Sergey Kuznetsov Capstone

Development of a Comprehensive Mutational Pannel as an Effective Tool for Personalized Diagnostic of Medullary Thyroid Carcinomas

 

Medullary Thyroid Carcinoma (MTC) originates from mutations in calcitonin-producing parafollicular C cells of the thyroid, is a rare malignancy, accounting for 3-4% of all thyroid carcinomas. It occurs in a hereditary form (HMTC, 25%) or in a sporadic form (SMTC, 75%). The prognosis for patients with MTC is poor, as the tumor metastasizes at early stages; and the only curative therapeutic option so far is radical surgery. Genetic analysis helps identify inherited cases at a stage where prophylactic surgery can be offered to carriers of such mutations to prevent the disease. This approach may also be used to determine better treatment options for patients who are already diagnosed with MTC.

The goal of this project was to develop a comprehensive mutational panel for the detection of clinically relevant mutations in MTC samples. A total of 143 mutations (nucleic acid variations) in 8 human genes were selected from numerous papers and public databases and included into the MTC mutational panel. The selection criteria were based on the coding mutations (mutations that occur in the coding area of the genes) that were reported to occur in MTC patients and considered functionally relevant. Some of the mutations included into the MTC mutational panel (such as BRAF gene V600E mutation) were also described in other types of thyroid cancer, but most of the mutations were unique to MTC (such as all RET gene mutations). The entire assay design was carried out using Sequenom's online design tools (ProxSNP and PreXTEND (https://www.mysequenom.com/Tools) and Assay Design software (v. 3.1)).

The final file comprised from 115 assays corresponding to all 143 mutations included into the MTC panel. This file will be further processed using the SEQUENOM® Mass-ARRAY iPLEX® platform for DNA genotyping of clinical samples by the cancer research scientists at the Abramson Cancer Center of the University of Pennsylvania.

 

References:

1. Millis, M. (2011, Summer). Medium-Throughput SNP Genotyping Using Mass Spectrometry: Multiplex SNP Genotyping Using the iPLEX® Gold Assay. Springer Protocols, 700. Retrieved August 20, 2012, from http://link.springer.com/protocol/10.1007%2F978-1-61737-954-3_5

This paper discusses and explains the basics of genotyping with the using MALDI-TOF Mass Spectrometry. This was the first paper that I have read right after being given the project. After fully interpreting this paper, I realized that I am going to be able to finish the project on my own. This paper served as a guide to me throughout the process of doing this project, as well as while writing the entire research report. In addition, this paper was used to make sure that I am not saying something that is factually incorrect.

 

2. Gabriel, S., Ziaugra, L., & Tabbaa, D. (2009, January 1). UNIT 2.12 SNP Genotyping Using the Sequenom MassARRAY iPLEX Platform. Current Protocols in Human Genetics.

This paper describes in details the SNP genotyping method based on the Sequenom MassARRAY platform. It includes two step protocol (initial locus-specific PCR reaction, followed by single base extension using mass-modified dideoxynucleotide terminators) an assay structure and how using MALDI-TOF mass spectrometry identify the SNP allele. The paper is mentioned in the corresponded section of the Introduction.

 

 

3.      Ricarte-Filho, J., Ryder, M., Ghossein, R., Fagin, J., Chitale, D., Rivera, M., et al. (2009, June 1). Mutational Profile of Advanced Primary and Metastatic Radioactive Iodine-Refractory Thyroid Cancers Reveals Distinct Pathogenetic Roles for BRAF, PIK3CA, and AKT1. CANCER RESEARCH. Retrieved July 1, 2012, from

http://cancerres.aacrjournals.org/content/69

The paper describes profiling of 111 mutations in RET, BRAF, NRAS, HRAS, KRAS, PIK3CA, AKT1 genes in clinical poorly differentiated, anaplastic and radioactive iodine-refractory differentiated thyroid cancers. The genotyping method is based on the Sequenom MassARRAY platform. It was shown that RAS mutations were prevalent in primary PDTC, whereas BRAF was more common in metastatic PDTC and ATC. PIK3CA or AKT1 mutations were rare. The paper is mentioned in the corresponded section of the Introduction.

 

4. MEN2 Database. (n.d.). AURP Scientific Resource for Research and Education.. Retrieved July 12, 2012, from http://arup.utah.edu/database/MEN2/MEN2_display.php

This database was only used for its mutations list during the collection phase of the project.

 

5. Human BLAT Search. (n.d.). UCSC Genome Browser. Retrieved August 17, 2012, from http://genome.ucsc.edu/cgi-bin/hgBlat

The Human BLAT database was used for alignment of nucleic sequences. The sequences retrieved from COSMIC were inputted in the BLAT Database to be aligned with the rest of the nucleic sequence, as only a small part of it could be gathered from COSMIC.

 

6. Catalogue of Somatic Mutations in Cancer - COSMIC. (n.d.). Wellcome Trust Sanger Institute. Retrieved July 10, 2012, from http://www.sanger.ac.uk/genetics/CGP/cosmic/

This database was used to retrieve the nucleic sequences that were used as the base to be inputted in the BLAT database. As it is impossible to use BLAT database with only knowing the position of the mutation, COSMIC was used to retrieve the minimal part of the sequence required to find the full nucleic sequence for any particular gene.

