosome 10, NCAPH on Figure 4. Copy number values calculated after completing data chromosome 2, SPINT2 on chromosome 19 in humans. All 3 Actin: y = -0.2563x + 11.445; R² = 1.0000 analysis (see calculations in Methods) seem to suggest that in genes have been studied in the role of other cancers. Melan-A: y = -0.2502x + 11.844; R² = 0.9975 metastatic melanoma samples (MM), HELLS expression levels are increased (4A) while SPINT2 levels are decreased (4B) in comparison Objective: To identify genetic fingerprints that may prove useful as Obtaining standard curves allowed us to confidently move on to qPCR to nevi and primary melanoma (PM) samples. It must be noted that for References Thank You diagnostic and/or prognostic biomarkers for melanoma progression. reactions, in which we measured the gene expression levels of HELLS, SPINT2, 2 samples of each nevi and PM had at least 1 of 3 repeats 1. Tsao, H., Atkins, M.B., Sober, A.J. Management of Cutaneous I thank Rhoda Alani, MD and SPINT2, and NCAPH in nevi (N), primary melanoma (PM), and metastatic Melanoma: N Engl J Med 351, 998-1012 (2004). unable to amplify in qPCR. On the other hand, 8 of the 12 MM samples 2. National Cancer Institute. Melanoma. (Accessed November 27, 2012, Byungwoo Ryu, PhD, my mentors in 1. Optimize quantitative RT-PCR (qPCR) conditions in detecting melanoma (MM). Unfortunately, although we could obtain a promising at http://www.cancer.gov/cancertopics/types/melanoma). the laboratory, for guiding and had at least 1 of 3 repeats not amplify. Nevi and PM samples were quite 3. Ryu, B., Kim, D.S., Deluca, A.M., Alani, R.M. Comprehensive expression encouraging me. I also thank the traces of HELLS, NCAPH, and SPINT2 (obtain standard curves). standard curve for NCAPH, we could not reproducibly detect NCAPH profiling of tumor cell lines identifies molecular signatures of small, perhaps contributing to the occasional 0 copy numbers (cT=0 ); Medical Student Summer Research expression in human tissue samples . It is likely that this is because there melanoma progression: PLoS One 2, e594 (2007). Program through the Boston 2. Quantify and compare the expression of HELLS, NCAPH, and however, MM samples were much larger, so the fact that the majority of 4. Dong, W., Chen, X., Xie, J., Sun, P., Wu, Y. Epigenetic inactivation and SPINT2 by qPCR in patient samples: nevi (benign birthmarks were only trace amounts of NCAPH, which resulted in variation in tumor suppressor activity of HAI-2/SPINT2 in gastric cancer: Int J University School of Medicine with MM samples had at least 1 of 3 repeats with 0 copy numbers is more Cancer 127, 1526-1534 (2010). special thanks to donators Karen and moles), primary melanoma, and metastatic melanoma. expression quantification (data not shown). likely due to the nature of MM rather than small volume of tissue. 5. National Center for Biotechnology Information. (Accessed January 22, Antman, MD and Jerry Serchuck, MD. 2013, at http://www.ncbi.nlm.nih.gov/gene). www.postersession.com .