 

7. Genetics of Endocrine and Neuroendocrine Neoplasias (PDQ®). (n.d.). National Cancer Institute. Retrieved July 12, 2012, from http://www.cancer.gov/cancertopics/pdq/genetics/medullarythyroid/HealthProfessional/Table4

This database was only used for its mutations list during the collection phase of the project.

 

8. OMIM Entry - # 171400 - MULTIPLE ENDOCRINE NEOPLASIA, TYPE IIA; MEN2A . (n.d.). OMIM - Online Mendelian Inheritance in Man . Retrieved July 19, 2012, from http://omim.org/entry/171400

This entry was used as the basis of understanding of the MEN 2A syndrome. It talks about the relationship between MTC and diseases such as pheochromocytoma, and about the basics of what MEN 2A syndrome actually is. In addition, it talks about what mutations are associated with the syndrome. It was specifically used for comparison with the MEN 2B and FMTC.

 

9. Jimenez, C., Hu, M. I., & Gagel, R. (2008, Spring). Management of Medullary Thyroid Carcinoma. Elsevier Saunders, ?, 15.

This MTC review was the first of many that I have read in the duration of this project. This review provided me with the basic information about MTC without which any attempt at actually finishing this project would have been obsolete. Many parts of the introduction are referred to this paper, as it was very influential. Similarly to many other sources, mutations were taken from this publication during the first phase of the project.

 

10. MacConaill, L Profiling Critical Cancer Gene Mutations in Clinical Tumor Samples. PLoS ONE (2009). http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007887

This publication was one of many that were used only for its mutations. No part, except for the abstract, which contributed to the overall idea of the research report, has been read.

 

11. Ehsan Alvandi, Seyed Mohammad Akrami, Mohsen Chiani, Mehdi Hedayati, Babak Noori Nayer, Mohammad Reza Mohajeri Tehrani,et al. (2011, April 5). Molecular Analysis of the RET Proto-Oncogene Key Exons in Patients with Medullary Thyroid Carcinoma: A Comprehensive Study of the Iranian Population. Thyroid, 1. Retrieved September 1, 2012, from http://online.liebertpub.com/doi/abs/10.10

This publication was one of many that were used only for its mutations, and or one small piece of information. No part, except for the abstract, which contributed to the overall idea of the research report, has been read.

 

12. Moura, M., Cavaco, B., Pinto, A., & Leite, V. (2011, February 16). High Prevalence of RAS Mutations in RET-Negative Sporadic Medullary Thyroid Carcinomas. JCEM ONLINE, 95, 6.

This paper has shown a study where 64% of the patients that had Sporadic MTC were found to have a BRAF mutation in position 600. This is very unusual as this mutation is considered to be PTC specific. In addition, this publication has given additional context to this research report. Similarly to many other sources, mutations were taken from this publication during the first phase of the project.

 

13. OMIM Entry - # 162300 - MULTIPLE ENDOCRINE NEOPLASIA, TYPE IIB; MEN2B . (n.d.). OMIM - Online Mendelian Inheritance in Man . Retrieved June 19, 2012, from http://omim.org/entry/162300 

This entry was used as the basis of understanding of the MEN 2B syndrome. It talks about the relationship between MTC and diseases such as pheochromocytoma, and about the basics of what MEN 2B syndrome actually is. In addition, this entry talks about what mutations are associated with the syndrome. It was specifically used for comparison with the MEN 2A and FMTC.

 

14. hybridization, f. i., & (1989), I. e. (n.d.). OMIM Entry - + 164761 - REARRANGED DURING TRANSFECTION PROTOONCOGENE; RET . OMIM - Online Mendelian Inheritance in Man . Retrieved July 19, 2012, from http://omim.org/entry/164761

This entry was used as the basis of understanding MTC, and why the mutations RET gene are found in so many cases of MTC. Unfortunately, as with many other publications, no definitive answer was given. This entry was also used as a guide, to make sure that what I say about mutations in RET gene is factually correct.

 

15. RT-PCR., & (2003), M. e. (n.d.). OMIM Entry - # 155240 - THYROID CARCINOMA, FAMILIAL MEDULLARY; MTC . OMIM - Online Mendelian Inheritance in Man. Retrieved July 19, 2012, from http://omim.org/entry/155240

This entry was used as the basis of understanding of the FMTC syndrome. It talks about the relationship between MTC and diseases such as pheochromocytoma, and about the basics of what FMTC syndrome actually is. In addition, this entry talks about what mutations are associated with the syndrome. It was specifically used for comparison with the MEN 2B and MEN 2A.

 

16. Hazard, J., Hawk, W., & Crile, G. (1959, January 1). MEDULLARY (SOLID) CARCINOMA OF THE THYROID—A CLINICOPATHOLOGIC ENTITY. JCEM, 19. Retrieved June 26, 2012, from http://jcem.endojournals.org/content/19/1/152

This publication was the first time MTC was classified. The paper itself was not read by this applicant, however, due to its historic relevance, it was referred to in the first paragraph of the introduction.

 

 

17. Cakir, M., & Grossman, A. (2009, May 25). Medullary Thyroid Cancer: Molecular Biology and Novel Molecular Therapies. Neuro Endocrinology, 25.

This publication, alongside many MTC reviews, was used as the guideline for this research report. Many facts, such as information about Sporadic and Hereditary MTC were confirmed by this publication. Similarly to many other sources, mutations were taken from this publication during the first phase of the project.